<td>Under development: Not open for comment. Do not cite</td>
<td>Open for citation & comment</td>
<td></td>
<td></td>
<td></td>
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</div>
<div id="abstract">
<h2>Abstract</h2>
<p><span style="background-color:transparent; color:#000000; font-family:Calibri,sans-serif; font-size:11pt">The focus of this AOP is on inactivation of Glycogen synthase kinase 3 beta (Gsk3b) by different chemicals which leads to defects in developing inner ear of zebrafish. Inactivation of Gsk3b leads to repressed expression of </span><em>gbx2</em><span style="background-color:transparent; color:#000000; font-family:Calibri,sans-serif; font-size:11pt"> (KE1) which consequently increases expression of two genes </span><em>foxi1 </em><span style="background-color:transparent; color:#000000; font-family:Calibri,sans-serif; font-size:11pt">(KE2) and </span><em>six1b </em><span style="background-color:transparent; color:#000000; font-family:Calibri,sans-serif; font-size:11pt">(KE3). Increase in </span><em>six1b</em><span style="background-color:transparent; color:#000000; font-family:Calibri,sans-serif; font-size:11pt"> expression leads to inhibited expression of eya1 (KE4). Changes on molecular level (MIE-KE4) leads to changes at cellular level such as increased cell death in developing inner ear (KE5).</span><em> </em><span style="background-color:transparent; color:#000000; font-family:Calibri,sans-serif; font-size:11pt">Alterations in inner ear (KE6) translate to (AO) decrease in population trajectory through reduced hearing (KE7) and increased mortality (AO). An overall assessment of this AOP shows that there is low to moderate biological plausibility to suggest a qualitative link between the repression of Gsk3b expression to the KE4-cell death within developing inner ear and high evidence linking KE5 to increased mortality (AO). Currently there is not enough data for an appropriate assessment of essentiality of KEs and empirical support. KEs on molecular level have some uncertainties like </span><em>foxi1</em><span style="background-color:transparent; color:#000000; font-family:Calibri,sans-serif; font-size:11pt"> loss of function experiment resulting in no expression of six1b in otic placode and inconsistencies between zebrafish and mouse (<em>six1b</em> and <em>eya1</em> role in otic placode development).</span></p>
</div>
<div id="background">
<h3>Background</h3>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:"Times New Roman",serif">The motivation behind building the AOP was methodological. Our team has recently developed molecular causal networks for developmental cardiotoxicity and neurotoxicity in zebrafish (</span></span></span><a href="https://doi.org/10.1021/acs.chemrestox.0c00095" title="DOI URL">doi.org/10.1021/acs.chemrestox.0c00095</a><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:"Times New Roman",serif">). These networks are highly curated, but rather large, going from adverse outcomes on the organ level upstream to wherever evidence takes us (many times finishing at what would be called MIEs). As there are many causal networks already present on the </span><a href="http://causalbionet.com/" style="color:#0563c1; text-decoration:underline"><span style="font-family:"Times New Roman",serif">http://causalbionet.com/</span></a><span style="font-family:"Times New Roman",serif"> (mostly for humans and for lung conditions), we were wondering how the rich knowledge available in causal pathways could be translated to AOPs. The AOP described in this document is one such example. </span></span></span></p>
<h2>AOP Development Strategy</h2>
<div id="context">
<h3>Context</h3>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:"Times New Roman",serif">The motivation behind building the AOP was methodological. Our team has recently developed molecular causal networks for developmental cardiotoxicity and neurotoxicity in zebrafish (</span></span></span><a href="https://doi.org/10.1021/acs.chemrestox.0c00095" title="DOI URL">doi.org/10.1021/acs.chemrestox.0c00095</a><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:"Times New Roman",serif">). These networks are highly curated, but rather large, going from adverse outcomes on the organ level upstream to wherever evidence takes us (many times finishing at what would be called MIEs). As there are many causal networks already present on the </span><a href="http://causalbionet.com/" style="color:#0563c1; text-decoration:underline"><span style="font-family:"Times New Roman",serif">http://causalbionet.com/</span></a><span style="font-family:"Times New Roman",serif"> (mostly for humans and for lung conditions), we were wondering how the rich knowledge available in causal pathways could be translated to AOPs. The AOP described in this document is one such example. </span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-family:"Calibri",sans-serif">6-bromoindirubin- 3′-oxime (6BIO), a hemi-synthetic derivative of indirubins found in edible mollusks and plants, is a potent inhibitor of Glycogen synthase kinase 3β (Gsk-3β) </span><span style="font-family:"Calibri",sans-serif">(Sklirou <em>et al.</em>, 2017)</span><span style="font-family:"Calibri",sans-serif">. In zebrafish embryos treated with BIO, a selective GSK3 inhibitor that activates Wnt signaling, gbx2 expression was specifically repressed in the telencephalon, but was unaffected or weakly activated in the isthmus and OV </span><span style="font-family:"Calibri",sans-serif">(Wang <em>et al.</em>, 2018)</span><span style="font-family:"Calibri",sans-serif">.</span></span></span></p>
</div>
<div id="overall_assessment">
<h2>Overall Assessment of the AOP</h2>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">An overall assessment of this AOP shows that there is low to moderate biological plausibility to suggest a qualitative link between the inactivation of Gsk3b to the KE4-cell death within developing inner ear and high evidence linking KE5 to increased mortality (AO). Biological plausibility is considered moderate because there is ample evidence from gain- and loss- of function experiments and knock out animal models that support the relationships between key events which are consistent with current biological knowledge, but there is mostly indirect evidence linking KEs on molecular level. KEs on molecular level have some uncertainties like <em>foxi1</em> loss of function experiment resulting in no expression of <em>six1b</em> in otic placode (due to absence of otic placode) and inconsistencies across species (zebrafish, mouse). The evidence for essentiality of the KEs is mostly missing therefore the overall assessment of essentiality is low. The same goes for empirical support, currently there is no evidence for empirical support. Additional studies are needed to obtain data for empirical support, therefore, the empirical support of KERs is considered is low.</span></span></p>
<p dir="ltr"><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Life stage:</strong> The current AOP is applicable from 2-8 cell stage (1,25 hpf; start of <em>Gsk3b</em> expression in zebrafish) (Valenti, 2015) up to 96 hpf wich is the expression limit of <em>six1b</em> in the developing inner ear (Webb & Shirey, 2003).</span></span></p>
<p dir="ltr"><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Taxonomic:</strong> This AOP is based on experimental evidence from studies on zebrafish, but is potentially also relevant to other vertebrates, because of conservation of all involved key events (Wnt signalling-Gsk3b, Gbx2, Eya1). But there are certain differences especially between zebrafish and mouse. Foxi1 gene is critical for zebrafish otic induction (Solomon et al., 2003), while it is not essential for this process in mice (Hulander et al., 2003). Interactions between Six1b and other members ofthe Pax–Six–Eya–Dach gene network, such as Eya1, also seem to differ between mouse and zebrafish (Li et al., 2003; Zheng et al., 2003).</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Sex: </strong>Sex differences are typically not investigated in tests using early life stages of zebrafish and it is currently unclear whether sex-related differences are important in this AOP.</span></span></p>
<td><span style="font-family:Arial,Helvetica,sans-serif"><strong>Support for Essenti<span style="font-size:16px">ality of KEs</span></strong></span></td>
<td>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px"><strong>Defining Question</strong>: Are downstream KEs and/or the AO prevented if an upstream KE is blocked?</span></span></p>
<ul>
<li><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>High (Strong):</strong> Direct evidence from specifically designed experimental studies illustrating essentiality for at least one of the important KEs (e.g. stop/reversibility studies, antagonism, knock out models, etc.).</span></span></li>
<li><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Moderate:</strong> Indirect evidence that sufficient modification of an expected modulating factor attenuates or augments a KE leading to increase in KE down or AO.</span></span></li>
<li><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Low (Weak):</strong> No or contradictory experimental evidence of the essentiality of any of the KEs.</span></span></li>
<td><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Low:</strong> No experimental evidence of essentiality.</span></span></td>
</tr>
<tr>
<td><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>KE1:</strong> Repression of <em>gbx2</em> expression</span></span></td>
<td><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Low:</strong> No experimental evidence of essentiality.</span></span></td>
<td><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>High:</strong> When <em>foxi1</em> is knock down no expression of <em>six1b</em> is detected in otocyst (Bricaud and Collazo, 2006).</span></span></td>
<td><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Moderate:</strong> <em>Six1b</em> gain/loss-of-function experiment results indicate that in both cases normal development of inner ear is affected (KE5) (Bricaud and Collazo, 2006).</span></span></td>
<td><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Low:</strong> No experimental evidence of essentiality.</span></span></td>
<td><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>High:</strong> One of key players in normal development of sensory organs (KE6) (Whitfield et al., 2002; Kozlowski et al., 2005).</span></span></td>
<td><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Low:</strong> No experimental evidence of essentiality.</span></span></td>
<td><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Moderate:</strong> One of the factors that are responsible for higher rate of mortality in fish (KE8) (Kasumyan, 2009).</span></span></td>
<td><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>High:</strong> Inability to perceive the environment leads to increase in mortality (Besson et al., 2020).</span></span></td>
</tr>
<tr>
<td><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>AO:</strong> Decrease of population trajectory</span></span></td>
<td><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>High:</strong> decrease in population trajectory is an imminent result of increased mortality (Rearick et al., 2018).</span></span></td>
<td><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Support for Biological Plausibility of KERs</strong></span></span></td>
<td>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Defining Question:</strong> Is there a mechanistic relationship between KEup and KEdown consistent with established biological knowledge?</span></span></p>
<ul>
<li><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>High (Strong)</strong>: Extensive understanding of the KER based on extensive previous documentation and broad acceptance.</span></span></li>
<li><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Moderate</strong>: KER is plausible based on analogy to accepted biological relationships, but scientific understanding is incomplete.</span></span></li>
<li><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Low (Weak)</strong>: Empirical support for association between KEs, but the structural or functional relationship between them is not understood.</span></span></li>
</ul>
</td>
</tr>
<tr>
<td><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>KER1:</strong> Gsk3b inactivation leads to repression of <em>gbx2</em> expression</span></span></td>
<td><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>High: </strong>There is extensive evidence linking inhibition of Gsk3b to activation of canonical Wnt pathway for which Gbx2 is representative marker.</span></span></td>
</tr>
<tr>
<td><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>KER2:</strong> Repression of <em>gbx2</em> expression leads to increased <em>foxi1</em> expression </span></span></td>
<td><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Moderate: </strong>Extensive evidence that Gbx2 represses many developmental regulatory genes such as <em>foxi1</em>, but multifunctional nature of Gbx2 is still unknown.</span></span></td>
<td><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Low:</strong> Relationship was confirmed with loss-of-function experiment, but the connection could be secondary to the overall absence of otic placode.</span></span></td>
<td><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Low: </strong>Mutual regulation and interactions of both entities have not yet been well researched and described. Inconsistencies in zebrafish and mouse models.</span></span></td>
<td><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>High:</strong> Extensive evidence of relationship in vertebrate models.</span></span></td>
</tr>
<tr>
<td><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>KER6:</strong> Increased cell death leads to altered inner ear development</span></span></td>
<td><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>High:</strong> Extensive understanding that inner ear development depends on correct regulation of cell death in precursor cells and tissues.</span></span></td>
</tr>
<tr>
<td><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>KER7:</strong> Altered inner ear development leads to reduced hearing</span></span></td>
<td><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>High:</strong> Extensive understanding of defects in the development of inner ear and outcomes suggestive of deafness.</span></span></td>
</tr>
<tr>
<td><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>KER8:</strong> Reduced hearing leads to increased mortality</span></span></td>
<td><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>High:</strong> Extensive understanding that defective hearing decreases survival in natural setting.</span></span></td>
</tr>
<tr>
<td><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>KER9:</strong> Increased mortality leads to decrease of population trajectory</span></span></td>
<td><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>High:</strong> Extensive understanding that increased mortality on individual level decreases population trajectory.</span></span></td>
</tr>
</tbody>
</table>
<p><strong>Empirical support:</strong> Currently there is no sufficient evidence to estimate the weight of the evidence of empirical support for KERs in this AOP. Further more specific research on the relationships between the entities involved in the AOP is needed.</p>
<h3>Quantitative Consideration</h3>
<p>Data to support the quantitative understanding of this AOP is currently lacking.</p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Besson, M. <em>et. al. </em>2020. „Anthropogenic stressors impact fish sensory development and survival via thyroid disruption“. <em>Nature Communications 2020 11:1</em> 11(1): 1–10.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Bricaud, O., Leslie, A. C., & Gonda, S. (2006). Development/Plasticity/Repair The Transcription Factor six1 Inhibits Neuronal and Promotes Hair Cell Fate in the Developing Zebrafish (Danio rerio) Inner Ear. <em>Journal of Neuroscience</em>, <em>26</em>(41), 10438–10451. https://doi.org/10.1523/JNEUROSCI.1025-06.2006</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Hulander, M., Kiernan, A., Blomqvist, S., Carlsson, P., Samuelsson, E., Johansson, B., Steel, K., & Enerbäck, S. (2003). Lack of pendrin expression leads to deafness and expansion of the endolymphatic compartment in inner ears of Foxi1null mutant mice 2013. <em>Development</em>, <em>130</em>, 2013–2025. https://doi.org/10.1242/dev.00376</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Kasumyan, A. O. 2009. „Acoustic signaling in fish“. <em>Journal of Ichthyology 2009 49:11</em> 49(11): 963–1020.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Kozlowski, D. J., Whitfield, T. T., Hukriede, N. A., Lam, W. K., & Weinberg, E. S. (2005). The zebrafish dog-eared mutation disrupts eya1, a gene required for cell survival and differentiation in the inner ear and lateral line. <em>Developmental Biology</em>, <em>277</em>(1), 27–41. https://doi.org/10.1016/j.ydbio.2004.08.033</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Li, X., Oghi, K. A., Zhang, J., Krones, A., Bush, K. T., Glass, C. K., Nigam, S. K., Aggarwal, A. K., Maas, R., Rose, D. W., & Rosenfeld, M. G. (2003). Eya protein phosphatase activity regulates Six1-Dach-Eya transcriptional effects in mammalian organogenesis. <em>Nature</em>, <em>426</em>(6964), 247–254. https://doi.org/10.1038/nature02083</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Rearick, Daniel C., Jessica Ward, Paul Venturelli and Heiko Schoenfuss. 2018. „Environmental oestrogens cause predation-induced population decline in a freshwater fish“. <em>Royal Society Open Science</em> 5(10).</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Sklirou, A. D. <em>et al.</em> (2017) ‘6-bromo-indirubin-3′-oxime (6BIO), a Glycogen synthase kinase-3β inhibitor, activates cytoprotective cellular modules and suppresses cellular senescence-mediated biomolecular damage in human fibroblasts’, <em>Sci Rep</em>, 7, p. 11713. doi: 10.1038/s41598-017-11662-7.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Solomon, K. S., Kudoh, T., Dawid, I. B., & Fritz, A. (2003). Zebrafish foxi1 mediates otic placode formation and jaw development. <em>Development</em>, <em>130</em>(5), 929–940. https://doi.org/10.1242/dev.00308</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Valenti, Fabio <em>et al.</em> 2015. „The Increase in Maternal Expression of axin1 and axin2 Contribute to the Zebrafish Mutant Ichabod Ventralized Phenotype“. <em>Journal of Cellular Biochemistry</em> 116(3): 418–30.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Wang, Z. <em>et al.</em> (2018) ‘The role of gastrulation brain homeobox 2 (gbx2) in the development of the ventral telencephalon in zebrafish embryos’, <em>Differentiation</em>, 99(September 2017), pp. 28–40. doi: 10.1016/j.diff.2017.12.005.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Webb, J. F., & Shirey, J. E. (2003). Postembryonic Development of the Cranial Lateral Line Canals and Neuromasts in Zebrafish. <em>Developmental Dynamics</em>, <em>228</em>(3), 370–385. https://doi.org/10.1002/dvdy.10385</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Whitfield, T. T., Riley, B. B., Chiang, M. Y., & Phillips, B. (2002). Development of the zebrafish inner ear. <em>Developmental Dynamics</em>, <em>223</em>(4), 427–458. https://doi.org/10.1002/dvdy.10073</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Zheng, W., Huang, L., Wei, Z.-B., Silvius, D., Tang, B., & Pin-Xian, X. (2003). The role of Six1 in mammalian auditory system development. <em>Development</em>, <em>130</em>, 3989–4000. https://doi.org/10.1242/dev.00628</span></span></p>
<td><a href="/aops/410">Aop:410 - Repression of Gbx2 expression leads to defects in developing inner ear and consequently to increased mortality</a></td>
<td><a href="/aops/410">Aop:410 - GSK3beta inactivation leading to increased mortality via defects in developing inner ear</a></td>
<p>Phosphorylation of GSK3beta is induced, which means the inactivation of GSK3beta, in <em>Homo sapiens</em> (<a href="#_ENREF_32" title="Huang, 2019 #97">Huang et al., 2019</a>).</p>
<p>Phosphorylation of GSK3beta is induced, which means the inactivation of GSK3beta, in <em>Homo sapiens</em> (<a href="#_ENREF_32" title="Huang, 2019 #97">Huang et al., 2019</a>). Evidence for this KE is also provided for zebrafish (Anichtchik et al., 2008; Wang et al. 2018)</p>
<h4>Key Event Description</h4>
<p>・Glycogen synthase kinase 3beta (GSK3 beta) is inhibited by CHIR99021 (<a href="#_ENREF_52" title="Li, 2017 #39">C. H. Li et al., 2017</a>; <a href="#_ENREF_55" title="Liu, 2016 #38">C. C. Liu et al., 2016</a>; <a href="#_ENREF_82" title="Sineva, 2010 #37">Sineva & Pospelov, 2010</a>).</p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">The protein encoded by <em>gsk3b</em> gene is a serine-threonine kinase belonging to the glycogen synthase kinase subfamily. It is a negative regulator of glucose homeostasis and is involved in energy metabolism, inflammation, ER-stress, mitochondrial dysfunction, and apoptotic pathways. Defects in this gene have been associated with Parkinson disease and Alzheimer disease (<em>GSK3B Gene - GeneCards</em>). GSK3b has been identified within mitochondria (Hoshi et al., 1996), as well as in the cytoplasm (Anichtchik <em>et al.</em>, 2008).</span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">GSK3b kinase is constitutively active in resting cells and undergoes a rapid and transient inhibition in response to a number of external signals. GSK3b activity is regulated by site-specific phosphorylation. Full activity of GSK3b generally requires phosphorylation at tyrosine 216 (Tyr216), and conversely, phosphorylation at serine 9 (Ser9) inhibits GSK3b activity. Phosphorylation of Ser9 is the most common and important regulatory mechanism. Many kinases are capable of phosphorylating Ser9, including p70 S6 kinase, extracellular signal-regulated kinases (ERKs), p90Rsk (also called MAP-KAP kinase-1), protein kinase B (also called Akt), certain isoforms of protein kinase C (PKC) and cyclic AMP-dependent protein kinase (protein kinase A, PKA). In opposition to the inhibitory modulation of GSK3b that occurs by serine phosphorylation, tyrosine phosphorylation of GSK3b increases the enzyme’s activity </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Grimes and Jope, 2001; Luo, 2012)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">.</span></span></p>
<p>・Glycogen synthase kinase 3beta (GSK3 beta) is inhibited by CHIR99021 (<a href="#_ENREF_52" title="Li, 2017 #39">C. H. Li et al., 2017</a>; <a href="#_ENREF_55" title="Liu, 2016 #38">C. C. Liu et al., 2016</a>; <a href="#_ENREF_82" title="Sineva, 2010 #37">Sineva & Pospelov, 2010</a>).</p>
<p>・Glycogen synthase kinase 3beta (GSK3 beta) is inhibited by BIO (6-bromoindirubin-3’-oxime) (<a href="#_ENREF_63" title="Mohammed, 2016 #41">Mohammed et al., 2016</a>; <a href="#_ENREF_82" title="Sineva, 2010 #37">Sineva & Pospelov, 2010</a>).</p>
<p>・Kenpaullone is a dual inhibitor for GSK3 alpha/beta and HPK1/GCK-like kinase (<a href="#_ENREF_113" title="Yang, 2013 #48">Y. M. Yang et al., 2013</a>; <a href="#_ENREF_114" title="Yao, 1999 #49">Yao et al., 1999</a>).</p>
<p>・CHIR and BIO treatments lead to a slight upregulation of the primary transcripts of the miR-302-367 cluster and miR-181 family of miRNAs, which activate Wnt/beta-catenin signaling (<a href="#_ENREF_110" title="Wu, 2015 #56">Y. Wu et al., 2015</a>).</p>
<p>・CHIR98014 inhibits GSK3beta (<a href="#_ENREF_26" title="Guerrero, 2014 #53">Guerrero et al., 2014</a>; <a href="#_ENREF_53" title="Lian, 2014 #52">Lian et al., 2014</a>).</p>
<h4>How it is Measured or Detected</h4>
<p>Inactivation of GSK3 beta is measured by Wnt/beta-catenin activity assay, in which the vector containing the firefly luciferase gene controlled by TCF/LEF binding sites is transfected in the cells (<a href="#_ENREF_65" title="Naujok, 2014 #50">Naujok et al., 2014</a>). Phosphorylation of GSK3beta at residue Ser9 leads to the inactivation of GSK3beta. Phosphorylation of GSK3 beta is measured by immunoblotting with anti-phospho-GSK3beta (<a href="#_ENREF_32" title="Huang, 2019 #97">Huang et al., 2019</a>).</p>
<h4>References</h4>
<p style="margin-left:36.0pt">Guerrero, F., Herencia, C., Almaden, Y., Martinez-Moreno, J. M., Montes de Oca, A., Rodriguez-Ortiz, M. E., . . . Munoz-Castaneda, J. R. (2014). TGF-beta prevents phosphate-induced osteogenesis through inhibition of BMP and Wnt/beta-catenin pathways. <em>PLoS One, 9</em>(2), e89179. Retrieved from <a href="https://www.ncbi.nlm.nih.gov/pubmed/24586576">https://www.ncbi.nlm.nih.gov/pubmed/24586576</a>. doi:10.1371/journal.pone.0089179</p>
<p style="margin-left:40px"><span style="font-size:18px"><span style="font-family:"Calibri",sans-serif">Anichtchik, O. et al. (2008) ‘Loss of PINK1 function affects development and results in neurodegeneration in zebrafish’, Journal of Neuroscience, 28(33), pp. 8199–8207. doi: 10.1523/JNEUROSCI.0979-08.2008</span></span></p>
<p style="margin-left:40px"><span style="font-size:18px"><span style="font-family:"Calibri",sans-serif">Grimes, C. A. and Jope, R. S. (2001) ‘The multifaceted roles of glycogen synthase kinase 3β in cellular signaling’, Progress in Neurobiology, 65(4), pp. 391–426. doi: 10.1016/S0301-0082(01)00011-9</span></span></p>
<p style="margin-left:40px"><span style="font-size:18px"><span style="font-family:"Calibri",sans-serif">GSK3B Gene - GeneCards | GSK3B Protein | GSK3B Antibody (no date). Available at: https://www.genecards.org/cgi-bin/carddisp.pl?gene=GSK3B (Accessed: 3 October 2021)</span></span></p>
<p style="margin-left:36.0pt">Huang, J. Q., Wei, F. K., Xu, X. L., Ye, S. X., Song, J. W., Ding, P. K., . . . Gong, L. Y. (2019). SOX9 drives the epithelial-mesenchymal transition in non-small-cell lung cancer through the Wnt/beta-catenin pathway. <em>J Transl Med, 17</em>(1), 143. Retrieved from <a href="https://www.ncbi.nlm.nih.gov/pubmed/31060551">https://www.ncbi.nlm.nih.gov/pubmed/31060551</a>. doi:10.1186/s12967-019-1895-2</p>
<p style="margin-left:40px">Guerrero, F., Herencia, C., Almaden, Y., Martinez-Moreno, J. M., Montes de Oca, A., Rodriguez-Ortiz, M. E., . . . Munoz-Castaneda, J. R. (2014). TGF-beta prevents phosphate-induced osteogenesis through inhibition of BMP and Wnt/beta-catenin pathways. <em>PLoS One, 9</em>(2), e89179. Retrieved from <a href="https://www.ncbi.nlm.nih.gov/pubmed/24586576">https://www.ncbi.nlm.nih.gov/pubmed/24586576</a>. doi:10.1371/journal.pone.0089179</p>
<p style="margin-left:36.0pt">Li, C. H., Liu, C. W., Tsai, C. H., Peng, Y. J., Yang, Y. H., Liao, P. L., . . . Kang, J. J. (2017). Cytoplasmic aryl hydrocarbon receptor regulates glycogen synthase kinase 3 beta, accelerates vimentin degradation, and suppresses epithelial-mesenchymal transition in non-small cell lung cancer cells. <em>Arch Toxicol, 91</em>(5), 2165-2178. Retrieved from <a href="https://www.ncbi.nlm.nih.gov/pubmed/27752740">https://www.ncbi.nlm.nih.gov/pubmed/27752740</a>. doi:10.1007/s00204-016-1870-0</p>
<p style="margin-left:40px"><span style="font-size:18px"><span style="font-family:"Calibri",sans-serif">Hoshi, M. <em>et al.</em> (1996) <em>Regulation of mitochondrial pyruvate dehydrogenase activity by tau protein kinase I/glycogen synthase kinase 3p3 in brain</em>, <em>Neurobiology</em></span></span></p>
<p style="margin-left:36.0pt">Lian, X., Bao, X., Al-Ahmad, A., Liu, J., Wu, Y., Dong, W., . . . Palecek, S. P. (2014). Efficient differentiation of human pluripotent stem cells to endothelial progenitors via small-molecule activation of WNT signaling. <em>Stem Cell Reports, 3</em>(5), 804-816. Retrieved from <a href="https://www.ncbi.nlm.nih.gov/pubmed/25418725">https://www.ncbi.nlm.nih.gov/pubmed/25418725</a>. doi:10.1016/j.stemcr.2014.09.005</p>
<p style="margin-left:40px">Huang, J. Q., Wei, F. K., Xu, X. L., Ye, S. X., Song, J. W., Ding, P. K., . . . Gong, L. Y. (2019). SOX9 drives the epithelial-mesenchymal transition in non-small-cell lung cancer through the Wnt/beta-catenin pathway. <em>J Transl Med, 17</em>(1), 143. Retrieved from <a href="https://www.ncbi.nlm.nih.gov/pubmed/31060551">https://www.ncbi.nlm.nih.gov/pubmed/31060551</a>. doi:10.1186/s12967-019-1895-2</p>
<p style="margin-left:36.0pt">Liu, C. C., Cai, D. L., Sun, F., Wu, Z. H., Yue, B., Zhao, S. L., . . . Yan, D. W. (2016). FERMT1 mediates epithelial–mesenchymal transition to promote colon cancer metastasis via modulation of β-catenin transcriptional activity. <em>Oncogene, 36</em>, 1779. Retrieved from <a href="https://doi.org/10.1038/onc.2016.339">https://doi.org/10.1038/onc.2016.339</a>. doi:10.1038/onc.2016.339</p>
<p style="margin-left:40px">Li, C. H., Liu, C. W., Tsai, C. H., Peng, Y. J., Yang, Y. H., Liao, P. L., . . . Kang, J. J. (2017). Cytoplasmic aryl hydrocarbon receptor regulates glycogen synthase kinase 3 beta, accelerates vimentin degradation, and suppresses epithelial-mesenchymal transition in non-small cell lung cancer cells. <em>Arch Toxicol, 91</em>(5), 2165-2178. Retrieved from <a href="https://www.ncbi.nlm.nih.gov/pubmed/27752740">https://www.ncbi.nlm.nih.gov/pubmed/27752740</a>. doi:10.1007/s00204-016-1870-0</p>
<p style="margin-left:40px">Lian, X., Bao, X., Al-Ahmad, A., Liu, J., Wu, Y., Dong, W., . . . Palecek, S. P. (2014). Efficient differentiation of human pluripotent stem cells to endothelial progenitors via small-molecule activation of WNT signaling. <em>Stem Cell Reports, 3</em>(5), 804-816. Retrieved from <a href="https://www.ncbi.nlm.nih.gov/pubmed/25418725">https://www.ncbi.nlm.nih.gov/pubmed/25418725</a>. doi:10.1016/j.stemcr.2014.09.005</p>
<p style="margin-left:36.0pt">Mohammed, M. K., Shao, C., Wang, J., Wei, Q., Wang, X., Collier, Z., . . . Lee, M. J. (2016). Wnt/beta-catenin signaling plays an ever-expanding role in stem cell self-renewal, tumorigenesis and cancer chemoresistance. <em>Genes Dis, 3</em>(1), 11-40. Retrieved from <a href="https://www.ncbi.nlm.nih.gov/pubmed/27077077">https://www.ncbi.nlm.nih.gov/pubmed/27077077</a>. doi:10.1016/j.gendis.2015.12.004</p>
<p style="margin-left:40px">Liu, C. C., Cai, D. L., Sun, F., Wu, Z. H., Yue, B., Zhao, S. L., . . . Yan, D. W. (2016). FERMT1 mediates epithelial–mesenchymal transition to promote colon cancer metastasis via modulation of β-catenin transcriptional activity. <em>Oncogene, 36</em>, 1779. Retrieved from <a href="https://doi.org/10.1038/onc.2016.339">https://doi.org/10.1038/onc.2016.339</a>. doi:10.1038/onc.2016.339 <a href="https://www.nature.com/articles/onc2016339#supplementary-information">https://www.nature.com/articles/onc2016339 - supplementary-information</a></p>
<p style="margin-left:36.0pt">Naujok, O., Lentes, J., Diekmann, U., Davenport, C., & Lenzen, S. (2014). Cytotoxicity and activation of the Wnt/beta-catenin pathway in mouse embryonic stem cells treated with four GSK3 inhibitors. <em>BMC Res Notes, 7</em>, 273. Retrieved from <a href="https://www.ncbi.nlm.nih.gov/pubmed/24779365">https://www.ncbi.nlm.nih.gov/pubmed/24779365</a>. doi:10.1186/1756-0500-7-273</p>
<p style="margin-left:40px"><span style="font-size:18px"><span style="font-family:"Calibri",sans-serif">Luo, J. (2012) ‘The role of GSK3beta in the development of the central nervous system’, Front. Biol, 7(3), pp. 212–220. doi:10.1007/s11515-012-1222-2</span></span></p>
<p style="margin-left:36.0pt">Sineva, G. S., & Pospelov, V. A. (2010). Inhibition of GSK3beta enhances both adhesive and signalling activities of beta-catenin in mouse embryonic stem cells. <em>Biol Cell, 102</em>(10), 549-560. Retrieved from <a href="https://www.ncbi.nlm.nih.gov/pubmed/20626347">https://www.ncbi.nlm.nih.gov/pubmed/20626347</a>. doi:10.1042/BC20100016</p>
<p style="margin-left:40px">Mohammed, M. K., Shao, C., Wang, J., Wei, Q., Wang, X., Collier, Z., . . . Lee, M. J. (2016). Wnt/beta-catenin signaling plays an ever-expanding role in stem cell self-renewal, tumorigenesis and cancer chemoresistance. <em>Genes Dis, 3</em>(1), 11-40. Retrieved from <a href="https://www.ncbi.nlm.nih.gov/pubmed/27077077">https://www.ncbi.nlm.nih.gov/pubmed/27077077</a>. doi:10.1016/j.gendis.2015.12.004</p>
<p style="margin-left:36.0pt">Tang, Y. Y., Sheng, S. Y., Lu, C. G., Zhang, Y. Q., Zou, J. Y., Lei, Y. Y., . . . Hong, H. (2018). Effects of Glycogen Synthase Kinase-3beta Inhibitor TWS119 on Proliferation and Cytokine Production of TILs From Human Lung Cancer. <em>J Immunother, 41</em>(7), 319-328. Retrieved from <a href="https://www.ncbi.nlm.nih.gov/pubmed/29877972">https://www.ncbi.nlm.nih.gov/pubmed/29877972</a>. doi:10.1097/CJI.0000000000000234</p>
<p style="margin-left:40px">Naujok, O., Lentes, J., Diekmann, U., Davenport, C., & Lenzen, S. (2014). Cytotoxicity and activation of the Wnt/beta-catenin pathway in mouse embryonic stem cells treated with four GSK3 inhibitors. <em>BMC Res Notes, 7</em>, 273. Retrieved from <a href="https://www.ncbi.nlm.nih.gov/pubmed/24779365">https://www.ncbi.nlm.nih.gov/pubmed/24779365</a>. doi:10.1186/1756-0500-7-273</p>
<p style="margin-left:36.0pt">Wu, Y., Liu, F., Liu, Y., Liu, X., Ai, Z., Guo, Z., & Zhang, Y. (2015). GSK3 inhibitors CHIR99021 and 6-bromoindirubin-3'-oxime inhibit microRNA maturation in mouse embryonic stem cells. <em>Sci Rep, 5</em>, 8666. Retrieved from <a href="https://www.ncbi.nlm.nih.gov/pubmed/25727520">https://www.ncbi.nlm.nih.gov/pubmed/25727520</a>. doi:10.1038/srep08666</p>
<p style="margin-left:40px">Sineva, G. S., & Pospelov, V. A. (2010). Inhibition of GSK3beta enhances both adhesive and signalling activities of beta-catenin in mouse embryonic stem cells. <em>Biol Cell, 102</em>(10), 549-560. Retrieved from <a href="https://www.ncbi.nlm.nih.gov/pubmed/20626347">https://www.ncbi.nlm.nih.gov/pubmed/20626347</a>. doi:10.1042/BC20100016</p>
<p style="margin-left:36.0pt">Yang, Y. M., Gupta, S. K., Kim, K. J., Powers, B. E., Cerqueira, A., Wainger, B. J., . . . Rubin, L. L. (2013). A small molecule screen in stem-cell-derived motor neurons identifies a kinase inhibitor as a candidate therapeutic for ALS. <em>Cell Stem Cell, 12</em>(6), 713-726. Retrieved from <a href="https://www.ncbi.nlm.nih.gov/pubmed/23602540">https://www.ncbi.nlm.nih.gov/pubmed/23602540</a>. doi:10.1016/j.stem.2013.04.003</p>
<p style="margin-left:40px">Tang, Y. Y., Sheng, S. Y., Lu, C. G., Zhang, Y. Q., Zou, J. Y., Lei, Y. Y., . . . Hong, H. (2018). Effects of Glycogen Synthase Kinase-3beta Inhibitor TWS119 on Proliferation and Cytokine Production of TILs From Human Lung Cancer. <em>J Immunother, 41</em>(7), 319-328. Retrieved from <a href="https://www.ncbi.nlm.nih.gov/pubmed/29877972">https://www.ncbi.nlm.nih.gov/pubmed/29877972</a>. doi:10.1097/CJI.0000000000000234</p>
<p>Yao, Z., Zhou, G., Wang, X. S., Brown, A., Diener, K., Gan, H., & Tan, T. H. (1999). A novel human STE20-related protein kinase, HGK, that specifically activates the c-Jun N-terminal kinase signaling pathway. <em>J Biol Chem, 274</em>(4), 2118-2125. Retrieved from <a href="https://www.ncbi.nlm.nih.gov/pubmed/9890973">https://www.ncbi.nlm.nih.gov/pubmed/9890973</a>.</p>
<p style="margin-left:40px"><span style="font-size:18px"><span style="font-family:"Calibri",sans-serif">Wang, Z. <em>et al.</em> (2018) ‘The role of gastrulation brain homeobox 2 (gbx2) in the development of the ventral telencephalon in zebrafish embryos’, <em>Differentiation</em>, 99(December 2017), pp. 28–40. doi: 10.1016/j.diff.2017.12.005</span></span></p>
<p style="margin-left:40px">Wu, Y., Liu, F., Liu, Y., Liu, X., Ai, Z., Guo, Z., & Zhang, Y. (2015). GSK3 inhibitors CHIR99021 and 6-bromoindirubin-3'-oxime inhibit microRNA maturation in mouse embryonic stem cells. <em>Sci Rep, 5</em>, 8666. Retrieved from <a href="https://www.ncbi.nlm.nih.gov/pubmed/25727520">https://www.ncbi.nlm.nih.gov/pubmed/25727520</a>. doi:10.1038/srep08666</p>
<p style="margin-left:40px">Yang, Y. M., Gupta, S. K., Kim, K. J., Powers, B. E., Cerqueira, A., Wainger, B. J., . . . Rubin, L. L. (2013). A small molecule screen in stem-cell-derived motor neurons identifies a kinase inhibitor as a candidate therapeutic for ALS. <em>Cell Stem Cell, 12</em>(6), 713-726. Retrieved from <a href="https://www.ncbi.nlm.nih.gov/pubmed/23602540">https://www.ncbi.nlm.nih.gov/pubmed/23602540</a>. doi:10.1016/j.stem.2013.04.003</p>
<p style="margin-left:40px">Yao, Z., Zhou, G., Wang, X. S., Brown, A., Diener, K., Gan, H., & Tan, T. H. (1999). A novel human STE20-related protein kinase, HGK, that specifically activates the c-Jun N-terminal kinase signaling pathway. <em>J Biol Chem, 274</em>(4), 2118-2125. Retrieved from <a href="https://www.ncbi.nlm.nih.gov/pubmed/9890973">https://www.ncbi.nlm.nih.gov/pubmed/9890973</a>.</p>
<h3>List of Key Events in the AOP</h3>
<h4><a href="/events/1902">Event: 1902: Repression of Gbx2 expression</a></h4>
<h5>Short Name: Repression of Gbx2 expression</h5>
<td><a href="/aops/410">Aop:410 - Repression of Gbx2 expression leads to defects in developing inner ear and consequently to increased mortality</a></td>
<td>MolecularInitiatingEvent</td>
<td><a href="/aops/410">Aop:410 - GSK3beta inactivation leading to increased mortality via defects in developing inner ear</a></td>
<tr><th scope="col">Level of Biological Organization</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>Molecular</td></tr>
</tbody>
</table>
</div>
<h3>Evidence for Perturbation by Stressor</h3>
<h4>Overview for Molecular Initiating Event</h4>
<ul>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Zebrafish embryos were treated with chemical inhibitors or activators of various signaling pathways, such as the Wnt, FGF, retinoic acid (RA), HH, BMP, Nodal, and Notch pathways, and examined gbx2 expression in the telencephalon. First, embryos were treated with chemicals from 14 hpf to 18 hpf, immediately before the advent of gbx2 expression in the telencephalon, and then gbx2 expression was examined in this brain region . In embryos treated with BIO, a selective GSK3 inhibitor that activates Wnt signaling (Sato et al., 2004), gbx2 expression was specifically repressed in the telencephalon, but was unaffected or weakly activated in the isthmus and otic vesicle (OV). In embryos where FGF signaling was inhibited by SU5402, gbx2 was downregulated in the telencephalon and MHB, but its expression in the OV was little affected. Retinoic acid (RA) treatment strongly repressed gbx2 expression in the telencephalon, but not in the MHB and OV. These results suggest that gbx2-dependent telencephalon development is regulated by Wnt, FGF, and RA signaling (Z. Wang et al., 2018). </span></span></li>
<li><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">To clarify the critical stages of previous study for gbx2 regulation in the telencephalon, chemical treatment started between 14 and 17 hpf and gbx2 expression was examined at 18 hpf. Alternatively, chemical treatment was started at 14 hpf and then embryos were washed between 15 and 18 hpf, cultured in the absence of chemicals, and gbx2 expression was examined at 18 hpf. Resuoults showed that the downregulation of gbx2 by BIO grew less significant as the start time was delayed, and the repression of gbx2 by BIO in the telencephalon became less prominent when the chemicals were removed earlier, suggesting that Wnt signaling remains effective throughout the 4-h period (14–18 hpf) and that the repressive effect of BIO is reversible. Similarly, SU5402 mediated repression of gbx2 expression in the telencephalon and MHB became less significant as the treatment start time was delayed from 14 hpf to 17 hpf, and gbx2 expression was gradually restored with earlier removal of the chemical, showing that FGF signaling is continuously required for gbx2 expression in the telencephalon. Essentially the same results were obtained with RA treatment in terms of gbx2 expression in the telencephalon </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Z. Wang et al., 2018)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">.</span></span></li>
</ul>
<h4>BIO (6-bromoindirubin-3’-oxime) </h4>
<p><p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Embryos were treated with chemicals from 14 hpf to 18 hpf, immediately before the advent of gbx2 expression in the telencephalon, and then gbx2 expression was examined in this brain region . In embryos treated with BIO, a selective GSK3 inhibitor that activates Wnt signaling (Sato et al., 2004), gbx2 expression was specifically repressed in the telencephalon, but was unaffected or weakly activated in the isthmus and otic vesicle (OV). </span></span></p>
</p>
<h4>Retinoic acid</h4>
<p><p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Zebrafish embryos were treated with chemicals from 14 hpf to 18 hpf, immediately before the advent of gbx2 expression in the telencephalon, and then gbx2 expression was examined in this brain region. Retinoic acid (RA) treatment strongly repressed gbx2 expression in the telencephalon, but not in the MHB and OV. </span></span></p>
</p>
<h4>su5402</h4>
<p><p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Zebrafish embryos were treated with chemicals from 14 hpf to 18 hpf, immediately before the advent of gbx2 expression in the telencephalon, and then gbx2 expression was examined in this brain region. In embryos where FGF signaling was inhibited by SU5402, gbx2 was downregulated in the telencephalon and MHB, but its expression in the OV was little affected (Z. Wang et al., 2018). </span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">The gastrulation brain homebox (Gbx) group of transcription factor genes, composed of two genes, gbx1 and gbx2, in vertebrates, is also present in invertebrates (Chiang et al., 1995), and can be regarded as widely conserved among animals (Wang et al., 2018). Gbx2 functions in a variety of developmental processes after midbrain-hindbrain boundary (MHB) establishment. (Burroughs-Garcia et al., 2011) data demonstrate that the role of gbx2 in anterior hindbrain development is functionally conserved between zebrafish and mice. This gene was shown to be required for neural crest (NC) formation in mice </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(B. Li et al., 2009; Roeseler et al., 2012)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">. In Xenopus gbx2 is the earliest factor for specifying neural crest (NC) cells, and that gbx2 is directly regulated by NC inducing signaling pathways, such as Wnt/β-catenin signaling (Li et al., 2009).</span></span></p>
<h4>Key Event Description</h4>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">During vertebrate brain development, the gastrulation brain homeobox 2 gene (gbx2) is expressed in the forebrain (Z. Wang et al., 2018). The genes encoding the Gbx-type homeodomain transcription factors have been identified in a variety of vertebrates, and are primarily implicated in the regulation of various aspects of vertebrate brain development (Nakayama et al., 2017). Gbx2 exhibits DNA-binding transcription factor activity, RNA polymerase II-specific. Involved in cerebellum development; iridophore differentiation; and telencephalon regionalization. Predicted to localize to nucleus. Is expressed in several structures, including midbrain hindbrain boundary neural keel; midbrain hindbrain boundary neural rod; midbrain neural rod; nervous system; and presumptive rhombomere 1. Orthologous to human GBX2 (gastrulation brain homeobox 2) (<em>ZFIN Gene: Gbx2</em>, n.d.)</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">During vertebrate brain development, the gastrulation brain homeobox 2 gene (gbx2) is expressed in the forebrain (Z. Wang et al., 2018). The genes encoding the Gbx-type homeodomain transcription factors have been identified in a variety of vertebrates, and are primarily implicated in the regulation of various aspects of vertebrate brain development (Nakayama et al., 2017). Gbx2 exhibits DNA-binding transcription factor activity, RNA polymerase II-specific. Involved in cerebellum development; iridophore differentiation; and telencephalon regionalization. Predicted to localize to nucleus. Is expressed in several structures, including midbrain hindbrain boundary neural keel; midbrain hindbrain boundary neural rod; midbrain neural rod; nervous system; and presumptive rhombomere 1. Orthologous to human GBX2 (gastrulation brain homeobox 2) (<em>ZFIN Gene: Gbx2</em>, n.d.)</span></span><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">. Gbx2 is one of the key downstream markers of FGF and WNT signaling </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Wang <em>et al.</em>, 2018)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">, representative marker of anteroposterior (AP) axis patterning and midbrain specification </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Kim <em>et al.</em>, 2018)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">.</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Retinoids such as retinoic acid (RA) are chemopreventive and chemotherapeutic agents. One source of RA is vitamin A, derived from dietary β-carotene. RA regulates cell proliferation, differentiation, and morphogenesis (X. J. Wang et al., 2007). It inhibits tumorigenesis through suppression of cell growth and stimulation of cellular differentiation (Soprano et al., 2004). Also, RA promotes apoptosis (Atencia et al., 1997; Herget et al., 2000), and this property may contribute to its antitumor properties. The effects of retinoids are mediated by specific nuclear receptors, namely, retinoic acid receptors (RAR-α, -β, and -γ) and retinoid X receptors (RXR- α, - β, and - γ) (Rochette-Egly & Chambon, 2001). RXRs form heterodimers with RARs or other nuclear hormone receptors and function as transcriptional regulators. Retinoids can either activate or repress gene expression through RAR/RXR heterodimers interacting with other transcription factors, such as AP-1, estrogen receptor α, and NF-κB activities (Shaulian & Karin, 2002). Retinoic acid has been shown to repress Gbx2 expression in talencephalon in Zebrafish and other vertebrate models in early stages of development.</span></span></p>
<p> </p>
<h4>How it is Measured or Detected</h4>
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Repression of expression can be measured with reverse transcription polymerase chain reaction (RT-PCR). This technique is primarily used to measure the amount of specific RNA which is achieved by monitoring the amplification reaction using fluorescence, a technique called real-time PCR or quantitative PCR (qPCR) (Wong & Medrano, 2005). Combined RT-PCR and qPCR are routinely used for analysis of gene expression. Measuring changes in Gbx2 expression are described in detail in </span></span>(Rhinn <em>et al.</em>, 2003; Nakayama <em>et al.</em>, 2017; Wang <em>et al.</em>, 2018).</span></span></p>
<h4>References</h4>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Atencia, R., García-Sanz, M., Pérez-Yarza, G., Asumendi, A., Hilario, E., & Aréchaga, J. (1997). A structural analysis of cytoskeleton components during the execution phase of apoptosis. <em>Protoplasma</em>, <em>198</em>(3–4), 163–169. https://doi.org/10.1007/BF01287565</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Chiang, C., Young, K. E., & Beachy, P. A. (1995). Control of Drosophila tracheal branching by the novel homeodomain gene unplugged, a regulatory target for genes of the bithorax complex. <em>Development</em>, <em>121</em>(11), 3901–3912.</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Herget, T., Esdar, C., Oehrlein, S. A., Heinrich, M., Schützei, S., Maelicke, A., & Van Echten-Deckert, G. (2000). Production of ceramides causes apoptosis during early neural differentiation in vitro. <em>Journal of Biological Chemistry</em>, <em>275</em>(39), 30344–30354. https://doi.org/10.1074/jbc.M000714200</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Li, B., Kuriyama, S., Moreno, M., & Mayor, R. (2009). The posteriorizing gene Gbx2 is a direct target of Wnt signalling and the earliest factor in neural crest induction. <em>Development</em>, <em>136</em>(19), 3267–3278. https://doi.org/10.1242/dev.036954</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Luu, B., Ellisor, D., & Zervas, M. (2011). The Lineage Contribution and Role of Gbx2 in Spinal Cord Development. <em>PLoS ONE</em>, <em>6</em>. https://doi.org/10.1371/journal.pone.0020940</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Nakayama, Y., Inomata, C., Yuikawa, T., Tsuda, S., & Yamasu, K. (2017). Comprehensive analysis of target genes in zebrafish embryos reveals gbx2 involvement in neurogenesis. <em>Developmental Biology</em>, <em>430</em>(1), 237–248. https://doi.org/10.1016/j.ydbio.2017.07.015</span></span></p>
<p style="margin-left:40px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Chiang, C., Young, K. E., & Beachy, P. A. (1995). Control of Drosophila tracheal branching by the novel homeodomain gene unplugged, a regulatory target for genes of the bithorax complex. <em>Development</em>, <em>121</em>(11), 3901–3912.</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Rochette-Egly, C., & Chambon, P. (2001). F9 embryocarcinoma cells: A cell autonomous model to study the functional selectivity of RARs and RXRs in retinoid signaling. <em>Histology and Histopathology</em>, <em>16</em>(3), 909–922. https://doi.org/10.14670/HH-16.909</span></span></p>
<p style="margin-left:40px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Li, B., Kuriyama, S., Moreno, M., & Mayor, R. (2009). The posteriorizing gene Gbx2 is a direct target of Wnt signalling and the earliest factor in neural crest induction. <em>Development</em>, <em>136</em>(19), 3267–3278. https://doi.org/10.1242/dev.036954</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Roeseler, D. A., Sachdev, S., Buckley, D. M., Joshi, T., & Wu, D. K. (2012). Elongation Factor 1 alpha1 and Genes Associated with Usher Syndromes Are Downstream Targets of GBX2. <em>PLoS ONE</em>, <em>7</em>(11), 47366. https://doi.org/10.1371/journal.pone.0047366</span></span></p>
<p style="margin-left:40px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Luu, B., Ellisor, D., & Zervas, M. (2011). The Lineage Contribution and Role of Gbx2 in Spinal Cord Development. <em>PLoS ONE</em>, <em>6</em>. https://doi.org/10.1371/journal.pone.0020940</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Shaulian, E., & Karin, M. (2002). AP-1 as a regulator of cell life and death. <em>Nature Cell Biology</em>, <em>4</em>(5), E131–E136. https://doi.org/10.1038/ncb0502-e131</span></span></p>
<p style="margin-left:40px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Nakayama, Y., Inomata, C., Yuikawa, T., Tsuda, S., & Yamasu, K. (2017). Comprehensive analysis of target genes in zebrafish embryos reveals gbx2 involvement in neurogenesis. <em>Developmental Biology</em>, <em>430</em>(1), 237–248. https://doi.org/10.1016/j.ydbio.2017.07.015</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Soprano, D. R., Qin, P., & Soprano, K. J. (2004). Retinoic acid receptors and cancers. <em>Annual Review of Nutrition</em>, <em>24</em>, 201–221. https://doi.org/10.1146/annurev.nutr.24.012003.132407</span></span></p>
<p style="margin-left:40px"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif">Rhinn, M. <em>et al.</em> (2003) ‘Cloning, expression and relationship of zebrafish gbx1 and gbx2 genes to Fgf signaling’, <em>Mechanisms of Development</em>, 120(8), pp. 919–936. doi: 10.1016/S0925-4773(03)00135-7.</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Wang, X. J., Hayes, J. D., Henderson, C. J., & Roland Wolf, C. (2007). Identification of retinoic acid as an inhibitor of transcription factor Nrf2 through activation of retinoic acid receptor alpha. <em>Proc Natl Acad Sci U S A</em>, <em>104</em>(49), 19589–19594. www.pnas.org/cgi/content/full/</span></span></p>
<p style="margin-left:40px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Roeseler, D. A., Sachdev, S., Buckley, D. M., Joshi, T., & Wu, D. K. (2012). Elongation Factor 1 alpha1 and Genes Associated with Usher Syndromes Are Downstream Targets of GBX2. <em>PLoS ONE</em>, <em>7</em>(11), 47366. https://doi.org/10.1371/journal.pone.0047366</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Wang, Z., Nakayama, Y., Tsuda, S., & Yamasu, K. (2018). The role of gastrulation brain homeobox 2 (gbx2) in the development of the ventral telencephalon in zebrafish embryos. <em>Differentiation</em>, <em>99</em>(December 2017), 28–40. https://doi.org/10.1016/j.diff.2017.12.005</span></span></p>
<p style="margin-left:40px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Wang, Z., Nakayama, Y., Tsuda, S., & Yamasu, K. (2018). The role of gastrulation brain homeobox 2 (gbx2) in the development of the ventral telencephalon in zebrafish embryos. <em>Differentiation</em>, <em>99</em>(December 2017), 28–40. https://doi.org/10.1016/j.diff.2017.12.005</span></span></p>
<p><em><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"> ZFIN Gene: gbx2</span></span></em><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">. (n.d.). Retrieved April 12, 2021, from https://zfin.org/ZDB-GENE-020509-2</span></span></p>
<p><em><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"> ZFIN Gene: gbx2</span></span></em><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">. (n.d.). Retrieved April 12, 2021, from https://zfin.org/ZDB-GENE-020509-2</span></span></p>
<td><a href="/aops/410">Aop:410 - Repression of Gbx2 expression leads to defects in developing inner ear and consequently to increased mortality</a></td>
<td><a href="/aops/410">Aop:410 - GSK3beta inactivation leading to increased mortality via defects in developing inner ear</a></td>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Foxi I class genes have been described in zebrafish (Hans et al., 2004; Solomon et al., 2003), humans (Larsson et al., 1995; Pierrou et al., 1994), mouse (Hulander et al., 1998; Overdier et al., 1997), rat (Clevidence et al., 1993) and Xenopus (Lef et al., 1994, 1996). However, it is unclear whether zebrafish foxi1 is orthologous to any one of these genes. The Xenopus FoxI1c (Lef et al., 1996), FoxI1a and FoxI1b genes (Lef et al., 1994) share the highest degree of sequence conservation with the zebrafish gene. The expression pattern of the two Xenopus pseudoallelic variants FoxI1a/b does not suggest functional similarity to zebrafish foxi1. Of the three Xenopus FoxI genes, FoxI1c (XFD-10) is most similar to foxi1 in sequence. However, Xenopus FoxI1c was reported to be expressed in the neuroectoderm and somites but not in the otic placode, unlike the pattern for foxi1 reported in (Lef et al., 1996). (Pohl et al., 2002) report provides a more detailed description of Xenopus FoxI1c, which suggests that this gene is expressed in preplacodal tissue and the branchial arches, similar to observations for zebrafish foxi1. Thus, it appears probable that Xenopus FoxI1c represents the ortholog of zebrafish foxi1 (Solomon et al., 2003).</span></span></p>
<h4>Key Event Description</h4>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Foxi1 exhibits DNA-binding transcription factor activity. Involved in several processes, including animal organ development; epidermal cell fate specification; and neuron development. Predicted to localize to nucleus. Is expressed in several structures, including ectoderm; epibranchial ganglion; head; neural crest; and neurogenic field. Human ortholog(s) of this gene implicated in autosomal recessive nonsyndromic deafness 4. Orthologous to human FOXI1 (forkhead box I1) (<em>ZFIN Gene: Foxi1</em>, n.d.). The zebrafish Foxi1 protein shares 52% identity with Xenopus FoxI1c and 40% with human FOXI1; the forkhead domains are 95% and 94% identical, respectively (Solomon et al., 2003).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Foxi1 exhibits DNA-binding transcription factor activity. Involved in several processes, including animal organ development; epidermal cell fate specification; and neuron development. Predicted to localize to nucleus. Is expressed in several structures, including ectoderm; epibranchial ganglion; head; neural crest; and neurogenic field. Human ortholog(s) of this gene implicated in autosomal recessive nonsyndromic deafness. Orthologous to human FOXI1 (forkhead box I1) (<em>ZFIN Gene: Foxi1</em>, n.d.). The zebrafish Foxi1 protein shares 52% identity with Xenopus FoxI1c and 40% with human FOXI1; the forkhead domains are 95% and 94% identical, respectively (Solomon et al., 2003).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Zebrafish Foxi1 is expressed in nonneural ectoderm. Based on double in situ labeling with otx2, the anterior-most region of foxi1 expression lies just posterior to the midbrain hindbrain boundary. At the three-somite stage, the two domains of foxi1 expression become more compact, but are still located in approximately the same position lateral to the hindbrain (Solomon et al., 2003).</span></span></p>
<h4>How it is Measured or Detected</h4>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Inhibition of expression can be measured with reverse transcription polymerase chain reaction (RT-PCR). This technique is primarily used to measure the amount of specific RNA which is achieved by monitoring the amplification reaction using fluorescence, a technique called real-time PCR or quantitative PCR (qPCR) (Wong & Medrano, 2005). Combined RT-PCR and qPCR are routinely used for analysis of gene expression.</span></span></p>
<h4>References</h4>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Clevidence, D. E., Overdier, D. G., Taot, W., Qian, X., Pani, L., Lait, E., & Costa, R. H. (1993). Identification of nine tissue-specific transcription factors of the hepatocyte nuclear factor 3/forkhead DNA-binding-domain family (tissue-specific transcription factors/gene family/differentiation). In <em>Proc. Natl. Acad. Sci. USA</em> (Vol. 90).</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Hulander, M., Wurst, W., Carlsson, P., & Enerbäck, S. (1998). The winged helix transcription factor FKh10 is required for normal development of the inner ear. <em>Nature Genetics</em>, <em>20</em>(4), 374–376. https://doi.org/10.1038/3850</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Larsson, C., Hellqvist, M., Pierrou, S., White, I., Enerback, S. and, & Carlsson, P. (1995). Chromosomal Localization of Six Human Forkhead Genes, freac-1 (FKHL5), -3 (FKHL7), -4 (FKHL8), -5 (FKHL9), -6 (FKHL10), and -8 (FKHL12). <em>Genomics</em>, <em>30</em>, 464–469.</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Lef, J., Clement, J. H., Oschwald, R., Köster, M., & Knöchel, W. (1994). Spatial and temporal transcription patterns of the forkhead related XFD-2/XFD-2′ genes in Xenopus laevis embryos. <em>Mechanisms of Development</em>, <em>45</em>(2), 117–126. https://doi.org/10.1016/0925-4773(94)90025-6</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Lef, J., Dege, P., Scheucher, M., Forsbach-Birk, V., Clement, J. H., & Knöchel, W. (1996). A fork head related multigene family is transcribed in Xenopus laevis embryos. <em>International Journal of Developmental Biology</em>, <em>40</em>(1), 245–253. https://doi.org/10.1387/ijdb.8735935</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Overdier, D. G., Ye, H., Peterson, R. S., Clevidence, D. E., & Costa, R. H. (1997). The Winged Helix Transcriptional Activator HFH-3 Is Expressed in the Distal Tubules of Embryonic and Adult Mouse Kidney*. In <em>THE JOURNAL OF BIOLOGICAL CHEMISTRY</em> (Vol. 272, Issue 21). https://doi.org/10.1074/jbc.272.21.13725</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Pierrou, S., Hellqvist, M., Samuelsson, L., Enerbäck, S., & Carlsson, P. (1994). Cloning and characterization of seven human forkhead proteins: Binding site specificity and DNA bending. <em>EMBO Journal</em>, <em>13</em>(20), 5002–5012. https://doi.org/10.1002/j.1460-2075.1994.tb06827.x</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Pohl, B. S., Knöchel, S., Dillinger, K., & Knöchel, W. (2002). Sequence and expression of FoxB2 (XFD-5) and FoxI1c (XFD-10) in Xenopus embryogenesis. <em>Mechanisms of Development</em>, <em>117</em>(1–2), 283–287. https://doi.org/10.1016/S0925-4773(02)00184-3</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Solomon, K. S., Kudoh, T., Dawid, I. B., & Fritz, A. (2003). Zebrafish foxi1 mediates otic placode formation and jaw development. <em>Development</em>, <em>130</em>(5), 929–940. https://doi.org/10.1242/dev.00308</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Wong, M. L., & Medrano, J. F. (2005). <em>Real-time PCR for mRNA quantitation</em>. <em>39</em>(1), 75–85. https://doi.org/10.2144/05391RV01</span></span></p>
<p><em><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"> ZFIN Gene: foxi1</span></span></em><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">. (n.d.). Retrieved April 12, 2021, from https://zfin.org/ZDB-GENE-030505-1</span></span></p>
<td><a href="/aops/410">Aop:410 - Repression of Gbx2 expression leads to defects in developing inner ear and consequently to increased mortality</a></td>
<td><a href="/aops/410">Aop:410 - GSK3beta inactivation leading to increased mortality via defects in developing inner ear</a></td>
<tr><th scope="col">Level of Biological Organization</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>Molecular</td></tr>
</tbody>
</table>
</div>
<h4>Domain of Applicability</h4>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Evidence was provided for vertebrates ((Brodbeck & Englert, 2004; Heanue et al., 1999; Li et al., 2003; Wawersik & Maas, 2000) and Drosophila (Bui et al., 2000). </span></span></p>
<h4>Key Event Description</h4>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Six1b is predicted to have DNA-binding transcription factor activity, RNA polymerase II-specific and RNA polymerase II cis-regulatory region sequence-specific DNA binding activity. Involved in several processes, including muscle organ development; nervous system development; and regulation of skeletal muscle cell proliferation. Human ortholog(s) of this gene implicated in autosomal dominant nonsyndromic deafness; branchiootorenal syndrome; and nephroblastoma. Orthologous to human SIX1 (SIX homeobox 1) (<em>ZFIN Gene: Six1b</em>, n.d.). </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Six1b is a Member of the Pax–Six1b–Eya–Dach ( paired box–sine oculis homeobox–eyes absent– dachshund) gene regulatory network, involved in the development of numerous organs and tissues (Bessarab et al., 2004; Bricaud et al., 2006). It has been proposed to play an important role in inner ear development (Baker & Bronner-Fraser, 2001; Whitfield et al., 2002). Six1b expression appears to be regulated by pax2b and also by foxi1 (forkhead box I1) as expected for an early inducer of the otic placode (Bricaud et al., 2006). </span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Six1b promotes hair cell fate and, conversely, inhibits neuronal fate by differentially affecting cell proliferation and cell death in these lineages. Gain/loss-of-function experiment results indicate that, when six1 is overexpressed, not only are fewer neural progenitors formed but many of these progenitors do not go on to differentiate into neurons </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Bricaud et al., 2006)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">.</span></span></p>
<h4>How it is Measured or Detected</h4>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Inhibition of expression can be measured with reverse transcription polymerase chain reaction (RT-PCR). This technique is primarily used to measure the amount of specific RNA which is achieved by monitoring the amplification reaction using fluorescence, a technique called real-time PCR or quantitative PCR (qPCR) (Wong & Medrano, 2005). Combined RT-PCR and qPCR are routinely used for analysis of gene expression.</span></span></p>
<h4>References</h4>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Baker, C. V. H., & Bronner-Fraser, M. (2001). Vertebrate cranial placodes. I. Embryonic induction. <em>Developmental Biology</em>, <em>232</em>(1), 1–61. https://doi.org/10.1006/dbio.2001.0156</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Bessarab, D. A., Chong, S., & Korzh, V. (2004). <em>Expression of Zebrafish six1 During Sensory Organ Development and Myogenesis</em>. <em>June</em>, 781–786. https://doi.org/10.1002/dvdy.20093</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Bricaud, O., Leslie, A. C., & Gonda, S. (2006). Development/Plasticity/Repair The Transcription Factor six1 Inhibits Neuronal and Promotes Hair Cell Fate in the Developing Zebrafish (Danio rerio) Inner Ear. <em>Journal of Neuroscience</em>, <em>26</em>(41), 10438–10451. https://doi.org/10.1523/JNEUROSCI.1025-06.2006</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Brodbeck, S., & Englert, C. (2004). Genetic determination of nephrogenesis: The Pax/Eya/Six gene network. <em>Pediatric Nephrology</em>, <em>19</em>(3), 249–255. https://doi.org/10.1007/s00467-003-1374-z</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Heanue, T. A., Reshef, R., Davis, R. J., Mardon, G., Oliver, G., Tomarev, S., Lassar, A. B., & Tabin, C. J. (1999). <em>Synergistic regulation of vertebrate muscle development by Dach2, Eya2, and Six1, homologs of genes required for Drosophila eye formation</em>. www.genesdev.org</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Li, X., Oghi, K. A., Zhang, J., Krones, A., Bush, K. T., Glass, C. K., Nigam, S. K., Aggarwal, A. K., Maas, R., Rose, D. W., & Rosenfeld, M. G. (2003). Eya protein phosphatase activity regulates Six1-Dach-Eya transcriptional effects in mammalian organogenesis. <em>Nature</em>, <em>426</em>(6964), 247–254. https://doi.org/10.1038/nature02083</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Wawersik, S., & Maas, R. L. (2000). Vertebrate eye development as modeled in Drosophila. In <em>Human Molecular Genetics</em> (Vol. 9, Issue 6). http://hgu.mrc.ac.uk/Softdata/PAX6/</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Whitfield, T. T., Riley, B. B., Chiang, M. Y., & Phillips, B. (2002). Development of the zebrafish inner ear. <em>Developmental Dynamics</em>, <em>223</em>(4), 427–458. https://doi.org/10.1002/dvdy.10073</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Wong, M. L., & Medrano, J. F. (2005). <em>Real-time PCR for mRNA quantitation</em>. <em>39</em>(1), 75–85. https://doi.org/10.2144/05391RV01</span></span></p>
<p><em><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"> ZFIN Gene: six1b</span></span></em><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">. (n.d.). Retrieved April 12, 2021, from https://zfin.org/ZDB-GENE-040426-230</span></span></p>
<td><a href="/aops/410">Aop:410 - Repression of Gbx2 expression leads to defects in developing inner ear and consequently to increased mortality</a></td>
<td><a href="/aops/410">Aop:410 - GSK3beta inactivation leading to increased mortality via defects in developing inner ear</a></td>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Evidence was provided zebrafish (Kozlowski et al., 2005), Drosophila and vertebrates (Li et al., 2003; Zimmerman et al., 1997), and human (Abdelhak et al., 1997)</span></span></p>
<h4>Key Event Description</h4>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Eya1 is predicted to have protein tyrosine phosphatase activity. Involved in adenohypophysis development; otic vesicle morphogenesis; and otolith development. Predicted to localize to nucleus. Is expressed in several structures, including adenohypophyseal placode; brain; ectoderm; head; and lateral line system. Orthologous to human EYA1 (EYA transcriptional coactivator and phosphatase 1) (<em>ZFIN Gene: Eya1</em>, n.d.).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Eyes absent (Eya) genes regulate organogenesis in both vertebrates and invertebrates. Mutations in human EYA1 cause congenital Branchio-Oto-Renal (BOR) syndrome and hereditary syndromic deafness, while targeted inactivation of murine Eya1 impairs early developmental processes in multiple organs, including ear, kidney and skeletal system (Kozlowski et al., 2005; Xu et al., 2002).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">In zebrafish, the eya1 gene is widely expressed in placode-derived sensory organs during embryogenesis. Eya1 function appears to be primarily required for survival of sensory hair cells in the developing ear and lateral line neuromasts (Kozlowski et al., 2005).</span></span></p>
<h4>How it is Measured or Detected</h4>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Inhibition of expression can be measured with reverse transcription polymerase chain reaction (RT-PCR). This technique is primarily used to measure the amount of specific RNA which is achieved by monitoring the amplification reaction using fluorescence, a technique called real-time PCR or quantitative PCR (qPCR) </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Wong & Medrano, 2005)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">. Combined RT-PCR and qPCR are routinely used for analysis of gene expression</span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Inhibition of expression can be measured with reverse transcription polymerase chain reaction (RT-PCR). This technique is primarily used to measure the amount of specific RNA which is achieved by monitoring the amplification reaction using fluorescence, a technique called real-time PCR or quantitative PCR (qPCR) </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Wong & Medrano, 2005)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">. Combined RT-PCR and qPCR are routinely used for analysis of gene expression.</span></span></p>
<h4>References</h4>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Abdelhak, S., Kalatzis, V., Heilig, R., Compain, S., Samoson, D., Vincent, C., Weil, D., Cruaud, C., Sahly, I., Leibovici, M., Bitner-Glindzicz, M., & Francis, M. (1997). A human homologue of the Drosophila eyes absent gene underlies branchio-oto-renal (BOR) syndrome and identifies a novel gene family. <em>Nature Genetics</em>, <em>15</em>, 157–167. https://doi.org/10.1038/ng0297-157</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Kozlowski, D. J., Whitfield, T. T., Hukriede, N. A., Lam, W. K., & Weinberg, E. S. (2005). The zebrafish dog-eared mutation disrupts eya1, a gene required for cell survival and differentiation in the inner ear and lateral line. <em>Developmental Biology</em>, <em>277</em>(1), 27–41. https://doi.org/10.1016/j.ydbio.2004.08.033</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Li, X., Oghi, K. A., Zhang, J., Krones, A., Bush, K. T., Glass, C. K., Nigam, S. K., Aggarwal, A. K., Maas, R., Rose, D. W., & Rosenfeld, M. G. (2003). Eya protein phosphatase activity regulates Six1-Dach-Eya transcriptional effects in mammalian organogenesis. <em>Nature</em>, <em>426</em>(6964), 247–254. https://doi.org/10.1038/nature02083</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Wong, M. L., & Medrano, J. F. (2005). <em>Real-time PCR for mRNA quantitation</em>. <em>39</em>(1), 75–85. https://doi.org/10.2144/05391RV01</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Xu, P.-X., Weiming, Z., Laclef, C., Maire, P., Maas L., R., Peters, H., & Xin, X. (2002). Eya1is required for the morphogenesis of mammalian thymus, parathyroid and thyroid. <em>Development</em>, <em>129</em>, 3033–3044.</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><em>ZFIN Gene: eya1</em>. (n.d.). Retrieved April 12, 2021, from https://zfin.org/ZDB-GENE-990712-18</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Zimmerman, J. E., Bui, Q. T., Kur Steingrimsson, E. [, Nagle, D. L., Fu, W., Genin, A., Spinner, N. B., Copeland, N. G., Jenkins, N. A., Bucan, M., & Bonini, N. M. (1997). Cloning and Characterization of Two Vertebrate Homologs of the Drosophila eyes absent Gene. <em>Development</em>, <em>124</em>(23), 4819–4826.</span></span></p>
<td><a href="/aops/264">Aop:264 - Uncoupling of oxidative phosphorylation leading to growth inhibition via increased cell death</a></td>
<td><a href="/aops/291">Aop:291 - Mitochondrial ATP synthase antagonism leading to growth inhibition (2)</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/266">Aop:266 - Uncoupling of oxidative phosphorylation leading to growth inhibition via oxidative DNA damage</a></td>
<td><a href="/aops/287">Aop:287 - Mitochondrial complex III antagonism leading to growth inhibition (2)</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/267">Aop:267 - Uncoupling of oxidative phosphorylation leading to growth inhibition via increased lipid peroxidation</a></td>
<td><a href="/aops/368">Aop:368 - Cytochrome oxidase inhibition leading to increased nasal lesions</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/268">Aop:268 - Uncoupling of oxidative phosphorylation leading to growth inhibition via increased protein oxidation</a></td>
<td><a href="/aops/377">Aop:377 - Dysregulated prolonged Toll Like Receptor 9 (TLR9) activation leading to Multi Organ Failure involving Acute Respiratory Distress Syndrome (ARDS)</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/291">Aop:291 - Mitochondrial ATP synthase antagonism leading to growth inhibition (2)</a></td>
<td><a href="/aops/410">Aop:410 - GSK3beta inactivation leading to increased mortality via defects in developing inner ear</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/287">Aop:287 - Mitochondrial complex III antagonism leading to growth inhibition (2)</a></td>
<td><a href="/aops/418">Aop:418 - Aryl hydrocarbon receptor activation leading to impaired lung function through AHR-ARNT toxicity pathway</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/368">Aop:368 - Cytochrome oxidase inhibition leading to olfactory nasal lesions</a></td>
<td><a href="/aops/464">Aop:464 - Calcium overload in dopaminergic neurons of the substantia nigra leading to parkinsonian motor deficits</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/377">Aop:377 - Dysregulated prolonged Toll Like Receptor 9 (TLR9) activation leading to Acute Respiratory Distress Syndrome (ARDS) and Multiple Organ Dysfunction (MOD)</a></td>
<td><a href="/aops/468">Aop:468 - Binding of SARS-CoV-2 to ACE2 leads to hyperinflammation (via cell death)</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/410">Aop:410 - Repression of Gbx2 expression leads to defects in developing inner ear and consequently to increased mortality</a></td>
<td><a href="/aops/482">Aop:482 - Deposition of energy leading to occurrence of bone loss</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/418">Aop:418 - Aryl hydrocarbon receptor activation leading to impaired lung function through AHR-ARNT toxicity pathway</a></td>
<p><p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Autophagy can be initiated by a variety of stressors, most notably by nutrient deprivation (caloric restriction) or can result from signals present during cellular differentiation and embryogenesis and on the surface of damaged organelles </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Mizushima et al., 2008)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">.</span></span></p>
</p>
<h4>Gentamicin</h4>
<p><p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Gentamicin causes significant inner ear sensory hair cell death and auditory dysfunction in zebrafish </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Uribe et al., 2013)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">.</span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">The process of cell death is highly conserved within multi‐cellular organisms. </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Lockshin & Zakeri, 2004)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">.</span></span></p>
<p>The process of cell death is highly conserved within multi‐cellular organisms. (Lockshin & Zakeri, 2004). </p>
<p> </p>
<p><strong>Taxonomic applicability: </strong>Increased cell death is applicable to all animals. This includes vertebrates such as humans, mice and rats (Alberts et al., 2002). </p>
<p><strong>Life stage applicability: </strong>There is sufficient data to show the KE is applicable across different on life stages. </p>
<p><strong>Sex applicability:</strong> This key event is not sex specific (Forger and de Vries, 2010; Ortona Matarrese, and Malorni, 2014). </p>
<p><strong>Evidence for perturbation by a stressor:</strong> Multiple studies show that cell death can be increased or disrupted by many types of stressors including ionizing radiation and altered gravity (Zhu et al., 2016). </p>
<h4>Key Event Description</h4>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Cell death is part of normal development and maturation cycle, and is the component of many response patterns of living tissues to xenobiotic agents (i.e.. microorganisms and chemicals) and to endogenous modulations, such as inflammation and disturbed blood supply (Kanduc et al., 2002). Many physiological processes require cell death for their function (e.g.., embryonal development and immune selection of B and T cells) (Bertheloot et al., 2021). Defects in cells that result in their inappropriate survival or untimely death can negatively impact development or contribute to a variety of human pathologies, including cancer, AIDS, autoimmune disorders, and chronic infection. Cell death may also occur following exposure to environmental toxins or cytotoxic chemicals. Although this is often harmful, it can be beneficial in some cases, such as in the treatment of cancer (Crowley et al., 2016). </span></span></p>
<p>Cell death is part of normal development and maturation cycle, and is the component of many response patterns of living tissues to xenobiotic agents (i.e.. microorganisms and chemicals) and to endogenous modulations, such as inflammation and disturbed blood supply (Kanduc et al., 2002). Many physiological processes require cell death for their function (e.g.., embryonal development and immune selection of B and T cells) (Bertheloot et al., 2021). Defects in cells that result in their inappropriate survival or untimely death can negatively impact development or contribute to a variety of human pathologies, including cancer, AIDS, autoimmune disorders, and chronic infection. Cell death may also occur following exposure to environmental toxins or cytotoxic chemicals. Although this is often harmful, it can be beneficial in some cases, such as in the treatment of cancer (Crowley et al., 2016). </p>
<p>Cell death can be divided into: programmed cell death (cell death as a normal component of development) and non-programmed cell death (uncontrolled death of the cell). Although this simplistic view has blurred the intricate mechanisms separating these forms of cell death, studies have and will uncover new effectors, cell death pathways and reveal a more complex and intertwined landscape of processes involving cell death (Bertheloot et al., 2021). </p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Cell death can be </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">divided into: programmed cell death (cell death as a normal component of development) and non-programmed cell death (uncontrolled death of the cell). Although this simplistic view has blurred the intricate mechanisms separating these forms of cell death, studies have and will uncover new effectors, cell death pathways and reveal a more complex and intertwined landscape of processes involving cell death </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Bertheloot et al., 2021).</span></span></p>
<p><em>Programmed cell death:</em> is a form of cell death in which the dying cell plays an active part in its own demise (Cotter & Al-Rubeai, 1995). </p>
<p><span style="font-size:18px"><em><span style="font-family:"Calibri",sans-serif">Programmed cell death:</span></em></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"> is a form of cell death in which the dying cell plays an active part in its own demise </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Cotter & Al-Rubeai, 1995)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">.</span></span></p>
<p><u><strong>Apoptosis</strong></u> At a morphological level, it is characterized by cell shrinkage rather than the swelling seen in necrotic cell death. It is characterized by a number of characteristic morphological changes in the structure of the cell, together with a number of enzyme‐dependent biochemical processes. The result of it being the clearance of cells from the body, with minimal damage to surrounding tissues. An essential feature of apoptosis is the release of cytochrome c from mitochondria, regulated by a balance between proapoptotic and antiapoptotic proteins of the BCL-2 family, initiator caspases (caspase-8, -9 and -10) and effector caspases (caspase-3, -6 and -7). Apoptosis culminates in the breakdown of the nuclear membrane by caspase-6, the cleavage of many intracellular proteins (e.g., PARP and lamin), membrane blebbing, and the breakdown of genomic DNA into nucleosomal structures (Bertheloot et al., 2021). Mechanistically, two main pathways contribute to the caspase activation cascade downstream of mitochondrial cytochrome c release: </p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong><u>Apoptosis</u></strong> At a morphological level, it is characterized by cell shrinkage rather than the swelling seen in necrotic cell death. It is characterized by a number of characteristic morphological changes in the structure of the cell, together with a number of enzyme‐dependent biochemical processes. The result of it being the clearance of cells from the body, with minimal damage to surrounding tissues. An essential feature of apoptosis is the release of cytochrome c from mitochondria, regulated by a balance between proapoptotic and antiapoptotic proteins of the BCL-2 family, initiator caspases (caspase-8, -9 and -10) and effector caspases (caspase-3, -6 and -7). Apoptosis culminates in the breakdown of the nuclear membrane by caspase-6, the cleavage of many intracellular proteins (e.g., PARP and lamin), membrane blebbing, and the breakdown of genomic DNA into nucleosomal structures (Bertheloot et al., 2021). Mechanistically, two main pathways contribute to the caspase activation cascade downstream of mitochondrial cytochrome c release: </span></span></p>
<ul>
<li>
<p>Intrinsic pathway is triggered by dysregulation of or imbalance in intracellular homeostasis by toxic agents or DNA damage. It is characterized by mitochondrial outer membrane permeabilization (MOMP), which results in the release of cytochrome c into the cytosol. </p>
</li>
</ul>
<ul>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><u>Intrinsic pathway</u> is triggered by dysregulation of or imbalance in intracellular homeostasis by toxic agents or DNA damage. It is characterized by mitochondrial outer membrane permeabilization (MOMP), which results in the release of cytochrome c into the cytosol.</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><u>Extrinsic pathway</u> is initiated by activation of cell surface death receptors. Proapoptotic death receptors include TNFR1/2, Fas and the TNF-related apoptosis-inducing ligand (TRAIL) receptors DR4 and DR5.</span></span></li>
<li>
<p>Extrinsic pathway is initiated by activation of cell surface death receptors. Proapoptotic death receptors include TNFR1/2, Fas and the TNF-related apoptosis-inducing ligand (TRAIL) receptors DR4 and DR5. </p>
</li>
</ul>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><u>Other pathways of programmed cell death are called »non-apoptotic programmed cell-death« or »caspase-independent programmed cell-death« </u>(Blank & Shiloh, 2007)<u>.</u></span></span></p>
<p>Other pathways of programmed cell death are called »non-apoptotic programmed cell-death« or »caspase-independent programmed cell-death« (Blank & Shiloh, 2007). </p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong><u>Necroptosis:</u></strong> This type of regulated cell death, occurs following the activation of the tumor necrosis receptor (TNFR1) by TNFα. Activation of other cellular receptors triggers necroptosis. These receptors include death receptors (i.e., Fas/FasL), Toll-like receptors (TLR4 and TLR3) and cytosolic nucleic acid sensors such as RIG-I and STING, which induce type I interferon (IFN-I) and TNFα production and thus promote necroptosis in an autocrine feedback loop. Most of these pathways trigger NFκB- dependent proinflammatory and prosurvival signals. </span></span></p>
<p><u><strong>Necroptosis:</strong></u> This type of regulated cell death, occurs following the activation of the tumour necrosis receptor (TNFR1) by TNFα. Activation of other cellular receptors triggers necroptosis. These receptors include death receptors (i.e., Fas/FasL), Toll-like receptors (TLR4 and TLR3) and cytosolic nucleic acid sensors such as RIG-I and STING, which induce type I interferon (IFN-I) and TNFα production and thus promote necroptosis in an autocrine feedback loop. Most of these pathways trigger NFκB- dependent proinflammatory and prosurvival signals. </p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong><u>Pyroptosis</u></strong>is a type of cell death culminating in the loss of plasma membrane integrity and induced by activation of so-called inflammasome sensors. These include the Nod-like receptor (NLR) family, the DNA receptor Absent in Melanoma 2 (AIM2) and the Pyrin receptor.</span></span></p>
<p><u><strong>Pyroptosis</strong></u>is a type of cell death culminating in the loss of plasma membrane integrity and induced by activation of so-called inflammasome sensors. These include the Nod-like receptor (NLR) family, the DNA receptor Absent in Melanoma 2 (AIM2) and the Pyrin receptor. </p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong><u>Autophagy:</u></strong> is a process where cellular components such as macro proteins or even whole organelles are sequestered into lysosomes for degradation (Mizushima et al., 2008; Shintani & Klionsky, 2004). The lysosomes are then able to digest these substrates, the components of which can either be recycled to create new cellular structures and/or organelles or alternatively can be further processed and used as a source of energy (D’Arcy, 2019).</span></span></p>
<p><u><strong>Autophagy:</strong></u> is a process where cellular components such as macro proteins or even whole organelles are sequestered into lysosomes for degradation (Mizushima et al., 2008; Shintani & Klionsky, 2004). The lysosomes are then able to digest these substrates, the components of which can either be recycled to create new cellular structures and/or organelles or alternatively can be further processed and used as a source of energy (D’Arcy, 2019). </p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong><u>Anoikis</u></strong>is apoptosis induced by loss of attachment to substrate or to other cells (anoikis). Anoikis overlaps with apoptosis in molecular terms, but is classified as a separate entity because of its specific form od induction (Blank & Shiloh, 2007). Induction of anoikis occurs when cells lose attachment to ECM, or adhere to an inappropriate type of ECM, the latter being the more relevant <em>in vivo </em>(Gilmore, 2005).</span></span></p>
<p><u><strong>Anoikis</strong></u>is apoptosis induced by loss of attachment to substrate or to other cells (anoikis). Anoikis overlaps with apoptosis in molecular terms, but is classified as a separate entity because of its specific form of induction (Blank & Shiloh, 2007). Induction of anoikis occurs when cells lose attachment to ECM, or adhere to an inappropriate type of ECM, the latter being the more relevant in vivo (Gilmore, 2005). </p>
<p><strong><u><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Cornification</span></span></u></strong><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">: is programmed cell death of keratinocytes. Cell death in the context of cornification involves distinct enzyme classes such as transglutaminases, proteases, DNases and others </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Eckhart et al., 2013)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">.</span></span></p>
<p><u><strong>Cornification: </strong></u>is programmed cell death of keratinocytes. Cell death in the context of cornification involves distinct enzyme classes such as transglutaminases, proteases, DNases and others (Eckhart et al., 2013). </p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:18px"><em>Non-programmed cell death:</em></span> occurs accidentally in an unplanned manner.</span></span></p>
<p><u><strong>Non-programmed cell death</strong></u>: occurs accidentally in an unplanned manner. </p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong><u>Necrosis</u></strong> is generally characterized to be the uncontrolled death of the cell, usually following a severe insult, resulting in spillage of the contents of the cell into surrounding tissues and subsequent damage thereof (D’Arcy, 2019).</span></span></p>
<p><u><strong>Necrosis</strong></u> is generally characterized to be the uncontrolled death of the cell, usually following a severe insult, resulting in the spillage of the contents of the cell into surrounding tissues and subsequent damage thereof (D’Arcy, 2019). </p>
<p> </p>
<p> </p>
<p> </p>
<h4>How it is Measured or Detected</h4>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong>Assays for Quantitating Cell Death:</strong></span></span></p>
<p><strong> Assays for Quantitating Cell Death: </strong></p>
<ul>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Cell death can be measured by staining a sample of cells with trypan blue, assay is described in protocol: Measuring Cell Death by Trypan Blue Uptake and Light Microscopy (Crowley, Marfell, Christensen, et al., 2015d). Or with propidium Iodide, assay is described in protocol: Measuring Cell Death by Propidium Iodide (PI) Uptake and Flow Cytometry (Crowley & Waterhouse, 2015a) </span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">TUNEL technique: in situ terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling can be used to detect apoptotic cells (Bever & Fekete, 1999; Uribe et al., 2013).</span></span></li>
<li>
<p>Cell death can be measured by staining a sample of cells with trypan blue, assay is described in protocol: Measuring Cell Death by Trypan Blue Uptake and Light Microscopy (Crowley, Marfell, Christensen, et al., 2015d). Or with propidium Iodide, assay is described in protocol: Measuring Cell Death by Propidium Iodide (PI) Uptake and Flow Cytometry (Crowley & Waterhouse, 2015a) </p>
</li>
</ul>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong>Assays for Quantitating Cell Survival </strong></span></span></p>
<ul>
<li>
<p>TUNEL technique: in situ terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labelling can be used to detect apoptotic cells (Bever & Fekete, 1999; Uribe et al., 2013). </p>
</li>
</ul>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Colony-forming assay can be used to define the number of cells in a population that are capable of proliferating and forming large groups of cells. Described in Protocol: Measuring Survival of Adherent Cells with the Colony-Forming Assay (Crowley, Christensen, & Waterhouse, 2015c); Measuring Survival of Hematopoietic Cancer Cells with the Colony-Forming Assay in Soft Agar (Crowley & Waterhouse, 2015b).</span></span></p>
<p><strong>Assays for Quantitating Cell Survival </strong> </p>
<p><em><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong>ASSAYS TO DISTINGUISH APOPTOSIS FROM NECROSIS AND OTHER DEATH MODALITIES</strong></span></span></em></p>
<p>Colony-forming assay can be used to define the number of cells in a population that are capable of proliferating and forming large groups of cells. Described in Protocol: Measuring Survival of Adherent Cells with the Colony-Forming Assay (Crowley, Christensen, & Waterhouse, 2015c); Measuring Survival of Hematopoietic Cancer Cells with the Colony-Forming Assay in Soft Agar (Crowley & Waterhouse, 2015b). </p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong><u>Detecting Nuclear Condensation:</u></strong> The nucleus is generally round in healthy cells but fragmented in apoptotic cells. Dyes such as Giemsa or hematoxylin, which are purple in color and therefore easily viewed using light microscopy, are commonly used to stain the nucleus. Other features of apoptosis and necrosis, such as plasma membrane blebbing or rupture, can be identified by staining the cytoplasm with eosin. Eosin is pinkish in color and can also be viewed using light microscopy. Hematoxylin and eosin are, therefore, commonly used together to stain cells. Assay is described in Protocol: Morphological Analysis of Cell Death by Cytospinning Followed by Rapid Staining (Crowley, Marfell, & Waterhouse, 2015c); Analyzing Cell Death by Nuclear Staining with Hoechst 33342 (Crowley, Marfell, & Waterhouse, 2015a).</span></span></p>
<p><strong><em>ASSAYS TO DISTINGUISH APOPTOSIS FROM NECROSIS AND OTHER DEATH MODALITIES </em></strong></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong><u>Detection of DNA Fragmentation: </u></strong>Apoptotic cells with fragmented DNA can be identified and distinguished from live cells by staining with Propidium Iodide (PI) and measuring DNA content by flow cytometry. This assay is described in Protocol: Measuring the DNA Content of Cells in Apoptosis and at Different Cell-Cycle Stages by Propidium Iodide Staining and Flow Cytometry (Crowley, Chojnowski, & Waterhouse, 2015a).<strong><u> TUNEL technique </u></strong>can also be used<strong>:</strong> in situ terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling can be used to detect apoptotic cells (Bever & Fekete, 1999; Crowley, Marfell, & Waterhouse, 2015b; Uribe et al., 2013).</span></span></p>
<p><strong>Detecting Nuclear Condensation: </strong>The nucleus is generally round in healthy cells but fragmented in apoptotic cells. Dyes such as Giemsa or hematoxylin, which are purple in colour and therefore easily viewed using light microscopy, are commonly used to stain the nucleus. Other features of apoptosis and necrosis, such as plasma membrane blebbing or rupture, can be identified by staining the cytoplasm with eosin. Eosin is pinkish in colour and can also be viewed using light microscopy. Hematoxylin and eosin are, therefore, commonly used together to stain cells. Assay is described in Protocol: Morphological Analysis of Cell Death by Cytospinning Followed by Rapid Staining (Crowley, Marfell, & Waterhouse, 2015c); Analyzing Cell Death by Nuclear Staining with Hoechst 33342 (Crowley, Marfell, & Waterhouse, 2015a). </p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong><u>Detecting Phosphatidylserine Exposure:</u></strong> Apoptosis is also characterized by exposure of phosphatidylserine (PS) on the outside of apoptotic cells, which acts as a signal that triggers removal of the dying cell by phagocytosis. Annexin V, can selectively bind to PS to label apoptotic cells in which PS is exposed. Purified annexin V can be conjugated to various fluorochromes, which can then be visualized by fluorescence microscopy or detected by flow cytometry. This assay is described in protocol: Quantitation of Apoptosis and Necrosis by Annexin V Binding, Propidium Iodide Uptake, and Flow Cytometry (Crowley, Marfell, Scott, et al., 2015e). </span></span></p>
<p><strong>Detection of DNA Fragmentation</strong>: Apoptotic cells with fragmented DNA can be identified and distinguished from live cells by staining with Propidium Iodide (PI) and measuring DNA content by flow cytometry. This assay is described in Protocol: Measuring the DNA Content of Cells in Apoptosis and at Different Cell-Cycle Stages by Propidium Iodide Staining and Flow Cytometry (Crowley, Chojnowski, & Waterhouse, 2015a). TUNEL technique can also be used: in situ terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labelling can be used to detect apoptotic cells (Bever & Fekete, 1999; Crowley, Marfell, & Waterhouse, 2015b; Uribe et al., 2013). </p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong><u>Detecting Caspase Activity:</u></strong> antibodies that specifically recognize the cleaved fragments of caspases and their substrates can be used to specifically detect caspase activity in apoptotic cells by immunocytochemistry. Flow cytometry (using primary antibodies conjugated to fluorescent molecules, or by counter staining with fluorescently labeled antibodies against the primary antibody) can then be used to quantitate the number of apoptotic cells. This assay is described in protocol: Detecting Cleaved Caspase-3 in Apoptotic Cells by Flow Cytometry (Crowley & Waterhouse, 2015a).</span></span></p>
<p><strong>Detecting Phosphatidylserine Exposure: </strong>Apoptosis is also characterized by exposure of phosphatidylserine (PS) on the outside of apoptotic cells, which acts as a signal that triggers removal of the dying cell by phagocytosis. Annexin V, can selectively bind to PS to label apoptotic cells in which PS is exposed. Purified annexin V can be conjugated to various fluorochromes, which can then be visualized by fluorescence microscopy or detected by flow cytometry. This assay is described in protocol: Quantitation of Apoptosis and Necrosis by Annexin V Binding, Propidium Iodide Uptake, and Flow Cytometry (Crowley, Marfell, Scott, et al., 2015e). </p>
<p><strong><u><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Detecting Mitochondrial Damage:</span></span></u></strong><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"> flow cytometry can be used to quantitate the number of cells that have reduced mitochondrial transmembrane potential, which is commonly associated with cytochrome c release during apoptosis. For this assay see protocol: Measuring Mitochondrial Transmembrane Potential by TMRE Staining </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Crowley, Christensen, & Waterhouse, 2015b)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">.</span></span></p>
<p><strong>Detecting Caspase Activity:</strong> antibodies that specifically recognize the cleaved fragments of caspases and their substrates can be used to specifically detect caspase activity in apoptotic cells by immunocytochemistry. Flow cytometry (using primary antibodies conjugated to fluorescent molecules, or by counter staining with fluorescently labelled antibodies against the primary antibody) can then be used to quantitate the number of apoptotic cells. This assay is described in protocol: Detecting Cleaved Caspase-3 in Apoptotic Cells by Flow Cytometry (Crowley & Waterhouse, 2015a). </p>
<p><strong>Detecting Mitochondrial Damage:</strong> flow cytometry can be used to quantitate the number of cells that have reduced mitochondrial transmembrane potential, which is commonly associated with cytochrome c release during apoptosis. For this assay see protocol: Measuring Mitochondrial Transmembrane Potential by TMRE Staining (Crowley, Christensen, & Waterhouse, 2015b). </p>
<p> </p>
<p>Listed below are common methods for detecting the KE, however there may be other comparable methods that are not listed. </p>
<p> </p>
<p>Measures of apoptotic cytomorphological alterations: </p>
<p>Apoptotic cells exhibit electron dense nuclei, nuclear fragmentation, intact cell membrane up to the disintegration phase, disorganized cytoplasmic organelles, large clear vacuoles, blebs at cell surface, and apoptotic bodies, which can be visualized with various methods. (Elmore, 2007; Watanabe et al., 2002) </p>
<table border="1">
<tbody>
<tr>
<td>
<p><strong>Method of Measurement </strong></p>
<p>Transmission electron microscopy (TEM) / Scanning electron microscopy (SEM)/ Fluorescence microscopy </p>
</td>
<td>
<p>Martinez, Reif, and Pappas, 2010; Watanabe et al., 2002 </p>
</td>
<td>
<p>TEM and SEM can image the cytomorphological alterations caused by apoptosis. </p>
</td>
<td>
<p>No </p>
</td>
</tr>
<tr>
<td colspan="3" rowspan="1">
<p>Stains: </p>
</td>
<td>
<p> </p>
</td>
</tr>
<tr>
<td>
<p>Hematoxylin with eosin </p>
</td>
<td>
<p>Elmore, 2007 </p>
</td>
<td>
<p>Hematoxylin stains nuclei blue and eosin stains the cytoplasm/extracellular matrix pink, allowing for the visualization of the cytomorphological alterations of cells. </p>
</td>
<td>
<p>No </p>
</td>
</tr>
<tr>
<td>
<p>Toluidine blue or methylene blue </p>
</td>
<td>
<p>Watanabe et al., 2002 </p>
</td>
<td>
<p>Toluidine blue stains cellular nuclei, and identifies malignant tissue, which has an increased DNA content and a higher nuclear-to-cytoplasmic ratio. </p>
<p>Methylene blue stain applied to a healthy cell sample results in a colourless stain. This is due to the cell's enzymes, which reduce the methylene blue, thereby, reducing its colour. Methylene blue stain applied to a dead cell sample turns blue. </p>
</td>
<td>
<p>No </p>
</td>
</tr>
<tr>
<td>
<p>DAPI </p>
</td>
<td>
<p>Crowley, Marfell, and Waterhouse, 2016 </p>
</td>
<td>
<p>Binds strongly to adenine–thymine-rich regions in the DNA. DAPI can stain live and fixed cells. It passes less efficiently through the membrane in live cells. </p>
</td>
<td>
<p>Yes </p>
</td>
</tr>
<tr>
<td>
<p>Hoescht 33342 </p>
</td>
<td>
<p>Crowley, Marfell, and Waterhouse, 2016 </p>
</td>
<td>
<p>Binds to DNA in live and fixed cells, used to measure DNA condensation. </p>
</td>
<td>
<p>Yes </p>
</td>
</tr>
<tr>
<td>
<p>Acridine Orange (AO) </p>
</td>
<td>
<p>Watanabe et al., 2002 </p>
</td>
<td>
<p>Interacts with DNA/RNA through intercalation/electrostatic interaction, is able to penetrate cell membranes. Stains live cells green and dead cells red. </p>
</td>
<td>
<p>No </p>
</td>
</tr>
<tr>
<td>
<p>Nile blue sulphate </p>
</td>
<td>
<p>Watanabe et al., 2002 </p>
</td>
<td>
<p>Stains cell nuclei and lysosomes, indicating apoptotic bodies. </p>
<p>Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) assay </p>
</td>
<td>
<p>Kressel and Groscurth, 1994 </p>
</td>
<td>
<p>Apoptosis is detected with the TUNEL method to assay the endonuclease cleavage products by enzymatically end-labelling the DNA strand breaks. </p>
</td>
<td>
<p>Yes </p>
<p> </p>
</td>
</tr>
<tr>
<td>
<p>Nicoletti Assay (SubG1 cell fragment measurement) </p>
</td>
<td>
<p>Nicoletti et al., 1991 </p>
</td>
<td>
<p>Measures DNA content in nuclei at the pre-G1 phase of the cell cycle (apoptotic nuclei have less DNA than nuclei in healthy cells). </p>
</td>
<td>
<p>No </p>
</td>
</tr>
<tr>
<td>
<p>Cell Death Detection ELISA kit </p>
</td>
<td>
<p>Parajuli, 2014 </p>
</td>
<td>
<p>Apoptotic nucleosomes are detected using the Cell Death Detection ELISA kit, which were calculated as absorbance subtraction at 405 nm and 490 nm. </p>
</td>
<td>
<p>No </p>
</td>
</tr>
</tbody>
</table>
<p> </p>
<p>Measurement of apoptotic markers through immunochemistry: </p>
<table border="1">
<tbody>
<tr>
<td>
<p><strong>Method of Measurement </strong></p>
<p>Western blot / immunofluorescence microscopy / immunohistochemistry </p>
</td>
<td>
<p>Elmore 2007; Martinez, Reif, and Pappas, 2010; Parajuli et al, 2014 </p>
</td>
<td>
<p>Apoptosis can be detected with the expression of various apoptotic markers by western blotting using antibodies. Markers can include: cytosolic cytochrome-c; caspases 2, 3, 6, 7, 8, 9, 10; Bax; Bcl-2 (apoptosis inhibitor); BIRC2; BIRC3; GAPDH; PARP; CDK2; CDK4; cyclin D1; p53; p63; p73; cytokeratin-18 </p>
</td>
<td>
<p>No </p>
</td>
</tr>
</tbody>
</table>
<p> </p>
<p>Measures of altered caspase activity: </p>
<table border="1">
<tbody>
<tr>
<td>
<p><strong>Method of Measurement </strong></p>
<p>Caspase-3 and caspase-9 activity is measured with the enzyme-catalyzed release of p-nitroanilide (pNA) and quantified at 405 nm </p>
</td>
<td>
<p> Wu, 2016 </p>
</td>
<td>
<p>Visualizes caspase-3 and caspase-9 activity </p>
</td>
<td>
<p>No </p>
</td>
</tr>
<tr>
<td>
<p>PhiPhiLux Assay </p>
</td>
<td>
<p>Watanabe et al., 2002 </p>
</td>
<td>
<p>The PhiPhiLux molecule becomes fluorescent once it is cleaved by caspase-3, indicating caspase activity. </p>
</td>
<td>
<p>No </p>
</td>
</tr>
<tr>
<td>
<p>Ferrocene reporter </p>
</td>
<td>
<p>Martinez, Reif, and Pappas, 2010 </p>
</td>
<td>
<p>An electrochemical method to detect apoptosis. Ferrocene is attached to a peptide. The peptide sequence is a caspase 3 cleavage site and the ferrocene acts as the electrochemical reporter. The more caspase cleavage that occurs, the more ferrocene molecules are cleaved, the stronger the signal. </p>
</td>
<td>
<p>No </p>
</td>
</tr>
<tr>
<td>
<p>Self-assembled monolayers for matrix assisted laser desorption ionization time-of-flight mass spectrometry (SAMDI-MS) assay </p>
</td>
<td>
<p>Martinez, Reif, and Pappas, 2010 </p>
<p>LCSM can monitor many mitochondrial events following staining of cells, such as: mitochondrial permeability transition, depolarization of the inner mitochondrial membrane, which may be indicative of apoptosis. </p>
</td>
<td>
<p>No </p>
<p> </p>
</td>
</tr>
<tr>
<td>
<p>Fluorescent, cationic, lipophilic mitochondrial dyes, such as: JC-1 dye, Rhodamine, DiOC6, Mitotracker red </p>
</td>
<td>
<p>Martinez, Reif, and Pappas, 2010; Sivandzade, Bhalerao, and Cucullo, 2019 </p>
</td>
<td>
<p>These mitochondrial dyes can indicate disintegration of the mitochondrial outer membrane’s electrochemical gradient, as different fluorescence is observed between healthy and apoptotic cells. In healthy cells the dye accumulates in aggregates, but in apoptotic cells missing the electrochemical membrane, the dye will spread out into the cytoplasm providing different fluorescent signals. </p>
</td>
<td>
<p>No </p>
</td>
</tr>
</tbody>
</table>
<p> </p>
<p>Other measures: </p>
<table border="1">
<tbody>
<tr>
<td>
<p><strong>Method of measurement </strong></p>
<p>A method to profile the gene expression of many apoptotic-related genes, for example: ligands, receptors, intracellular modulators, and transcription factors. </p>
</td>
<td>
<p>No </p>
</td>
</tr>
<tr>
<td>
<p>Fluorescence correlation spectroscopy (FCS) or dual-colour fluorescence cross-correlation spectroscopy (dcFCCS) </p>
</td>
<td>
<p>Martinez, Reif, and Pappas, 2010 </p>
</td>
<td>
<p>Used to measure protease activity. </p>
</td>
<td>
<p>No </p>
</td>
</tr>
<tr>
<td>
<p>Apoptosis is measured with Annexin V-FITC probes </p>
</td>
<td>
<p>Elmore, 2007; Wu et al., 2016 </p>
</td>
<td>
<p>A measure of apoptotic membrane alterations. Annexin-V detects externalized phosphatidylserine residues, a result of apoptosis. Can be conducted in conjunction with propidium iodide (PI) staining. The relative percentage of Annexin V-FITC-positive/PI-negative cells is analyzed by flow cytometry. </p>
</td>
<td>
<p>Yes </p>
</td>
</tr>
</tbody>
</table>
<h4>References</h4>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Bertheloot, D., Latz, E., & Franklin, B. S. (2021). Necroptosis, pyroptosis and apoptosis: an intricate game of cell death. <em>Cellular & Molecular Immunology</em>, <em>18</em>, 1106–1121. https://doi.org/10.1038/s41423-020-00630-3</span></span></p>
<p>Alberts, B. et al. (2002), “Programmed Cell Death (Apoptosis)”, in Molecular Biology of the Cell. 4th edition, Garland Science, New York, https://www.ncbi.nlm.nih.gov/books/NBK26873/ </p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Bever, M. M., & Fekete, D. M. (1999). Ventromedial focus of cell death is absent during development of Xenopus and zebrafish inner ears. <em>Journal of Neurocytology</em>, <em>28</em>(10–11), 781–793. https://doi.org/10.1023/a:1007005702187</span></span></p>
<p>Bertheloot, D., Latz, E., and B. S. Franklin (2021), “Necroptosis, pyroptosis and apoptosis: an intricate game of cell death”, Cellular & Molecular Immunology, 18, 1106–1121. https://doi.org/10.1038/s41423-020-00630-3 </p>
<p>Bever, M. M., and D. M. Fekete (1999), “Ventromedial focus of cell death is absent during development of Xenopus and zebrafish inner ears”, Journal of Neurocytology, 28(10–11), 781–793. https://doi.org/10.1023/a:1007005702187 </p>
<p>Blank, M., and Y.Shiloh (2007), “Cell Cycle Programs for Cell Death: Apoptosis is Only One Way to Go”, Cell Cycle, 6(6), 686–695. https://doi.org/10.4161/cc.6.6.3990 </p>
<p>Cotter, T. G., and M. Al-Rubeai (1995), “Cell death (apoptosis) in cell culture systems”, Trends in Biotechnology, 13(4), 150–155. https://doi.org/10.1016/S0167-7799(00)88926-X </p>
<p>Crowley, L. C., Chojnowski, G., and N. J Waterhouse (2015a), “Measuring the DNA content of cells in apoptosis and at different cell-cycle stages by propidium iodide staining and flow cytometry”, Cold Spring Harbor Protocols, 10, 905–910. https://doi.org/10.1101/pdb.prot087247 </p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Blank, M., & Shiloh, Y. (2007). Cell Cycle Programs for Cell Death: Apoptosis is Only One Way to Go. <em>Cell Cycle</em>, <em>6</em>(6), 686–695. https://doi.org/10.4161/cc.6.6.3990</span></span></p>
<p>Crowley, L. C., Christensen, M. E., and N. J Waterhouse (2015b), “Measuring mitochondrial transmembrane potential by TMRE staining”, Cold Spring Harbor Protocols, 12, 1092–1096. https://doi.org/10.1101/pdb.prot087361 </p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Cotter, T. G., & Al-Rubeai, M. (1995). Cell death (apoptosis) in cell culture systems. <em>Trends in Biotechnology</em>, <em>13</em>(4), 150–155. https://doi.org/10.1016/S0167-7799(00)88926-X</span></span></p>
<p>Crowley, L. C., Christensen, M. E., and N. J Waterhouse (2015c), “Measuring survival of adherent cells with the Colony-forming assay”, Cold Spring Harbor Protocols, 8, 721– 724. https://doi.org/10.1101/pdb.prot087171 </p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Crowley, L. C., Chojnowski, G., & Waterhouse, N. J. (2015a). Measuring the DNA content of cells in apoptosis and at different cell-cycle stages by propidium iodide staining and flow cytometry. <em>Cold Spring Harbor Protocols</em>, <em>10</em>, 905–910. https://doi.org/10.1101/pdb.prot087247</span></span></p>
<p>Crowley, L. C. et al. (2015d), “Measuring cell death by trypan blue uptake and light microscopy”, Cold Spring Harbor Protocols, 7, 643–646. https://doi.org/10.1101/pdb.prot087155 </p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Crowley, L. C., Christensen, M. E., & Waterhouse, N. J. (2015b). Measuring mitochondrial transmembrane potential by TMRE staining. <em>Cold Spring Harbor Protocols</em>, <em>12</em>, 1092–1096. https://doi.org/10.1101/pdb.prot087361</span></span></p>
<p>Crowley, L. C. et al. (2016), “Dead cert: Measuring cell death”, Cold Spring Harbor Protocols, 2016(12), 1064–1072. https://doi.org/10.1101/pdb.top070318 </p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Crowley, L. C., Christensen, M. E., & Waterhouse, N. J. (2015c). Measuring survival of adherent cells with the Colony-forming assay. <em>Cold Spring Harbor Protocols</em>, <em>8</em>, 721–724. https://doi.org/10.1101/pdb.prot087171</span></span></p>
<p>Crowley, L. C. et al. (2015e), “Quantitation of apoptosis and necrosis by annexin V binding, propidium iodide uptake, and flow cytometry”, Cold Spring Harbor Protocols, 11, 953–957. https://doi.org/10.1101/pdb.prot087288 </p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Crowley, L. C., Marfell, B. J., Christensen, M. E., & Waterhouse, N. J. (2015d). Measuring cell death by trypan blue uptake and light microscopy. <em>Cold Spring Harbor Protocols</em>, <em>7</em>, 643–646. https://doi.org/10.1101/pdb.prot087155</span></span></p>
<p>Crowley, L. C., Marfell, B. J., and N. J Waterhouse (2015a), “Analyzing cell death by nuclear staining with Hoechst 33342”, Cold Spring Harbor Protocols, 9, 778–781. https://doi.org/10.1101/pdb.prot087205 </p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Crowley, L. C., Marfell, B. J., Scott, A. P., Boughaba, J. A., Chojnowski, G., Christensen, M. E., & Waterhouse, N. J. (2016). Dead cert: Measuring cell death. <em>Cold Spring Harbor Protocols</em>, <em>2016</em>(12), 1064–1072. https://doi.org/10.1101/pdb.top070318</span></span></p>
<p>Crowley, L. C., Marfell, B. J., and N. J Waterhouse (2015b), “Detection of DNA fragmentation in apoptotic cells by TUNEL”, Cold Spring Harbor Protocols, 10, 900–905. https://doi.org/10.1101/pdb.prot087221 </p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Crowley, L. C., Marfell, B. J., Scott, A. P., & Waterhouse, N. J. (2015e). Quantitation of apoptosis and necrosis by annexin V binding, propidium iodide uptake, and flow cytometry. <em>Cold Spring Harbor Protocols</em>, <em>11</em>, 953–957. https://doi.org/10.1101/pdb.prot087288</span></span></p>
<p>Crowley, L. C., Marfell, B. J., and N. J Waterhouse (2015c), “Morphological analysis of cell death by cytospinning followed by rapid staining”, Cold Spring Harbor Protocols, 9, 773–777. https://doi.org/10.1101/pdb.prot087197 </p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Crowley, L. C., Marfell, B. J., & Waterhouse, N. J. (2015a). Analyzing cell death by nuclear staining with Hoechst 33342. <em>Cold Spring Harbor Protocols</em>, <em>9</em>, 778–781. https://doi.org/10.1101/pdb.prot087205</span></span></p>
<p>Crowley, L. C. and N. J Waterhouse (2015a), “Detecting cleaved caspase-3 in apoptotic cells by flow cytometry”, Cold Spring Harbor Protocols, 11, 958–962. https://doi.org/10.1101/pdb.prot087312 </p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Crowley, L. C., Marfell, B. J., & Waterhouse, N. J. (2015b). Detection of DNA fragmentation in apoptotic cells by TUNEL. <em>Cold Spring Harbor Protocols</em>, <em>10</em>, 900–905. https://doi.org/10.1101/pdb.prot087221</span></span></p>
<p>Crowley, L. C. and N. J Waterhouse (2015b), “Measuring survival of hematopoietic cancer cells with the Colony-forming assay in soft agar”, Cold Spring Harbor Protocols, 8, 725. https://doi.org/10.1101/pdb.prot087189 </p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Crowley, L. C., Marfell, B. J., & Waterhouse, N. J. (2015c). Morphological analysis of cell death by cytospinning followed by rapid staining. <em>Cold Spring Harbor Protocols</em>, <em>9</em>, 773–777. https://doi.org/10.1101/pdb.prot087197</span></span></p>
<p>D’Arcy, M. S. (2019), “Cell death: a review of the major forms of apoptosis, necrosis and autophagy”, Cell Biology International, 43(6), 582–592. https://doi.org/10.1002/cbin.11137 </p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Crowley, L. C., & Waterhouse, N. J. (2015a). Detecting cleaved caspase-3 in apoptotic cells by flow cytometry. <em>Cold Spring Harbor Protocols</em>, <em>11</em>, 958–962. https://doi.org/10.1101/pdb.prot087312</span></span></p>
<p>Eckhart, L. et al. (2013), “Cell death by cornification”, Biochimica et Biophysica Acta - Molecular Cell Research, 1833(12), 3471–3480. https://doi.org/10.1016/j.bbamcr.2013.06.010 </p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Crowley, L. C., & Waterhouse, N. J. (2015b). Measuring survival of hematopoietic cancer cells with the Colony-forming assay in soft agar. <em>Cold Spring Harbor Protocols</em>, <em>8</em>, 725. https://doi.org/10.1101/pdb.prot087189</span></span></p>
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<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Gilmore, A. P. (2005). Anoikis. <em>Cell Death and Differentiation</em>, <em>12</em>, 1473–1477. https://doi.org/10.1038/sj.cdd.4401723</span></span></p>
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<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Kanduc, D., Mittelman, A., Serpico, R., Sinigaglia, E., Sinha, A. A., Natale, C., Santacroce, R., Di Corcia, M. G., Lucchese, A., Dini, L., Pani, P., Santacroce, S., Simone, S., Bucci, R., & Farber, E. (2002). Cell death: apoptosis versus necrosis (review). <em>International Journal of Oncology</em>, <em>21</em>(1), 165–170. https://doi.org/10.3892/ijo.21.1.165</span></span></p>
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<p>Uribe, P. M. et al. (2013), “Aminoglycoside-Induced Hair Cell Death of Inner Ear Organs Causes Functional Deficits in Adult Zebrafish (Danio rerio)”, PLoS ONE, 8(3), 58755. https://doi.org/10.1371/journal.pone.0058755 </p>
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<p><p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Aminoglycoside antibiotics, like gentamicin, kill inner ear sensory hair cells in a variety of species including chickens, mice, and humans. The zebrafish (Danio rerio) has been used to study hair cell cytotoxicity in the lateral line organs of larval and adult animals. To assess the ototoxic effects of gentamicin, adult zebrafish received a single 250 mg/kg intraperitoneal injection of gentamicin and, 24 hours later, auditory evoked potential recordings (AEPs) revealed significant shifts in auditory thresholds compared to untreated controls </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Uribe <em>et al.</em>, 2013)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">.</span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Uribe, P. M. <em>et al.</em> (2013) ‘Aminoglycoside-Induced Hair Cell Death of Inner Ear Organs Causes Functional Deficits in Adult Zebrafish (Danio rerio)’, <em>PLoS ONE</em>, 8(3), p. 58755. doi: 10.1371/journal.pone.0058755.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">The zebrafish (Danio rerio), a genetically tractable vertebrate, lends itself particularly well as a model system in which to study the ear. Zebrafish do not possess outer or middle ears, but have a fairly typical vertebrate inner ear, the normal development and anatomy of which has been described in a series of atlas-type papers (Haddon and Lewis, 1996; Bang, Sewell and Malicki, 2001). Although the zebrafish ear does not contain a specialized hearing organ—there is no equivalent of the mammalian cochlea—many features are conserved with other vertebrate species (Whitfield, 2002).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Inner ear develops from an ectodermal thickening, the otic placode, visible on either side of the hindbrain from mid-somite stages. In the zebrafish, this placode cavitates to form a hollow ball of epithelium, the otic vesicle, from which all structures of the membranous labyrinth and the neurons of the statoacoustic (VIIIth) ganglion arise (Haddon and Lewis, 1996; Whitfield <em>et al.</em>, 2002). </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">The mature organ, found in all jawed vertebrates, has two functions: it serves as an auditory system, which detects sound waves, and as a vestibular system, which detects linear and angular accelerations, enabling the organism to maintain balance (Whitfield <em>et al.</em>, 1996).</span></span></p>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Direct observation of internal anatomic structures of zebrafish embryos. Defects visible under the dissecting microscope (Whitfield, 2002)</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Comparison of swimming patterns with wild-type fish. <em>Dog-eared</em> embryos are less responsive to vibrational stimuli, fail to maintain balance when swimming, and may circle when disturbed, a behavior characteristic of fish with vestibular defects (Nicolson <em>et al.</em>, 1998)</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">High-throughput behavioral screening method for detecting auditory response defects in zebrafish. Assay monitors a rapid escape reflex in response to a loud sound (Bang <em>et al.</em>, 2002).</span></span></li>
</ul>
<h4>References</h4>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Bang, P. I. <em>et al.</em> (2002) ‘High-throughput behavioral screening method for detecting auditory response defects in zebrafish’, <em>Journal of Neuroscience Methods</em>, 118(2), pp. 177–187. doi: 10.1016/S0165-0270(02)00118-8.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Bang, P. I., Sewell, W. F. and Malicki, J. J. (2001) ‘Morphology and cell type heterogeneities of the inner ear epithelia in adult and juvenile zebrafish (Danio rerio)’, <em>Journal of Comparative Neurology</em>, 438(2), pp. 173–190. doi: 10.1002/cne.1308.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Haddon, C. and Lewis, J. (1996) ‘Early ear development in the embryo of the zebrafish, Danio rerio’, <em>Journal of Comparative Neurology</em>, 365(1), pp. 113–128. doi: 10.1002/(SICI)1096-9861(19960129)365:1<113::AID-CNE9>3.0.CO;2-6.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Nicolson, T. <em>et al.</em> (1998) ‘Genetic analysis of vertebrate sensory hair cell mechanosensation: The zebrafish circler mutants’, <em>Neuron</em>, 20(2), pp. 271–283. doi: 10.1016/S0896-6273(00)80455-9.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Uribe, P. M. <em>et al.</em> (2013) ‘Aminoglycoside-Induced Hair Cell Death of Inner Ear Organs Causes Functional Deficits in Adult Zebrafish (Danio rerio)’, <em>PLoS ONE</em>, 8(3), p. 58755. doi: 10.1371/journal.pone.0058755.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Whitfield, T. T. <em>et al.</em> (1996) ‘Mutations affecting development of the zebrafish inner ear and lateral line’, <em>Development</em>, 123, pp. 241–254. doi: 10.1242/dev.123.1.241.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Whitfield, T. T. <em>et al.</em> (2002) ‘Development of the zebrafish inner ear’, <em>Developmental Dynamics</em>, 223(4), pp. 427–458. doi: 10.1002/dvdy.10073.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Whitfield, T. T. (2002) ‘Zebrafish as a Model for Hearing and Deafness’, <em>J Neurobiol</em>, 53, pp. 157–171. doi: 10.1002/neu.10123.</span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Whitfield, T. T. (2015) ‘Development of the inner ear’, <em>Current Opinion in Genetics and Development</em>, 32, pp. 112–118. doi: 10.1016/j.gde.2015.02.006.</span></span></p>
<td><a href="/aops/410">Aop:410 - Repression of Gbx2 expression leads to defects in developing inner ear and consequently to increased mortality</a></td>
<td><a href="/aops/410">Aop:410 - GSK3beta inactivation leading to increased mortality via defects in developing inner ear</a></td>
<li>A sense of hearing is known to exist in a wide range of vertebrates and invertebrates, although the organs and structures involved vary widely.</li>
</ul>
<h4>Key Event Description</h4>
<p>Hearing refers to the ability to perceive sound vibrations propagated as pressure changes through a medium such as air or water. Reduced hearing in the context of this key event can refer to reduction in the perceived volume of a sound relative to the amplitude of sound waves. Reduced hearing may also refer to a reduced range of frequencies that can be perceived.</p>
<h4>How it is Measured or Detected</h4>
<p>Hearing is generally measured behaviorally or electrophysiologically.</p>
<ul>
<li>Common behavioral tests involve transmission of pure tones of defined amplitude and frequency using and audiometer or PC and using a behavioral response (e.g., clicking a button; startle response) to determine whether the tone is perceived.</li>
</ul>
<p>Electrophysiological tests:</p>
<ul>
<li>Auditory brainstem response (ABR): Uses electrodes placed on the head to detect auditory evoked potentials from background electrical activity in the brain.</li>
</ul>
<p>Hearing tests in Fish:</p>
<ul>
<li>Through the mid-late 1980s conditioning and behavioral tests were most commonly employed in testing fish hearing. Methods reviewed by Fay (1988)</li>
<li>A high throughput behavioral test for detecting auditory response in fish has been described (Bang et al. 2002).</li>
<li>Invasive electrophysiological methods involving surgical insertion of electrodes into the auditory nerves have been employed.</li>
<li>Non-invasive recording of Auditory Evoked Potentials (AEPs; synonymous with ABRs) are now the most common approach for measuring hearing in fish. AEPs can be recorded via electrodes attached cutaneously to the head (see review by Ladich and Fay, 2013).</li>
</ul>
<h4>References</h4>
<ul>
<li>Fay RR (1988) Hearing in vertebrates: a psychophysics databook. Hill-Fay Associates, Winnetka, Ill</li>
<li>Ladich F, Fay RR. Auditory evoked potential audiometry in fish. Reviews in Fish Biology and Fisheries. 2013;23(3):317-364. doi:10.1007/s11160-012-9297-z.</li>
<li>Bang PI, Yelick PC, Malicki JJ, Sewell WF. High-throughput behavioral screening method for detecting auditory response defects in zebrafish. J Neurosci Methods. 2002 Aug 30;118(2):177-87. PubMed PMID: 12204308.</li>
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<td><a href="/aops/138">Aop:138 - Organic anion transporter (OAT1) inhibition leading to renal failure and mortality</a></td>
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<td><a href="/aops/363">Aop:363 - Thyroperoxidase inhibition leading to increased mortality via altered retinal layer structure</a></td>
<td><a href="/aops/363">Aop:363 - Thyroperoxidase inhibition leading to altered visual function via altered retinal layer structure</a></td>
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<td><a href="/aops/377">Aop:377 - Dysregulated prolonged Toll Like Receptor 9 (TLR9) activation leading to Acute Respiratory Distress Syndrome (ARDS) and Multiple Organ Dysfunction (MOD)</a></td>
<td><a href="/aops/377">Aop:377 - Dysregulated prolonged Toll Like Receptor 9 (TLR9) activation leading to Multi Organ Failure involving Acute Respiratory Distress Syndrome (ARDS)</a></td>
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<td><a href="/aops/364">Aop:364 - Thyroperoxidase inhibition leading to increased mortality via decreased eye size</a></td>
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<td><a href="/aops/365">Aop:365 - Thyroperoxidase inhibition leading to increased mortality via altered photoreceptor patterning</a></td>
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<td><a href="/aops/399">Aop:399 - Inhibition of Fyna leading to increased mortality via decreased eye size (Microphthalmos)</a></td>
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<td><a href="/aops/413">Aop:413 - Oxidation and antagonism of reduced glutathione leading to mortality via acute renal failure</a></td>
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<td><a href="/aops/410">Aop:410 - Repression of Gbx2 expression leads to defects in developing inner ear and consequently to increased mortality</a></td>
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<td><a href="/aops/450">Aop:450 - Inhibition of AChE and activation of CYP2E1 leading to sensory axonal peripheral neuropathy and mortality</a></td>
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<td><a href="/aops/536">Aop:536 - Estrogen receptor agonism leading to reduced survival and population growth due to renal failure</a></td>
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<td><a href="/aops/564">Aop:564 - DBDPE-induced inhibition of mitochondrial complex Ⅰ leading to population decline via neurotoxicity and metabotoxicity.</a></td>
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<td><a href="/aops/605">Aop:605 - Thyroid Peroxidase Inhibition Leading to Reduced, Swimming Performance via Hypomyelination</a></td>
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<td><a href="/aops/608">Aop:608 - Thyroid Hormone Excess Leading to Reduced, Swimming Performance via Hypomyelination</a></td>
<p>All living things are susceptible to mortality.</p>
<h4>Key Event Description</h4>
<p>Increased mortality refers to an increase in the number of individuals dying in an experimental replicate group or in a population over a specific period of time.</p>
<p><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Increased mortality refers to an increase in the number of individuals dying in an experimental replicate group or in a population over a specific period of time.</span></span></span></span></p>
<h4>How it is Measured or Detected</h4>
<p><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Calibri,sans-serif"><span style="color:#212529">Mortality of animals is generally observed as cessation of the heart beat, breathing (gill or lung movement) and locomotory movements. Mortality is typically measured by observation. Depending on the size of the organism, instruments such as microscopes may be used. The reported metric is mostly the mortality rate: the number of deaths in a given area or period, or from a particular cause.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="font-size:11pt"><span style="color:#212529"><span style="background-color:white">Mortality of animals is generally observed as cessation of the heart beat, breathing (gill or lung movement) and locomotory movements. Mortality is typically measured by observation. Depending on the size of the organism, instruments such as microscopes may be used. The reported metric is mostly the mortality rate: the number of deaths in a given area or period, or from a particular cause.</span></span></span></span></span></span></p>
<p><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Calibri,sans-serif"><span style="color:#212529">Depending on the species and the study setup, mortality can be measured:</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="font-size:11pt"><span style="color:#212529"><span style="background-color:white">Depending on the species and the study setup, mortality can be measured:</span></span></span></span></span></span></p>
<ul>
<li><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Calibri,sans-serif"><span style="color:#212529">in the lab by recording mortality during exposure experiments</span></span></span></span></li>
<li><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Calibri,sans-serif"><span style="color:#212529">in dedicated setups simulating a realistic situation such as mesocosms or drainable ponds for aquatic species</span></span></span></span></li>
<li><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Calibri,sans-serif"><span style="color:#212529">in the field, for example by determining age structure after one capture, or by capture-mark-recapture efforts. The latter is a method commonly used in ecology to estimate an animal population's size where it is impractical to count every individual.</span></span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:11pt"><span style="color:#212529"><span style="background-color:white">in the lab by recording mortality during exposure experiments</span></span></span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:11pt"><span style="color:#212529"><span style="background-color:white">in dedicated setups simulating a realistic situation such as mesocosms or drainable ponds for aquatic species</span></span></span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:11pt"><span style="color:#212529"><span style="background-color:white">in the field, for example by determining age structure after one capture, or by capture-mark-recapture efforts. The latter is a method commonly used in ecology to estimate an animal population's size where it is impractical to count every individual.</span></span></span></span></span></li>
</ul>
<h4>Regulatory Significance of the AO</h4>
<p>Increased mortality is one of the most common regulatory assessment endpoints, along with reduced growth and reduced reproduction.</p>
<h4><a href="/events/360">Event: 360: Decrease, Population growth rate</a></h4>
<h5>Short Name: Decrease, Population growth rate</h5>
<td><a href="/aops/23">Aop:23 - Androgen receptor agonism leading to reproductive dysfunction (in repeat-spawning fish)</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/25">Aop:25 - Aromatase inhibition leading to reproductive dysfunction</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/29">Aop:29 - Estrogen receptor agonism leading to reproductive dysfunction</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/30">Aop:30 - Estrogen receptor antagonism leading to reproductive dysfunction</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/100">Aop:100 - Cyclooxygenase inhibition leading to reproductive dysfunction via inhibition of female spawning behavior</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/122">Aop:122 - Prolyl hydroxylase inhibition leading to reproductive dysfunction via increased HIF1 heterodimer formation</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/123">Aop:123 - Unknown MIE leading to reproductive dysfunction via increased HIF-1alpha transcription</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/155">Aop:155 - Deiodinase 2 inhibition leading to increased mortality via reduced posterior swim bladder inflation</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/156">Aop:156 - Deiodinase 2 inhibition leading to increased mortality via reduced anterior swim bladder inflation</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/157">Aop:157 - Deiodinase 1 inhibition leading to increased mortality via reduced posterior swim bladder inflation </a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/158">Aop:158 - Deiodinase 1 inhibition leading to increased mortality via reduced anterior swim bladder inflation</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/159">Aop:159 - Thyroperoxidase inhibition leading to increased mortality via reduced anterior swim bladder inflation</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/101">Aop:101 - Cyclooxygenase inhibition leading to reproductive dysfunction via inhibition of pheromone release</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/102">Aop:102 - Cyclooxygenase inhibition leading to reproductive dysfunction via interference with meiotic prophase I /metaphase I transition</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/63">Aop:63 - Cyclooxygenase inhibition leading to reproductive dysfunction</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/103">Aop:103 - Cyclooxygenase inhibition leading to reproductive dysfunction via interference with spindle assembly checkpoint</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/292">Aop:292 - Inhibition of tyrosinase leads to decreased population in fish</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/310">Aop:310 - Embryonic Activation of the AHR leading to Reproductive failure, via epigenetic down-regulation of GnRHR</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/16">Aop:16 - Acetylcholinesterase inhibition leading to acute mortality</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/312">Aop:312 - Acetylcholinesterase Inhibition leading to Acute Mortality via Impaired Coordination & Movement</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/334">Aop:334 - Glucocorticoid Receptor Agonism Leading to Impaired Fin Regeneration</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/336">Aop:336 - DNA methyltransferase inhibition leading to population decline (1)</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/337">Aop:337 - DNA methyltransferase inhibition leading to population decline (2)</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/338">Aop:338 - DNA methyltransferase inhibition leading to population decline (3)</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/339">Aop:339 - DNA methyltransferase inhibition leading to population decline (4)</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/340">Aop:340 - DNA methyltransferase inhibition leading to transgenerational effects (1)</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/341">Aop:341 - DNA methyltransferase inhibition leading to transgenerational effects (2)</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/289">Aop:289 - Inhibition of 5α-reductase leading to impaired fecundity in female fish</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/297">Aop:297 - Inhibition of retinaldehyde dehydrogenase leads to population decline</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/346">Aop:346 - Aromatase inhibition leads to male-biased sex ratio via impacts on gonad differentiation</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/363">Aop:363 - Thyroperoxidase inhibition leading to altered visual function via altered retinal layer structure</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/349">Aop:349 - Inhibition of 11β-hydroxylase leading to decresed population trajectory </a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/348">Aop:348 - Inhibition of 11β-Hydroxysteroid Dehydrogenase leading to decreased population trajectory </a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/376">Aop:376 - Androgen receptor agonism leading to male-biased sex ratio</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/386">Aop:386 - Deposition of ionizing energy leading to population decline via inhibition of photosynthesis</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/387">Aop:387 - Deposition of ionising energy leading to population decline via mitochondrial dysfunction</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/388">Aop:388 - Deposition of ionising energy leading to population decline via programmed cell death</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/389">Aop:389 - Oxygen-evolving complex damage leading to population decline via inhibition of photosynthesis</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/364">Aop:364 - Thyroperoxidase inhibition leading to altered visual function via decreased eye size</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/365">Aop:365 - Thyroperoxidase inhibition leading to altered visual function via altered photoreceptor patterning</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/399">Aop:399 - Inhibition of Fyna leading to increased mortality via decreased eye size (Microphthalmos)</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/410">Aop:410 - GSK3beta inactivation leading to increased mortality via defects in developing inner ear</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/216">Aop:216 - Deposition of energy leading to population decline via DNA strand breaks and follicular atresia</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/238">Aop:238 - Deposition of energy leading to population decline via DNA strand breaks and oocyte apoptosis</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/299">Aop:299 - Deposition of energy leading to population decline via DNA oxidation and follicular atresia</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/311">Aop:311 - Deposition of energy leading to population decline via DNA oxidation and oocyte apoptosis</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/444">Aop:444 - Ionizing radiation leads to reduced reproduction in Eisenia fetida via reduced spermatogenesis and cocoon hatchability</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/138">Aop:138 - Organic anion transporter (OAT1) inhibition leading to renal failure and mortality</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/177">Aop:177 - Cyclooxygenase 1 (COX1) inhibition leading to renal failure and mortality</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/97">Aop:97 - 5-hydroxytryptamine transporter (5-HTT; SERT) inhibition leading to population decline</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/203">Aop:203 - 5-hydroxytryptamine transporter inhibition leading to decreased reproductive success and population decline</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/218">Aop:218 - Inhibition of CYP7B activity leads to decreased reproductive success via decreased locomotor activity</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/219">Aop:219 - Inhibition of CYP7B activity leads to decreased reproductive success via decreased sexual behavior</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/323">Aop:323 - PPARalpha Agonism Leading to Decreased Viable Offspring via Decreased 11-Ketotestosterone</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/536">Aop:536 - Estrogen receptor agonism leading to reduced survival and population growth due to renal failure</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/540">Aop:540 - Oxidative Stress in the Fish Ovary Leads to Reproductive Impairment via Reduced Vitellogenin Production</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/564">Aop:564 - DBDPE-induced inhibition of mitochondrial complex Ⅰ leading to population decline via neurotoxicity and metabotoxicity.</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/567">Aop:567 - Binding to plastoquinone B site leading to decreased population growth rate via photosystem II inhibition</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/592">Aop:592 - DBDPE-induced DNA strand breaks and LDH activity inhibition leading to population growth rate decline via energy metabolism disrupt and apoptosis</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/605">Aop:605 - Thyroid Peroxidase Inhibition Leading to Reduced, Swimming Performance via Hypomyelination</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/608">Aop:608 - Thyroid Hormone Excess Leading to Reduced, Swimming Performance via Hypomyelination</a></td>
<p>Consideration of population size and changes in population size over time is potentially relevant to all living organisms.</p>
<h4>Key Event Description</h4>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">A population can be defined as a group of interbreeding organisms, all of the same species, occupying a specific space during a specific time (Vandermeer and Goldberg 2003, Gotelli 2008). As the population is the biological level of organization that is often the focus of ecological risk</span> <span style="color:black">assessments, population growth rate (and hence population size over time) is important to consider within the context of applied conservation practices.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">If N is the size of the population and t is time, then the population growth rate (dN/dt) is proportional to the instantaneous rate of increase, r, which measures the per capita rate of population increase over a short time interval. Therefore, r, is a difference between the instantaneous birth rate (number of births per individual per unit of time; b) and the instantaneous death rate (number of deaths per individual per unit of time; d) [Equation 1]. Because r is an instantaneous rate, its units can be changed via division. For example, as there are 24 hours in a day, an r of 24 individuals/(individual x day) is equal to an r of 1 individual/(individual/hour) (Caswell 2001, Vandermeer and Goldberg 2003, Gotelli 2008, Murray and Sandercock 2020). </span></span></span></span></p>
<p style="margin-left:144px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Equation 1: r = b - d</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">This key event refers to scenarios where r < 0 (instantaneous death rate exceeds instantaneous birth rate).</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Examining r in the context of population growth rate:</span></span></span></span></p>
<p style="margin-left:48px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">● A population will decrease to extinction when the instantaneous death rate exceeds the instantaneous birth rate (r < 0). </span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black"> ● The smaller the value of r below 1, the faster the population will decrease to zero. </span></span></span></span></p>
<p style="margin-left:48px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">● A population will increase when resources are available and the instantaneous birth rate exceeds the instantaneous death rate (r > 0)</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black"> ● The larger the value that r exceeds 1, the faster the population can increase over time </span></span></span></span></p>
<p style="margin-left:48px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">● A population will neither increase or decrease when the population growth rate equals 0 (either due to N = 0, or if the per capita birth and death rates are exactly balanced). For example, the per capita birth and death rates could become exactly balanced due to density dependence and/or to the effect of a stressor that reduces survival and/or reproduction (Caswell 2001, Vandermeer and Goldberg 2003, Gotelli 2008, Murray and Sandercock 2020). </span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Effects incurred on a population from a chemical or non-chemical stressor could have an impact directly upon birth rate (reproduction) and/or death rate (survival), thereby causing a decline in population growth rate. </span></span></span></span></p>
<p style="margin-left:48px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">● Example of direct effect on r: Exposure to 17b-trenbolone reduced reproduction (i.e., reduced b) in the fathead minnow over 21 days at water concentrations ranging from 0.0015 to about 41 mg/L (Ankley et al. 2001; Miller and Ankley 2004). </span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Alternatively, a stressor could indirectly impact survival and/or reproduction. </span></span></span></span></p>
<p style="margin-left:48px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">● Example of indirect effect on r: Exposure of non-sexually differentiated early life stage fathead minnow to the fungicide prochloraz has been shown to produce male-biased sex ratios based on gonad differentiation, and resulted in projected change in population growth rate (decrease in reproduction due to a decrease in females and thus recruitment) using a population model. (Holbech et al., 2012; Miller et al. 2022)</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Density dependence can be an important consideration:</span></span></span></span></p>
<p style="margin-left:48px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">● The effect of density dependence depends upon the quantity of resources present within a landscape. A change in available resources could increase or decrease the effect of density dependence and therefore cause a change in population growth rate via indirectly impacting survival and/or reproduction. </span></span></span></span></p>
<p style="margin-left:48px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">● This concept could be thought of in terms of community level interactions whereby one species is not impacted but a competitor species is impacted by a chemical stressor resulting in a greater availability of resources for the unimpacted species. In this scenario, the impacted species would experience a decline in population growth rate. The unimpacted species would experience an increase in population growth rate (due to a smaller density dependent effect upon population growth rate for that species). </span> </span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Closed versus open systems:</span></span></span></span></p>
<p style="margin-left:48px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">● The above discussion relates to closed systems (there is no movement of individuals between population sites) and thus a declining population growth rate cannot be augmented by immigration. </span></span></span></span></p>
<p style="margin-left:48px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">● When individuals depart (emigrate out of a population) the loss will diminish population growth rate. </span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Population growth rate applies to all organisms, both sexes, and all life stages.</span></span></span></span></p>
<p> </p>
<h4>How it is Measured or Detected</h4>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Population growth rate (instantaneous growth rate) can be measured by sampling a population over an interval of time (i.e. from time t = 0 to time t = 1). The interval of time should be selected to correspond to the life history of the species of interest (i.e. will be different for rapidly growing versus slow growing populations). The population growth rate, r, can be determined by taking the difference (subtracting) between the initial population size, N</span><sub><span style="font-size:9pt"><span style="color:black">t=0 </span></span></sub><span style="color:black">(population size at time t=0), and the population size at the end of the interval, N</span><sub><span style="font-size:9pt"><span style="color:black">t=1 </span></span></sub><span style="color:black">(population size at time t = 1), and then subsequently dividing by the initial population size. </span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">The diversity of forms, sizes, and life histories among species has led to the development of a vast number of field techniques for estimation of population size and thus population growth over time (Bookhout 1994, McComb et al. 2021). </span></span></span></span></p>
<p style="margin-left:48px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">● For stationary species an observational strategy may involve dividing a habitat into units. After setting up the units, samples are performed throughout the habitat at a select number of units (determined using a statistical sampling design) over a time interval (at time t = 0 and again at time t = 1), and the total number of organisms within each unit are counted. The numbers recorded are assumed to be representative for the habitat overall, and can be used to estimate the population growth rate within the entire habitat over the time interval. </span></span></span></span></p>
<p style="margin-left:48px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">● For species that are mobile throughout a large range, a strategy such as using a mark-recapture method may be employed (i.e. tags, bands, transmitters) to determine a count over a time interval (at time = 0 and again at time =1). </span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Population growth rate can also be estimated using mathematical model constructs (for example, ranging from simple differential equations to complex age or stage structured matrix projection models and individual based modeling approaches), and may assume a linear or nonlinear population increase over time (Caswell 2001, Vandermeer and Goldberg 2003, Gotelli 2008, Murray and Sandercock 2020). The AOP framework can be used to support the translation of pathway-specific mechanistic data into responses relevant to population models and output from the population models, such as changing (declining) population growth rate, can be used to assess and manage risks of chemicals (Kramer et al. 2011). As such, this translational capability can increase the capacity and efficiency of safety assessments both for single chemicals and chemical mixtures (Kramer et al. 2011). </span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Some examples of modeling constructs used to investigate population growth rate:</span></span></span></span></p>
<p style="margin-left:48px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">● A modeling construct could be based upon laboratory toxicity tests to determine effect(s) that are then linked to the population model and used to estimate decline in population growth rate. Miller et al. (2007) used concentration–response data from short term reproductive assays with fathead minnow (<em>Pimephales promelas</em>) exposed to endocrine disrupting chemicals in combination with a population model to examine projected alterations in population growth rate. </span></span></span></span></p>
<p style="margin-left:48px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">● A model construct could be based upon a combination of effects-based monitoring at field sites (informed by an AOP) and a population model. Miller et al. (2015) applied a population model informed by an AOP to project declines in population growth rate for white suckers (Catostomus commersoni) using observed changes in sex steroid synthesis in fish exposed to a complex pulp and paper mill effluent in Jackfish Bay, Ontario, Canada. Furthermore, a model construct could be comprised of a series of quantitative models using KERs that culminates in the estimation of change (decline) in population growth rate. </span></span></span></span></p>
<p style="margin-left:48px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">● A quantitative adverse outcome pathway (qAOP) has been defined as a mathematical construct that models the dose–response or response–response relationships of all KERs described in an AOP (Conolly et al. 2017, Perkins et al. 2019). Conolly et al. (2017) developed a qAOP using data generated with the aromatase inhibitor fadrozole as a stressor and then used it to predict potential population‐level impacts (including decline in population growth rate). The qAOP modeled aromatase inhibition (the molecular initiating event) leading to reproductive dysfunction in fathead minnow (Pimephales promelas) using 3 computational models: a hypothalamus–pituitary–gonadal axis model (based on ordinary differential equations) of aromatase inhibition leading to decreased vitellogenin production (Cheng et al. 2016), a stochastic model of oocyte growth dynamics relating vitellogenin levels to clutch size and spawning intervals (Watanabe et al. 2016), and a population model (Miller et al. 2007).</span></span></span></span></p>
<p style="margin-left:48px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">● Dynamic energy budget (DEB) models offer a methodology that reverse engineers stressor effects on growth, reproduction, and/or survival into modular characterizations related to the acquisition and processing of energy resources (Nisbet et al. 2000, Nisbet et al. 2011). Murphy et al. (2018) developed a conceptual model to link DEB and AOP models by interpreting AOP key events as measures of damage-inducing processes affecting DEB variables and rates.</span></span></span></span></p>
<p style="margin-left:48px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">● Endogenous Lifecycle Models (ELMs), capture the endogenous lifecycle processes of growth, development, survival, and reproduction and integrate these to estimate and predict expected fitness (Etterson and Ankley, 2021). AOPs can be used to inform ELMs of effects of chemical stressors on the vital rates that determine fitness, and to decide what hierarchical models of endogenous systems should be included within an ELM (Etterson and Ankley, 2021).</span></span></span></span></p>
<p> </p>
<h4>Regulatory Significance of the AO</h4>
<p>Maintenance of sustainable fish and wildlife populations (i.e., adequate to ensure long-term delivery of valued ecosystem services) is a widely accepted regulatory goal upon which risk assessments and risk management decisions are based.</p>
<h4>References</h4>
<ul>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Ankley GT, Jensen KM, Makynen EA, Kahl MD, Korte JJ, Hornung MW, Henry TR, Denny JS, Leino RL, Wilson VS, Cardon MD, Hartig PC, Gray LE. 2003. Effects of the androgenic growth promoter 17b-trenbolone on fecundity and reproductive endocrinology of the fathead minnow. Environ. Toxicol. Chem. 22: 1350–1360.</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Bookhout TA. 1994. Research and management techniques for wildlife and habitats. The Wildlife Society, Bethesda, Maryland. 740 pp.</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Cheng WY, Zhang Q, Schroeder A, Villeneuve DL, Ankley GT, Conolly R. 2016. Computational modeling of plasma vitellogenin alterations in response to aromatase inhibition in fathead minnows. Toxicol Sci 154: 78–89.</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Conolly RB, Ankley GT, Cheng W-Y, Mayo ML, Miller DH, Perkins EJ, Villeneuve DL, Watanabe KH. 2017. Quantitative adverse outcome pathways and their application to predictive toxicology. Environ. Sci. Technol. 51: 4661-4672.</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Etterson MA, Ankley GT. 2021. Endogenous Lifecycle Models for Chemical Risk Assessment. Environ. Sci. Technol. 55: 15596-15608. </span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Gotelli NJ, 2008. A Primer of Ecology. Sinauer Associates, Inc., Sunderland, MA, USA.</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Holbech H, Kinnberg KL, Brande-Lavridsen N, Bjerregaard P, Petersen GI, Norrgren L, Orn S, Braunbeck T, Baumann L, Bomke C, Dorgerloh M, Bruns E, Ruehl-Fehlert C, Green JW, Springer TA, Gourmelon A. 2012 Comparison of zebrafish (<em>Danio rerio</em>) and fathead minnow <em>(Pimephales promelas</em>) as test species in the Fish Sexual Development Test (FSDT). Comp. Biochem. Physiol. C Toxicol. Pharmacol. 155: 407–415.</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Kramer VJ, Etterson MA, Hecker M, Murphy CA, Roesijadi G, Spade DJ, Stromberg JA, Wang M, Ankley GT. </span><span style="color:black">2011. Adverse outcome pathways and risk assessment: Bridging to population level effects. Environ. Toxicol. Chem. 30, 64-76.</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">McComb B, Zuckerberg B, Vesely D, Jordan C. 2021. Monitoring Animal Populations and their Habitats: A Practitioner's Guide. Pressbooks, Oregon State University, Corvallis, OR Version 1.13, 296 pp. </span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Miller DH, Villeneuve DL, Santana Rodriguez KJ, Ankley GT. 2022. A multidimensional matrix model for predicting the effect of male biased sex ratios on fish populations. Environmental Toxicology and Chemistry 41(4): 1066-1077.</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Miller DH, Tietge JE, McMaster ME, Munkittrick KR, Xia X, Griesmer DA, Ankley GT. 2015. </span><span style="color:black">Linking mechanistic toxicology to population models in forecasting recovery from chemical stress: A case study from Jackfish Bay, Ontario, Canada. Environmental Toxicology and Chemistry 34(7): 1623-1633.</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Miller DH, Jensen KM, Villeneuve DE, Kahl MD, Makynen EA, Durhan EJ, Ankley GT. 2007. </span><span style="color:black">Linkage of biochemical responses to population-level effects: A case study with vitellogenin in the fathead minnow (<em>Pimephales promelas</em>). Environ Toxicol Chem 26: 521–527.</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Miller DH, Ankley GT. 2004. Modeling impacts on populations: Fathead minnow (<em>Pimephales promelas</em>) exposure to the endocrine disruptor 17b-trenbolone as a case study. Ecotox Environ Saf 59: 1–9.</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Murphy CA, Nisbet RM, Antczak P, Garcia-Reyero N, Gergs A, Lika K, Mathews T, Muller EB, Nacci D, Peace A, Remien CH, Schultz IR, Stevenson LM, Watanabe KH. 2018. Incorporating suborganismal processes into dynamic energy budget models for ecological risk assessment. Integrated Environmental Assessment and Management 14(5): 615–624.</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Murray DL, Sandercock BK (editors). 2020. Population ecology in practice. Wiley-Blackwell, Oxford UK, 448 pp.</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Nisbet RM, Jusup M, Klanjscek T, Pecquerie L. 2011. Integrating dynamic energy budget (DEB) theory with traditional bioenergetic models. The Journal of Experimental Biology 215: 892-902.</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Nisbet RM, Muller EB, Lika K, Kooijman SALM. 2000. </span><span style="color:black">From molecules to ecosystems through dynamic energy budgets. J Anim Ecol 69: 913–926.</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Perkins EJ, Ashauer R, Burgoon L, Conolly R, Landesmann B,, Mackay C, Murphy CA, Pollesch N, Wheeler JR, Zupanic A, Scholzk S. 2019. Building and applying quantitative adverse outcome pathway models for chemical hazard and risk assessment. Environmental Toxicology and Chemistry 38(9): 1850–1865. </span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Vandermeer JH, Goldberg DE. 2003. Population ecology: first principles. Princeton University Press, Princeton NJ, 304 pp.</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Villeneuve DL, Crump D, Garcia-Reyero N, Hecker M, Hutchinson TH, LaLone CA, Landesmann B, Lattieri T, Munn S, Nepelska M, Ottinger MA, Vergauwen L, Whelan M. Adverse outcome pathway (AOP) development 1: Strategies and principles. Toxicol Sci. 2014: 142:312–320</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Watanabe KH, Mayo M, Jensen KM, Villeneuve DL, Ankley GT, Perkins EJ. 2016. Predicting fecundity of fathead minnows (<em>Pimephales promelas</em>) exposed to endocrine‐disrupting chemicals using a MATLAB(R)‐based model of oocyte growth dynamics. PLoS One 11: e0146594.</span></span></span></li>
</ul>
<h2>Appendix 2</h2>
<h2>List of Key Event Relationships in the AOP</h2>
<div id="evidence_supporting_links">
<h3>List of Adjacent Key Event Relationships</h3>
<div>
<h4><a href="/relationships/2485">Relationship: 2485: GSK3beta inactivation leads to Repression of Gbx2 expression</a></h4>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Evidence for this KER is provided for zebrafish </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Wang <em>et al.</em>, 2018)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"> and humans </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Grassilli <em>et al.</em>, 2014; Kim <em>et al.</em>, 2018)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">.</span></span></p>
<h4>Key Event Relationship Description</h4>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Wnt signaling is implicated in anteroposterior (AP) axis patterning and midbrain specification in both animal and human systems. GSK3 is a key enzyme mediating the canonical Wnt signaling. Inhibition of GSK3b (one of the isoforms of GSK3) leads to activation of canonical Wnt signal pathway </span></span><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif">(Grassilli <em>et al.</em>, 2014)</span></span><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">. Gbx2 is one of the representative AP markers and is downregulated in activation of Wnt signal pathway (GSK3b inhibition) (Kim <em>et al.</em>, 2018).</span></span></p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<ul>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Zebrafish embryos were treated with chemical inhibitors or activators of various signaling pathways, such as the Wnt, FGF, retinoic acid (RA), HH, BMP, Nodal, and Notch pathways, from 14 hpf to 18 hpf, immediately before the advent of <em>gbx2</em> expression in the telencephalon, and than <em>gbx2</em> expression was examined in the telencephalon. </span></span><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">In zebrafish embryos treated with BIO, a selective GSK3 inhibitor that activates Wnt signaling, <em>gbx2</em> expression was specifically repressed in the telencephalon, but was unaffected or weakly activated in the isthmus and OV (Wang <em>et al.</em>, 2018).</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Treatment of human ESC-derived NCPs with BIO (Gsk3b inhibitor) downregulated expression of <em>GBX2</em> in dose dependent manner (Kim <em>et al.</em>, 2018). Quantitative gene expression analysis following seven days of treatment revealed that the <em>GBX2</em> expression decreased as the BIO concentration increased (Kim <em>et al.</em>, 2018).</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">To confirm whether the effect of BIO on midbrain specification was indeed through the activation of canonical Wnt signal, other small molecules that inhibit GSK3 were tested in different modes of action, such as 1- AKP and LiCl on human ESC-derived NPCs. LiCl treatment elicited similar gene expression patterns (decreased expression of GBX2) as BIO treatment, although the fold changes in gene expression were lower than those of the other inhibitors. These data support that midbrain-specific gene expression results from the activation of canonical Wnt signal via GSK3 inhibition (Kim <em>et al.</em>, 2018).</span></span></li>
</ul>
<strong>Empirical Evidence</strong>
<p>No Data.</p>
<strong>Uncertainties and Inconsistencies</strong>
<p>No Data.</p>
<h4>Quantitative Understanding of the Linkage</h4>
<p>No Data.</p>
<strong>Response-response relationship</strong>
<p>No Data.</p>
<strong>Time-scale</strong>
<p><em>Gbx2</em> begins to express in telencephalon approximately 14-18hpf <span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">(Wang <em>et al.</em>, 2018).</span></span></p>
<pre style="text-align:left">
</pre>
<strong>Known modulating factors</strong>
<p>No Data.</p>
<strong>Known Feedforward/Feedback loops influencing this KER</strong>
<p>No Data.</p>
<h4>References</h4>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Grassilli, E. <em>et al.</em> (2014) ‘GSK3A is redundant with GSK3B in modulating drug resistance and chemotherapy-induced necroptosis’, <em>PLoS ONE</em>, 9(7), pp. 1–8. doi: 10.1371/journal.pone.0100947.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Kim, J. Y. <em>et al.</em> (2018) ‘Wnt signal activation induces midbrain specification through direct binding of the beta-catenin/TCF4 complex to the EN1 promoter in human pluripotent stem cells’, <em>Experimental & Molecular Medicine</em>, 50, p. 24. doi: 10.1038/s12276-018-0044-y.</span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Wang, Z. <em>et al.</em> (2018) ‘The role of gastrulation brain homeobox 2 (gbx2) in the development of the ventral telencephalon in zebrafish embryos’, <em>Differentiation</em>, 99(December 2017), pp. 28–40. doi: 10.1016/j.diff.2017.12.005.</span></span></p>
</div>
<div>
<h4><a href="/relationships/2436">Relationship: 2436: Repression of Gbx2 expression leads to foxi1 expression, increased</a></h4>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">The gastrulation brain homebox (Gbx) group of transcription factor genes, composed of two genes, gbx1 and gbx2, in vertebrates, is also present in invertebrates (Chiang et al., 1995), and can be regarded as widely conserved among animals (Wang et al., 2018). Gbx2 functions in a variety of developmental processes after midbrain-hindbrain boundary (MHB) establishment. (Burroughs-Garcia et al., 2011) data demonstrate that the role of gbx2 in anterior hindbrain development is functionally conserved between zebrafish and mice. This gene was shown to be required for neural crest (NC) formation in mice </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(B. Li et al., 2009; Roeseler et al., 2012)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">. In Xenopus gbx2 is the earliest factor for specifying neural crest (NC) cells, and that gbx2 is directly regulated by NC inducing signaling pathways, such as Wnt/β-catenin signaling (Li et al., 2009). </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Foxi I class genes have been described in zebrafish(Hans et al., 2004; Solomon et al., 2003), humans (Larsson et al., 1995; Pierrou et al., 1994), mouse (Hulander et al., 1998; Overdier et al., 1997), rat (Clevidence et al., 1993) and Xenopus (Lef et al., 1994, 1996). However, it is unclear whether zebrafish foxi1 is orthologous to any one of these genes. The Xenopus FoxI1c (Lef et al., 1996), FoxI1a and FoxI1b genes (Lef et al., 1994) share the highest degree of sequence conservation with the zebrafish gene. The expression pattern of the two Xenopus pseudoallelic variants FoxI1a/b does not suggest functional similarity to zebrafish foxi1. Of the three Xenopus FoxI genes, FoxI1c (XFD-10) is most similar to foxi1 in sequence. However, Xenopus FoxI1c was reported to be expressed in the neuroectoderm and somites but not in the otic placode, unlike the pattern for foxi1 reported in (Lef et al., 1996). (Pohl et al., 2002) report provides a more detailed description of Xenopus FoxI1c, which suggests that this gene is expressed in preplacodal tissue and the branchial arches, similar to observations for zebrafish foxi1. Thus, it appears probable that Xenopus FoxI1c represents the ortholog of zebrafish foxi1 (Solomon et al., 2003).</span></span></p>
<h4>Key Event Relationship Description</h4>
<p>Repression of Gbx2 expression leads to increased expression of foxi1.</p>
<h4>Evidence Supporting this KER</h4>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Gbx2 exhibits DNA-binding transcription factor activity, RNA polymerase II-specific. Involved in cerebellum development; iridophore differentiation; and telencephalon regionalization. Predicted to localize to nucleus. Is expressed in several structures, including midbrain hindbrain boundary neural keel; midbrain hindbrain boundary neural rod; midbrain neural rod; nervous system; and presumptive rhombomere 1 (<em>ZFIN Gene: Gbx2</em>, n.d.). After MHB establishment, murine gbx2 expression continues in the anterior hindbrain, suggesting later developmental roles for this gene. Li et al. (2002) showed different requirements for gbx2 in cerebellum formation depending on the loci along the mediolateral axis (J. Y. H. Li et al., 2002). In zebrafish, gbx2 expression persists in the isthmus until at least the hatching stage (Kikuta et al., 2003), and the roles of gbx2 are conserved in the developing anterior hindbrain, including nV cranial motor neurons, in zebrafish and mice (Burroughs-Garcia et al., 2011).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">A number of studies have shown that Gbx2 represses many developmental regulatory genes during MHB development including foxi1b (Nakamura, 2001; Rhinn & Brand, 2001; Simeone, 2000). Thus, Gbx2 may be a multifunctional transcriptional factor, although the mechanisms of the differential regulation of its activity during development are unknown (Nakayama et al., 2017). In (Nakayama et al., 2017) study Gbx2 has been shown to downregulate Foxi1 in zebrafish embryos.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Foxi1 exhibits DNA-binding transcription factor activity. Involved in several processes, including animal organ development; epidermal cell fate specification; and neuron development. Predicted to localize to nucleus. Is expressed in several structures, including ectoderm; epibranchial ganglion; head; neural crest; and neurogenic field (<em>ZFIN Gene: Foxi1</em>, n.d.).</span></span></p>
<strong>Biological Plausibility</strong>
<p><span style="font-size:14px">Foxi1 is one of the downstream genes regulated by gbx2 transcription factor. Downregulation of gbx2 leads to increased foxi1 expression in zebrafish embryos.</span></p>
<ul>
<li><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Nakayama et al., 2017)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"> sought to comprehensively identify the target genes of zebrafish gbx2 at the end of gastrulation by microarray analysis. Eight genes that had been shown by the microarray data to be downregulated (Group C, otx1b, otx2, hoxb5b, msi2b, neurog1; Group D, pou5f3; Group F, her5, foxi1) were indeed immediately downregulated in hsp-gbx2+ embryos. Most of the genes that were identified as upregulated or downregulated in the microarray analysis were confirmed by qPCR analysis. WISH (whole mount in situ hybridization) further confirmed the alterations in expression for 6 out of the 12 genes examined (otx2, otx1b, her5, hesx1, klf2a, and pou5f3). Failure to detect the expression alterations of the remaining genes with WISH is likely due to the non-quantitative nature of the WISH technique, which can only detect marked differences in expression levels. It is additionally possible that gbx2 induction affected broad and low-level expression that was undetectable by their conventional WISH technique. Still, the qPCR and WISH results together confirmed the reliability of the comprehensive microarray analysis </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Nakayama et al., 2017)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">.</span></span></li>
<strong>Uncertainties and Inconsistencies</strong>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Failure to detect the expression alterations of the remaining genes with WISH is likely due to the non-quantitative nature of the WISH technique, which can only detect marked differences in expression levels. It is additionally possible that gbx2 induction affected broad and low-level expression that was undetectable by their conventional WISH technique. Still, the qPCR and WISH results together confirmed the reliability of the comprehensive microarray analysis (Nakayama et al., 2017). </span></span></p>
<h4>Quantitative Understanding of the Linkage</h4>
<p>No Data</p>
<strong>Response-response relationship</strong>
<p>No Data</p>
<strong>Time-scale</strong>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Wang et al., 2018)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"> have shown that gbx2 is expressed in zebrafish (Danio rerio) embryos only after the late gastrula stage in the anterior hindbrain.</span></span></p>
<strong>Known modulating factors</strong>
<p>No Data</p>
<strong>Known Feedforward/Feedback loops influencing this KER</strong>
<p>No Data</p>
<h4>References</h4>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Burroughs-Garcia, J., Sittaramane, V., Chandrasekhar, A., & Waters, S. T. (2011). Evolutionarily conserved function of Gbx2 in anterior hindbrain development. <em>Developmental Dynamics</em>, <em>240</em>(4), 828–838. https://doi.org/10.1002/dvdy.22589</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Chiang, C., Young, K. E., & Beachy, P. A. (1995). Control of Drosophila tracheal branching by the novel homeodomain gene unplugged, a regulatory target for genes of the bithorax complex. <em>Development</em>, <em>121</em>(11), 3901–3912.</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Clevidence, D. E., Overdier, D. G., Taot, W., Qian, X., Pani, L., Lait, E., & Costa, R. H. (1993). Identification of nine tissue-specific transcription factors of the hepatocyte nuclear factor 3/forkhead DNA-binding-domain family (tissue-specific transcription factors/gene family/differentiation). In <em>Proc. Natl. Acad. Sci. USA</em> (Vol. 90).</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Hans, S., Liu, D., & Westerfield, M. (2004). Pax8 and Pax2a function synergistically in otic specification, downstream of the Foxi1 and Dlx3b transcription factors. <em>Development</em>, <em>131</em>(20), 5091–5102. https://doi.org/10.1242/dev.01346</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Hulander, M., Wurst, W., Carlsson, P., & Enerbäck, S. (1998). The winged helix transcription factor FKh10 is required for normal development of the inner ear. <em>Nature Genetics</em>, <em>20</em>(4), 374–376. https://doi.org/10.1038/3850</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Kikuta, H., Kanai, M., Ito, Y., & Yamasu, K. (2003). gbx2 Homeobox Gene Is Required for the Maintenance of the Isthmic Region in the Zebrafish Embryonic Brain. <em>Developmental Dynamics</em>, <em>228</em>(3), 433–450. https://doi.org/10.1002/dvdy.10409</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Larsson, C., Hellqvist, M., Pierrou, S., White, I., Enerback, S. and, & Carlsson, P. (1995). Chromosomal Localization of Six Human Forkhead Genes, freac-1 (FKHL5), -3 (FKHL7), -4 (FKHL8), -5 (FKHL9), -6 (FKHL10), and -8 (FKHL12). <em>Genomics</em>, <em>30</em>, 464–469.</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Lef, J., Clement, J. H., Oschwald, R., Köster, M., & Knöchel, W. (1994). Spatial and temporal transcription patterns of the forkhead related XFD-2/XFD-2′ genes in Xenopus laevis embryos. <em>Mechanisms of Development</em>, <em>45</em>(2), 117–126. https://doi.org/10.1016/0925-4773(94)90025-6</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Lef, J., Dege, P., Scheucher, M., Forsbach-Birk, V., Clement, J. H., & Knöchel, W. (1996). A fork head related multigene family is transcribed in Xenopus laevis embryos. <em>International Journal of Developmental Biology</em>, <em>40</em>(1), 245–253. https://doi.org/10.1387/ijdb.8735935</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Li, B., Kuriyama, S., Moreno, M., & Mayor, R. (2009). The posteriorizing gene Gbx2 is a direct target of Wnt signalling and the earliest factor in neural crest induction. <em>Development</em>, <em>136</em>(19), 3267–3278. https://doi.org/10.1242/dev.036954</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Li, J. Y. H., Lao, Z., & Joyner, A. L. (2002). Changing requirements for Gbx2 in development of the cerebellum and maintenance of the Mid/hindbrain organizer. <em>Neuron</em>, <em>36</em>(1), 31–43. https://doi.org/10.1016/S0896-6273(02)00935-2</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Nakamura, H. (2001). Regionalization of the optic tectum: Combinations of gene expression that define the tectum. <em>Trends in Neurosciences</em>, <em>24</em>(1), 32–39. https://doi.org/10.1016/S0166-2236(00)01676-3</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Nakayama, Y., Inomata, C., Yuikawa, T., Tsuda, S., & Yamasu, K. (2017). Comprehensive analysis of target genes in zebrafish embryos reveals gbx2 involvement in neurogenesis. <em>Developmental Biology</em>, <em>430</em>(1), 237–248. https://doi.org/10.1016/j.ydbio.2017.07.015</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Overdier, D. G., Ye, H., Peterson, R. S., Clevidence, D. E., & Costa, R. H. (1997). The Winged Helix Transcriptional Activator HFH-3 Is Expressed in the Distal Tubules of Embryonic and Adult Mouse Kidney*. In <em>THE JOURNAL OF BIOLOGICAL CHEMISTRY</em> (Vol. 272, Issue 21). https://doi.org/10.1074/jbc.272.21.13725</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Pierrou, S., Hellqvist, M., Samuelsson, L., Enerbäck, S., & Carlsson, P. (1994). Cloning and characterization of seven human forkhead proteins: Binding site specificity and DNA bending. <em>EMBO Journal</em>, <em>13</em>(20), 5002–5012. https://doi.org/10.1002/j.1460-2075.1994.tb06827.x</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Pohl, B. S., Knöchel, S., Dillinger, K., & Knöchel, W. (2002). Sequence and expression of FoxB2 (XFD-5) and FoxI1c (XFD-10) in Xenopus embryogenesis. <em>Mechanisms of Development</em>, <em>117</em>(1–2), 283–287. https://doi.org/10.1016/S0925-4773(02)00184-3</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Rhinn, M., & Brand, M. (2001). The midbrain-hindbrain boundary organizer. <em>Current Opinion in Neurobiology</em>, <em>11</em>(1), 34–42. https://doi.org/10.1016/S0959-4388(00)00171-9</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Roeseler, D. A., Sachdev, S., Buckley, D. M., Joshi, T., & Wu, D. K. (2012). Elongation Factor 1 alpha1 and Genes Associated with Usher Syndromes Are Downstream Targets of GBX2. <em>PLoS ONE</em>, <em>7</em>(11), 47366. https://doi.org/10.1371/journal.pone.0047366</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Simeone, A. (2000). Positioning the isthmic organizer - Where Otx2 and Gbx2 meet. <em>Trends in Genetics</em>, <em>16</em>(6), 237–240. https://doi.org/10.1016/S0168-9525(00)02000-X</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Solomon, K. S., Kudoh, T., Dawid, I. B., & Fritz, A. (2003). Zebrafish foxi1 mediates otic placode formation and jaw development. <em>Development</em>, <em>130</em>(5), 929–940. https://doi.org/10.1242/dev.00308</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Wang, Z., Nakayama, Y., Tsuda, S., & Yamasu, K. (2018). The role of gastrulation brain homeobox 2 (gbx2) in the development of the ventral telencephalon in zebrafish embryos. <em>Differentiation</em>, <em>99</em>(December 2017), 28–40. https://doi.org/10.1016/j.diff.2017.12.005</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><em>ZFIN Gene: foxi1</em>. (n.d.). Retrieved April 12, 2021, from https://zfin.org/ZDB-GENE-030505-1</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><em>ZFIN Gene: gbx2</em>. (n.d.). Retrieved April 12, 2021, from https://zfin.org/ZDB-GENE-020509-2</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Data was provided for zebrafish (Bricaud et al., 2006; Lleras-Forero & Streit, 2012), mice and chick (Hulander et al., 2003; Lleras-Forero & Streit, 2012)</span></span></p>
<h4>Key Event Relationship Description</h4>
<p>Increased foxi1 expression leads to increased six1b expression.</p>
<h4>Evidence Supporting this KER</h4>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">The forkhead family member, foxi1 is an important player not only in the induction of the otic placode (Solomon et al., 2003) but also in the proper activation of differentiation pathways in the inner ear (Hans et al., 2004). Foxi1 transcription factor regulate <em>six</em> and <em>eya</em> gene expression during anamniote preplacodal induction. When foxi1 is knocked down, the ear anlagen is either entirely missing or greatly reduced (Solomon et al., 2003) and no expression of six1b is detectable (Bricaud et al., 2006). With loss-of-function experiment (Bricaud et al., 2006) demonstrated that foxi1 can regulate, directly or indirectly, six1b transcription in developing zebrafish inner ear. Six1b acts early in both hair cell and neuronal lineages. When six1b is overexpressed, not only are fewer neural progenitors formed but many of these progenitors do not go on to differentiate into neurons. Gain-of-function, together with the six1b loss-of-function results, suggest that six1b is necessary and sufficient for the normal formation of hair cells in the anterior macula, although it inhibits neuronal fate in the developing inner ear (Bricaud et al., 2006).</span></span></p>
<strong>Biological Plausibility</strong>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Foxi1 is an early inducer of the otic placode and positively regulates the expression of six1b transcription factor. </span></span></p>
<ul>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">When foxi1 is knocked down, the ear anlagen is either entirely missing or greatly reduced (Solomon et al., 2003) and no expression of six1b is detectable in otocyst. Because, at 28 hpf, the lack of six1b expression could be secondary to the overall absence of the otic placode attributable to foxi1 loss-of-function, six1b expression was studied at either 28 hpf in embryos with less severe phenotype or at 16.5 hpf when the placode just arises. In both cases, no expression of six1b was detected (Bricaud et al., 2006).</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Overexpression of six1b during inner development was achieved by injecting a synthetic six1b mRNA at early stages. Such gain-of- function experiments gave the opposite phenotype to that seen after six1b loss-of-function. At 3 dpf, more hair cells are present. This overproduction of hair cells is detectable as early as 28 hpf, with an average of four hair cells observed instead of the two in wild-type embryos. We assayed for the presence of differentiated neurons at 3 dpf and neural precursors at 32 hpf with the neuronal markers HuC and neuroD, respectively. At 32 hpf in the six1b overexpressing embryos, fewer neuroD positive cells are detectable in the otic ganglion than in control embryos, suggesting that fewer neural progenitors are formed when six1b is overexpressed. At 3 dpf, the decrease in number of SAG neurons versus controls is even more dramatic. In extreme cases, SAG neurons are completely eliminated. These results indicate that, when six1b is overexpressed, not only are fewer neural progenitors formed but many ofthese progenitors do not go on to differentiate into neurons. In conclusion, these, together with the six1b loss-of-function results, suggest that six1b is necessary and sufficient for the normal formation of hair cells in the anterior macula, although it inhibits neuronal fate in the developing inner ear (Bricaud et al., 2006).</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Overexpression of six1b during inner development was achieved by injecting a synthetic six1b mRNA at early stages. Such gain-of- function experiments gave the opposite phenotype to that seen after six1b loss-of-function. At 3 dpf, more hair cells are present. This overproduction of hair cells is detectable as early as 28 hpf, with an average of four hair cells observed instead of the two in wild-type embryos. (Bricaud et al., 2006) assayed for the presence of differentiated neurons at 3 dpf and neural precursors at 32 hpf with the neuronal markers HuC and neuroD, respectively. At 32 hpf in the six1b overexpressing embryos, fewer neuroD positive cells are detectable in the otic ganglion than in control embryos, suggesting that fewer neural progenitors are formed when six1b is overexpressed. At 3 dpf, the decrease in number of SAG neurons versus controls is even more dramatic. In extreme cases, SAG neurons are completely eliminated. These results indicate that, when six1b is overexpressed, not only are fewer neural progenitors formed but many ofthese progenitors do not go on to differentiate into neurons. In conclusion, these, together with the six1b loss-of-function results, suggest that six1b is necessary and sufficient for the normal formation of hair cells in the anterior macula, although it inhibits neuronal fate in the developing inner ear.</span></span></li>
</ul>
<strong>Empirical Evidence</strong>
<p>No Data.</p>
<strong>Uncertainties and Inconsistencies</strong>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Foxi1 gene is critical for zebrafish otic induction (Solomon et al., 2003), while it is not essential for this process in mice (Hulander et al., 2003).</span></span></p>
<h4>Quantitative Understanding of the Linkage</h4>
<p>No Data.</p>
<strong>Response-response relationship</strong>
<p>No Data.</p>
<strong>Time-scale</strong>
<ul>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong>Expression of zebrafish six1b in the Inner Ear and Neuromasts: </strong>Expression of six1b was observed in the developing inner ear and neuromasts of the lateral line until 96 hpf, the latest stage analyzed in this study. Transcripts of six1b were detected in all five sensory patches of the inner ear as well as in the semicircular canals. Detected first at 48 hpf, six1b expression in neuromasts of the midbody lateral line reached its peak at 72 hpf with stronger staining at the basal region of the neuromast, where bodies of hair cells are localized (Webb & Shirey, 2003).</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong>Expression of zebrafish six1b in Muscles:</strong> Since the beginning of segmentation six1b was expressed in the somites. At 72 hpf, the expression of six1b became more pronounced in the ventral somites with stronger staining in the most ventral cells. It continued in the pectoral fin and ventral abdomen muscle. Six1b expression was also found in the muscles of the eye and the lower jaw. (Bricaud et al., 2006).</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Temporal changes in gene expression and the emergence of sensory placode progenitors. As development proceeds gene expression domains sharpen through mutually repressive interactions; in the head region, the neural crest and placode precursor specific transcripts begin to be expressed at early neurula stages. Initially their boundaries are fuzzy, but gene expression resolves to distinct domains by late neurula (black dashed lines). NP: neural plate; NC: neural crest; PPR: preplacodal region; Epi: future epidermis. Right: diagram of an embryo at early neurula stages; dashed lines indicate the medial boundaries of non-neural transcripts (Lleras-Forero & Streit, 2012).</span></span><img alt="Temporal changes in gene expression and the emergence of sensory placode progenitors. As development proceeds gene expression domains sharpen through mutually repressive interactions; in the head region, the neural crest and placode precursor specific transcripts begin to be expressed at early neurula stages. Initially their boundaries are fuzzy, but gene expression resolves to distinct domains by late neurula (black dashed lines). NP: neural plate; NC: neural crest; PPR: preplacodal region; Epi: future epidermis. Right: diagram of an embryo at early neurula stages; dashed lines indicate the medial boundaries of non-neural transcripts (Lleras-Forero & Streit, 2012)." src="https://aopwiki.org/system/dragonfly/production/2021/08/13/80uqg5idet_AOP2.png" style="height:598px; width:717px" /></li>
</ul>
<strong>Known modulating factors</strong>
<p>No Data.</p>
<strong>Known Feedforward/Feedback loops influencing this KER</strong>
<p>No Data.</p>
<h4>References</h4>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Bricaud, O., Leslie, A. C., & Gonda, S. (2006). Development/Plasticity/Repair The Transcription Factor six1 Inhibits Neuronal and Promotes Hair Cell Fate in the Developing Zebrafish (Danio rerio) Inner Ear. <em>Journal of Neuroscience</em>, <em>26</em>(41), 10438–10451. https://doi.org/10.1523/JNEUROSCI.1025-06.2006</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Hans, S., Liu, D., & Westerfield, M. (2004). Pax8 and Pax2a function synergistically in otic specification, downstream of the Foxi1 and Dlx3b transcription factors. <em>Development</em>, <em>131</em>(20), 5091–5102. https://doi.org/10.1242/dev.01346</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Hulander, M., Kiernan, A., Blomqvist, S., Carlsson, P., Samuelsson, E., Johansson, B., Steel, K., & Enerbäck, S. (2003). Lack of pendrin expression leads to deafness and expansion of the endolymphatic compartment in inner ears of Foxi1null mutant mice 2013. <em>Development</em>, <em>130</em>, 2013–2025. https://doi.org/10.1242/dev.00376</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Lleras-Forero, L., & Streit, A. (2012). Development of the sensory nervous system in the vertebrate head: The importance of being on time. <em>Current Opinion in Genetics and Development</em>, <em>22</em>(4), 315–322. https://doi.org/10.1016/j.gde.2012.05.003</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Solomon, K. S., Kudoh, T., Dawid, I. B., & Fritz, A. (2003). Zebrafish foxi1 mediates otic placode formation and jaw development. <em>Development</em>, <em>130</em>(5), 929–940. https://doi.org/10.1242/dev.00308</span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"> Webb, J. F., & Shirey, J. E. (2003). Postembryonic Development of the Cranial Lateral Line Canals and Neuromasts in Zebrafish. <em>Developmental Dynamics</em>, <em>228</em>(3), 370–385. </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">https://doi.org/10.1002</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">/dvdy.10385</span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Key event relationship described herein has been mostly studied on zebrafish model </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Bessarab et al., 2004; Bricaud et al., 2006)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">. Evidence was also provided for <em>Xenopus </em></span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Bever & Fekete, 1999; Kil & Collazo, 2001)</span></span><em><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">, Drosophila </span></span></em><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Brodbeck & Englert, 2004; Heanue et al., 1999; Li et al., 2003)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">, mouse </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Brodbeck & Englert, 2004; Li et al., 2003)</span></span></p>
<h4>Key Event Relationship Description</h4>
<p>Increase of six1b expression leads to inhibition of eya1.</p>
<h4>Evidence Supporting this KER</h4>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Retinoic acid is required for both, expression of preplacodal ectoderm (PPE) markers Six1b and Eya1 and for the definition of their posterior boundary of expression </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Schlosser, 2014)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">. Six1b and Eya1 are not only expressed in otic placodes, but initially mark the whole preplacodal region (PPR) </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Aghaallaei et al., 2007; Litsiou et al., 2005; Schlosser, 2006)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">. Six1b expression appears to be regulated by pax2b and also by foxi1 ( forkheadbox I1) as expected for an early inducer ofthe otic placode </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Bricaud et al., 2006)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">. In the inner ear, six1b expression is restricted to the ventral otocyst in which the first hair cells differentiate and prospective SAG neurons delaminate. six1b promotes formation of hair cells by increasing cell proliferation and independently inhibits neuronal development by inducing apoptosis (Bessarab et al., 2004; Bricaud et al., 2006). In zebrafish, the eya1 gene is widely expressed in placode-derived sensory organs during embryogenesis but Eya1 function appears to be primarily required for survival of sensory hair cells in the developing ear and lateral line neuromasts </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Kozlowski et al., 2005)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">. Eya and Six together with the Dach protein directly interact to form a functional transcription factor. In this complex, the DNA binding function is provided by the Six protein, Eya mediates transcriptional activation and Dach proteins appear to function as cofactors </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(López-Ríos et al., 2003)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">.<u> </u>A regulatory network of these proteins is thought to be active also during ear development </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Whitfield et al., 2002)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"> and vertebrate eye development </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Wawersik & Maas, 2000)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">. </span></span></p>
<strong>Biological Plausibility</strong>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Six1b is a transcription factor which inhibits expression of eya1. </span></span></p>
<ul>
<li><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">RT-PCR analysis first detected six1b mRNA at mid-gastrula and its expression level increased at the beginning of segmentation, when in situ hybridization first detected regionalized expression. Shortly after the tail bud stage, weak expression was observed in the horseshoe-shaped domain surrounding the anterior neural plate, corresponding to position of the cranial placode. During the segmentation period, expression of six1 was observed in the olfactory placode and in the region that later give rise to the otic vesicle as well as anterior and posterior lateral line placodes. These elements of expression resemble the patterns reported for zebrafish eya1 </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Bessarab et al., 2004; Sahly et al., 1999)</span></span></li>
<li><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">A regulatory network of DNA binding Six protein, eya1 transcriptional activator and Dach protein as cofactor is thought to be active during ear development </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Whitfield et al., 2002)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"> and vertebrate eye development </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Wawersik & Maas, 2000)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">. </span></span></li>
<li><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Six1b gain-of-function experiment results showed that overexpression of six1b in zebrafish developing inner ear inhibited expression of eya1 </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Bricaud et al., 2006)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">.</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Catalytically active phosphatase Eya1 in vertebrates cooperates with the DNA-binding protein Six1 to promote gene induction in response to sonic hedgehog (Shh) signaling and Eya1/Six1 together regulate Gli transcriptional activators (Eisner et al., 2015; Whitfield et al., 2002).</span></span></li>
</ul>
<strong>Empirical Evidence</strong>
<p>No Data</p>
<strong>Uncertainties and Inconsistencies</strong>
<ul>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Interactions between Six1b and other members ofthe Pax–Six–Eya–Dach gene network, such as Eya1, also seem to differ between mouse and zebrafish. Zebrafish six1b inhibits eya1 expression, although its own expression is independent of the function of eya1. In mouse, Eya1 positively regulates Six1b expression (Xu et al., 1999), although its own expression is Six1b independent (Li et al., 2003; Zheng et al., 2003). Not only may interactions between six1b and eya1 differ in zebrafish relative to mouse but so might the interactions between six1b and the pax2 genes.</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">six1b function seems restricted to the otic ganglia even though it is expressed in other ganglia. However,we cannot rule out more subtle effects of six1b in other cranial ganglia, such as controlling the type of receptors or neurotransmitters expressed by these neurons. The neural crest contribution to other placodes (Baker & Bronner-Fraser, 2001) could also make six1b function less obvious than in the SAG.</span></span></li>
</ul>
<h4>Quantitative Understanding of the Linkage</h4>
<p>No Data.</p>
<strong>Response-response relationship</strong>
<p>No Data.</p>
<strong>Time-scale</strong>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Six1b acts early in both hair cell and neuronal lineages. The lack of suitable markers for hair cell or SAG neuronal precursors means that assaying the identity of the dividing cells before they actually differentiate is currently not possible. Latest time point for six1b loss or gain-of-function rescue seems to be 15-48 hpf (Bricaud et al., 2006) which coicides with the initial wave of hair cell and neurnoal differentiation between 24-48 hpf observed during inner ear development (Haddon & Lewis, 1996).</span></span></p>
<strong>Known modulating factors</strong>
<p>No Data.</p>
<h4>References</h4>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Aghaallaei, N., Bajoghli, B., & Czerny, T. (2007). Distinct roles of Fgf8, Foxi1, Dlx3b and Pax8/2 during otic vesicle induction and maintenance in medaka. <em>Developmental Biology</em>, <em>307</em>(2), 408–420. https://doi.org/10.1016/j.ydbio.2007.04.022</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Baker, C. V. H., & Bronner-Fraser, M. (2001). Vertebrate cranial placodes. I. Embryonic induction. <em>Developmental Biology</em>, <em>232</em>(1), 1–61. https://doi.org/10.1006/dbio.2001.0156</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Bessarab, D. A., Chong, S., & Korzh, V. (2004). <em>Expression of Zebrafish six1 During Sensory Organ Development and Myogenesis</em>. <em>June</em>, 781–786. https://doi.org/10.1002/dvdy.20093</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Bever, M. M., & Fekete, D. M. (1999). Ventromedial focus of cell death is absent during development of Xenopus and zebrafish inner ears. <em>Journal of Neurocytology</em>, <em>28</em>(10–11), 781–793. https://doi.org/10.1023/a:1007005702187</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Bricaud, O., Leslie, A. C., & Gonda, S. (2006). Development/Plasticity/Repair The Transcription Factor six1 Inhibits Neuronal and Promotes Hair Cell Fate in the Developing Zebrafish (Danio rerio) Inner Ear. <em>Journal of Neuroscience</em>, <em>26</em>(41), 10438–10451. https://doi.org/10.1523/JNEUROSCI.1025-06.2006</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Brodbeck, S., & Englert, C. (2004). Genetic determination of nephrogenesis: The Pax/Eya/Six gene network. <em>Pediatric Nephrology</em>, <em>19</em>(3), 249–255. https://doi.org/10.1007/s00467-003-1374-z</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Eisner, A., Pazyra-Murphy, M. F., Durresi, E., Zhou, P., Zhao, X., Chadwick, E. C., Xu, P. X., Hillman, R. T., Scott, M. P., Greenberg, M. E., & Segal, R. A. (2015). The Eya1 phosphatase promotes shh signaling during hindbrain development and oncogenesis. <em>Developmental Cell</em>, <em>33</em>(1), 22–35. https://doi.org/10.1016/j.devcel.2015.01.033</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Haddon, C., & Lewis, J. (1996). Early ear development in the embryo of the zebrafish, Danio rerio. <em>Journal of Comparative Neurology</em>, <em>365</em>(1), 113–128. https://doi.org/10.1002/(SICI)1096-9861(19960129)365:1<113::AID-CNE9>3.0.CO;2-6</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Heanue, T. A., Reshef, R., Davis, R. J., Mardon, G., Oliver, G., Tomarev, S., Lassar, A. B., & Tabin, C. J. (1999). <em>Synergistic regulation of vertebrate muscle development by Dach2, Eya2, and Six1, homologs of genes required for Drosophila eye formation</em>. www.genesdev.org</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Kil, S. H., & Collazo, A. (2001). Origins of inner ear sensory organs revealed by fate map and time-lapse analyses. <em>Developmental Biology</em>, <em>233</em>(2), 365–379. https://doi.org/10.1006/dbio.2001.0211</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Kozlowski, D. J., Whitfield, T. T., Hukriede, N. A., Lam, W. K., & Weinberg, E. S. (2005). The zebrafish dog-eared mutation disrupts eya1, a gene required for cell survival and differentiation in the inner ear and lateral line. <em>Developmental Biology</em>, <em>277</em>(1), 27–41. https://doi.org/10.1016/j.ydbio.2004.08.033</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Lang, H., Bever, M. M., & Fekete, D. M. (2000). Cell Proliferation and Cell Death in the Developing Chick Inner Ear : <em>The Journal of Comparative Neurology</em>, <em>417</em>(May 1999), 205–220.</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Li, X., Oghi, K. A., Zhang, J., Krones, A., Bush, K. T., Glass, C. K., Nigam, S. K., Aggarwal, A. K., Maas, R., Rose, D. W., & Rosenfeld, M. G. (2003). Eya protein phosphatase activity regulates Six1-Dach-Eya transcriptional effects in mammalian organogenesis. <em>Nature</em>, <em>426</em>(6964), 247–254. https://doi.org/10.1038/nature02083</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Litsiou, A., Hanson, S., & Development, A. S. (2005). A balance of FGF, BMP and WNT signalling positions the future placode territory in the head. <em>Development</em>, <em>132</em>(21), 4895. https://doi.org/10.1242/dev.01964</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">López-Ríos, J., Tessmar, K., Loosli, F., Wittbrodt, J., & Bovolenta, P. (2003). Six3 and Six6 activity is modulated by members of the groucho family. <em>Development</em>, <em>130</em>, 185–195. https://doi.org/10.1242/dev.00185</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Schlosser, G. (2006). Induction and specification of cranial placodes. <em>Developmental Biology</em>, <em>294</em>(2), 303–351. https://doi.org/10.1016/j.ydbio.2006.03.009</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Schlosser, G. (2014). Early embryonic specification of vertebrate cranial placodes. <em>Wiley Interdisciplinary Reviews: Developmental Biology</em>, <em>3</em>(5), 349–363. https://doi.org/10.1002/wdev.142</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Wawersik, S., & Maas, R. L. (2000). Vertebrate eye development as modeled in Drosophila. In <em>Human Molecular Genetics</em> (Vol. 9, Issue 6). http://hgu.mrc.ac.uk/Softdata/PAX6/</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Whitfield, T. T., Riley, B. B., Chiang, M. Y., & Phillips, B. (2002). Development of the zebrafish inner ear. <em>Developmental Dynamics</em>, <em>223</em>(4), 427–458. https://doi.org/10.1002/dvdy.10073</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Xu, P. X., Adams, J., Peters, H., Brown, M. C., Heaney, S., & Maas, R. (1999). Eya1-deficient mice lack ears and kidneys and show abnormal apoptosis of organ primordia. <em>Nature Genetics</em>, <em>23</em>(1), 113–117. https://doi.org/10.1038/12722</span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"> Zheng, W., Huang, L., Wei, Z.-B., Silvius, D., Tang, B., & Pin-Xian, X. (2003). The role of Six1 in mammalian auditory system development. <em>Development</em>, <em>130</em>, 3989–4000. https://doi.org/10.1242/dev.00628</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Evidence was provided for zebrafish (Kozlowski et al., 2005; Sahly et al., 1999), other vertebrates and Drosophila (Li et al., 2003; Zimmerman et al., 1997) and mammals (Li et al., 2003).</span></span></p>
<h4>Key Event Relationship Description</h4>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Zebrafish Eya1 has a role in regulating apoptosis within developing otic vesicle.</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"> In mammals Eya1 dephosphorylates histone variant H2AX and thereby affects DNA repair and cell survival </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Cook et al., 2009)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">.</span></span></p>
<h4>Evidence Supporting this KER</h4>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Zebrafish eya1 has a role in development of the cristae, statoacoustic ganglia, and lateral line system. Primary consequence of loss of eya1 function in the zebrafish embryo is premature apoptosis in precursors to these structures. Apoptosis has also resulted from loss of eya gene function in Drosophila and mouse </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Bonini et al., 1993; Xu et al., 1999)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">, these findings may reflect a general mechanism of suppression of apoptosis by Eya proteins. Evidence also indicates a role of Eya protein in regulating genes controlling precursor cell proliferation and survival during mammalian organogenesis </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Li et al., 2003)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">.</span></span></p>
<strong>Biological Plausibility</strong>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Zebrafish Eya1 has a role in regulating apoptosis within developing otic vesicle.</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"> In mammals Eya1 dephosphorylates histone variant H2AX and thereby affects DNA repair and cell survival </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Cook et al., 2009)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">.</span></span></p>
<ul>
<li><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Increased levels of apoptosis occur in the migrating primordia of the posterior lateral line in <em>dog</em> (the zebrafish mutation <em>dog-eared</em> that is defective in formation of the inner ear and lateral line sensory systems) embryos and as well as in regions of the developing otocyst that are mainly fated to give rise to sensory cells of the cristae</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">. </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"> Because of the large number of apoptotic cells observed within the otic vesicle of <em>dog</em> mutants, it has been proposed that eya1 could act as a suppressor of apoptosis (Kozlowski et al., 2005). Eya1 could be required to prevent apoptosis in the hair cell lineage, whereas it could have opposite actions in the neuronal lineage </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Bricaud et al., 2006)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">.</span></span></li>
<li><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">With loss of eya1 function in the eye primordium of <em>Drosophila</em></span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">, the eye progenitor cells die by programmed cell death early in the differentiation process (Sahly et al., 1999)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">.</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Ectopic cell death in the developing otic vesicle is not restricted to prospective crista cells in the lateral wall. Acridine orange staining of <em>dog</em> embryos and wild-type siblings at several times during development revealed that cell death can occur throughout the <em>dog</em> otic vesicle. Ectopic cell death throughout the otic vesicle is the likely cause of the smaller otic vesicles observed in <em>dog</em> embryos during embryogenesis (Kozlowski et al., 2005). </span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">By 55 hpf, the expression of crista-specific genes is severely reduced or absent in <em>dog</em> embryos and crista sensory hair cell bundles are absent at 72 hpf, suggesting that they have failed to differentiate (Whitfield et al., 2002).</span></span></li>
</ul>
<strong>Empirical Evidence</strong>
<p>No Data.</p>
<strong>Uncertainties and Inconsistencies</strong>
<p>No Data.</p>
<h4>Quantitative Understanding of the Linkage</h4>
<p>No Data.</p>
<strong>Response-response relationship</strong>
<p>No Data.</p>
<strong>Time-scale</strong>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Zebrafish morphological defects of the otic vesicle are first obvious at 48 hpf, some 38 h after the onset of eya1 expression in the preplacodal domain, and 24 h after increased apoptosis is observed. By 48 hpf, otic vesicles of the weakest <em>dog</em> phenotypic class are slightly smaller and more oblong in shape than wild-type siblings. As the phenotypic severity increases, <em>dog</em> otic vesicles are less round at the anterior end, developing an indented or folded appearance. By 72 hpf, <em>dog</em> otic vesicles are visibly smaller than those of wild-type siblings and distortion of the anterior end of the vesicle is more pronounced. At 96 hpf, otic vesicles of the severe phenotypic class are significantly smaller than wild- type siblings and have a narrow, cylindrical appearance </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Kozlowski et al., 2005)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">. </span></span></p>
<strong>Known modulating factors</strong>
<p>No Data.</p>
<strong>Known Feedforward/Feedback loops influencing this KER</strong>
<p>No Data.</p>
<h4>References</h4>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Bever, M. M., & Fekete, D. M. (1999). Ventromedial focus of cell death is absent during development of Xenopus and zebrafish inner ears. <em>Journal of Neurocytology</em>, <em>28</em>(10–11), 781–793. https://doi.org/10.1023/a:1007005702187</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Bonini, N. M., Leiserson, W. M., & Benzer, S. (1993). The eyes absent gene: Genetic control of cell survival and differentiation in the developing Drosophila eye. <em>Cell</em>, <em>72</em>(3), 379–395. https://doi.org/10.1016/0092-8674(93)90115-7</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Bricaud, O., Leslie, A. C., & Gonda, S. (2006). Development/Plasticity/Repair The Transcription Factor six1 Inhibits Neuronal and Promotes Hair Cell Fate in the Developing Zebrafish (Danio rerio) Inner Ear. <em>Journal of Neuroscience</em>, <em>26</em>(41), 10438–10451. https://doi.org/10.1523/JNEUROSCI.1025-06.2006</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Cook, P. J., Ju, B. G., Telese, F., Wang, X., Glass, C. K., & Rosenfeld, M. G. (2009). Tyrosine dephosphorylation of H2AX modulates apoptosis and survival decisions. <em>Nature</em>, <em>458</em>(7238), 591–596. https://doi.org/10.1038/nature07849</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Kozlowski, D. J., Whitfield, T. T., Hukriede, N. A., Lam, W. K., & Weinberg, E. S. (2005). The zebrafish dog-eared mutation disrupts eya1, a gene required for cell survival and differentiation in the inner ear and lateral line. <em>Developmental Biology</em>, <em>277</em>(1), 27–41. https://doi.org/10.1016/j.ydbio.2004.08.033</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Li, X., Oghi, K. A., Zhang, J., Krones, A., Bush, K. T., Glass, C. K., Nigam, S. K., Aggarwal, A. K., Maas, R., Rose, D. W., & Rosenfeld, M. G. (2003). Eya protein phosphatase activity regulates Six1-Dach-Eya transcriptional effects in mammalian organogenesis. <em>Nature</em>, <em>426</em>(6964), 247–254. https://doi.org/10.1038/nature02083</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Rebay, I., Silver, S. J., & Tootle, T. L. (2005). New vision from Eyes absent: Transcription factors as enzymes. <em>Trends in Genetics</em>, <em>21</em>(3), 163–171. https://doi.org/10.1016/j.tig.2005.01.005</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Sahly, I., Andermann, P., & Petit, C. (1999). The zebrafish eya1 gene and its expression pattern during embryogenesis. <em>Development Genes and Evolution</em>, <em>209</em>(7), 399–410. https://doi.org/10.1007/s004270050270</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Tadjuidje, E., & Hegde, R. S. (2013). The Eyes Absent proteins in development and disease. <em>Cellular and Molecular Life Sciences</em>, <em>70</em>(11), 1897–1913. https://doi.org/10.1007/s00018-012-1144-9</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Whitfield, T. T., Riley, B. B., Chiang, M. Y., & Phillips, B. (2002). Development of the zebrafish inner ear. <em>Developmental Dynamics</em>, <em>223</em>(4), 427–458. https://doi.org/10.1002/dvdy.10073</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Xu, P. X., Adams, J., Peters, H., Brown, M. C., Heaney, S., & Maas, R. (1999). Eya1-deficient mice lack ears and kidneys and show abnormal apoptosis of organ primordia. <em>Nature Genetics</em>, <em>23</em>(1), 113–117. https://doi.org/10.1038/12722</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Zimmerman, J. E., Bui, Q. T., Kur Steingrimsson, E. [, Nagle, D. L., Fu, W., Genin, A., Spinner, N. B., Copeland, N. G., Jenkins, N. A., Bucan, M., & Bonini, N. M. (1997). Cloning and Characterization of Two Vertebrate Homologs of the Drosophila eyes absent Gene. <em>Development</em>, <em>124</em>(23), 4819–4826.</span></span></p>
</div>
<div>
<h4><a href="/relationships/2467">Relationship: 2467: Increase, Cell death leads to Altered, inner ear development</a></h4>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Evidence was provided for Zebrafish </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Whitfield <em>et al.</em>, 1996; Kozlowski <em>et al.</em>, 2005)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">, other vertebrates </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Schlosser <em>et al.</em>, 2008)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">, mice </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Johnson <em>et al.</em>, 1999; Xu <em>et al.</em>, 1999)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"> and human </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Bonini, Leiserson and Benzer, 1993)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">. </span></span></p>
<h4>Key Event Relationship Description</h4>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Increased cell death in otic vesicle leads to abnormal inner ear development.</span></span></p>
<h4>Evidence Supporting this KER</h4>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">The vertebrate inner ear develops from the otic placode, an ectodermal thickening that appears early in development and invaginates to form the otic vesicle </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Aghaallaei et al., 2007)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">. Eya1 gene was shown to regulate cell death during development of otic vesicle </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Abdelhak et al., 1997; Kozlowski et al., 2005; Schlosser, 2014; Whitfield et al., 2002; Zhou et al., 2017). Increased cell death resulted in smaller otic vesicle </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Kozlowski et al., 2005)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">. </span></span></p>
<strong>Biological Plausibility</strong>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Increased cell death in otic vesicle leads to sensory defects via malformations of inner ear and lateral line sensory systems (Kozlowski et al., 2005).</span></span></p>
<ul>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Increased levels of apoptosis occur in the migrating primordia of the posterior lateral line in <em><span style="font-family:"Calibri",sans-serif">dog</span></em> (the zebrafish mutation <em><span style="font-family:"Calibri",sans-serif">dog-eared</span></em> that is defective in formation of the inner ear and lateral line sensory systems) embryos and as well as in regions of the developing otocyst that are mainly fated to give rise to sensory cells of the cristae. Ectopic cell death throughout the otic vesicle is the likely cause of the smaller otic vesicles observed in <em><span style="font-family:"Calibri",sans-serif">dog</span></em> embryos during embryogenesis (Kozlowski et al., 2005).</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">After Six1 or Eya1 loss of function, the numbers of sensory receptors and neurons in the sense organs and ganglia derived from the olfactory, otic, lateral line, profundal/trigeminal, and epibranchial placodes are reduced, and only small, malformed sense organs develop that are abnormally patterned and functionally deficient (Schlosser, 2014). </span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Other cell types of the inner ear, including supporting cells and endolymph-producing cells, are also derived from the otic placode as are the sensory neurons of the vestibulocochlear ganglion, which innervate the hair cells. The lateral line placodes of fishes and amphibians also give rise to hair cells and supporting cells, which form small mechanosensory organs (neuromasts) distributed in lines along the body surface and involved in the detection of water movements. They also produce the sensory neurons innervating these receptor organs (Schlosser, 2014; Whitfield, 2002).</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><em>Dog-eared</em> zebrafish mutants exibit increased death in otic vesicle during development; loss of cristae; abnormal macuae and semicircular canal system (Kozlowski et al., 2005; Whitfield et al., 1996, 2002). Dog-eared mutants are zebrafish model for human branchio-oto renal syndrome (Whitfield, 2002).</span></span></li>
<li><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">BOR (branchio-oto-renal) syndrome in humans is characterized by branchial cleft abnormalities, otic developmental defects and renal malformations. To date, autosomal dominant mutations in the EYA1 (Eyes Absent 1) gene are the most common genetic cause of BOR. EYA1 is the human homologue of the Drosophila gene eya (eyes absent), in which null mutations result in eyeless fly embryos due to apoptotic loss of eye disc cells </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Bonini et al., 1993)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">. Subsequent studies reported homologues of the eya gene in vertebrates </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Duncan et al., 1997; Li et al., 2010)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">.</span></span></li>
</ul>
<strong>Empirical Evidence</strong>
<p>No Data.</p>
<strong>Uncertainties and Inconsistencies</strong>
<p>No Data.</p>
<h4>Quantitative Understanding of the Linkage</h4>
<p>No Data.</p>
<strong>Response-response relationship</strong>
<p>No Data.</p>
<strong>Time-scale</strong>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Z</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">ebrafish morphological defects of the otic vesicle are first obvious at 48 hpf, some 38 h after the onset of eya1 expression in the preplacodal domain, and 24 h after increased apoptosis is observed. By 48 hpf, otic vesicles of the weakest <em><span style="font-family:"Calibri",sans-serif">dog</span></em> phenotypic class are slightly smaller and more oblong in shape than wild-type siblings. As the phenotypic severity increases, <em><span style="font-family:"Calibri",sans-serif">dog</span></em> otic vesicles are less round at the anterior end, developing an indented or folded appearance. By 72 hpf, <em><span style="font-family:"Calibri",sans-serif">dog</span></em> otic vesicles are visibly smaller than those of wild-type siblings and distortion of the anterior end of the vesicle is more pronounced. At 96 hpf, otic vesicles of the severe phenotypic class are significantly smaller than wild- type siblings and have a narrow, cylindrical appearance (Kozlowski et al., 2005).</span></span></p>
<strong>Known modulating factors</strong>
<p>No Data.</p>
<strong>Known Feedforward/Feedback loops influencing this KER</strong>
<p>No Data.</p>
<h4>References</h4>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Abdelhak, S., Kalatzis, V., Heilig, R., Compain, S., Samoson, D., Vincent, C., Weil, D., Cruaud, C., Sahly, I., Leibovici, M., Bitner-Glindzicz, M., & Francis, M. (1997). A human homologue of the Drosophila eyes absent gene underlies branchio-oto-renal (BOR) syndrome and identifies a novel gene family. <em>Nature Genetics</em>, <em>15</em>, 157–167. https://doi.org/10.1038/ng0297-157</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Aghaallaei, N., Bajoghli, B., & Czerny, T. (2007). Distinct roles of Fgf8, Foxi1, Dlx3b and Pax8/2 during otic vesicle induction and maintenance in medaka. <em>Developmental Biology</em>, <em>307</em>(2), 408–420. https://doi.org/10.1016/j.ydbio.2007.04.022</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Bonini, N. M., Leiserson, W. M., & Benzer, S. (1993). The eyes absent gene: Genetic control of cell survival and differentiation in the developing Drosophila eye. <em>Cell</em>, <em>72</em>(3), 379–395. https://doi.org/10.1016/0092-8674(93)90115-7</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Duncan, M. K., Kos, L., Jenkins, N. A., Gilbert, D. J., Copeland, N. G., & Tomarev, S. I. (1997). Eyes absent: a gene family found in several metazoan phyla. In <em>Mammalian Genome</em> (Vol. 8). Spfinger-VerlagNew York Inc.</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Johnson, K. R., Cook, S. A., Erway, L. C., Matthews, A. N., Sanford, L. P., Paradies, N. E., & Friedman, R. A. (1999). Inner ear and kidney anomalies caused by IAP insertion in an intron of the Eya1 gene in a mouse model of BOR syndrome. In <em>Human Molecular Genetics</em> (Vol. 8, Issue 4).</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Kozlowski, D. J., Whitfield, T. T., Hukriede, N. A., Lam, W. K., & Weinberg, E. S. (2005). The zebrafish dog-eared mutation disrupts eya1, a gene required for cell survival and differentiation in the inner ear and lateral line. <em>Developmental Biology</em>, <em>277</em>(1), 27–41. https://doi.org/10.1016/j.ydbio.2004.08.033</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Li, Y., Manaligod, J. M., & Weeks, D. L. (2010). EYA1 mutations associated with the branchio-oto-renal syndrome result in defective otic development in Xenopus laevis. <em>Biol. Cell</em>, <em>102</em>, 277–292. https://doi.org/10.1042/BC20090098</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Schlosser, G. (2014). Early embryonic specification of vertebrate cranial placodes. <em>Wiley Interdisciplinary Reviews: Developmental Biology</em>, <em>3</em>(5), 349–363. https://doi.org/10.1002/wdev.142</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Schlosser, G., Awtry, T., Brugmann, S. A., Jensen, E. D., Neilson, K., Ruan, G., Stammler, A., Voelker, D., Yan, B., Zhang, C., Klymkowsky, M. W., & Moody, S. A. (2008). Eya1 and Six1 promote neurogenesis in the cranial placodes in a SoxB1-dependent fashion. <em>Developmental Biology</em>, <em>320</em>(1), 199–214. https://doi.org/10.1016/j.ydbio.2008.05.523</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Whitfield, T. T. (2002). Zebrafish as a Model for Hearing and Deafness. <em>J Neurobiol</em>, <em>53</em>, 157–171. https://doi.org/10.1002/neu.10123</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Whitfield, T. T., Granato, M., Van Eeden, F. J. M., Schach, U., Brand, M., Furutani-Seiki, M., Haffter, P., Hammerschmidt, M., Heisenberg, C. P., Jiang, Y. J., Kane, D. A., Kelsh, R. N., Mullins, M. C., Odenthal, J., & Nüsslein-Volhard, C. (1996). Mutations affecting development of the zebrafish inner ear and lateral line. <em>Development</em>, <em>123</em>, 241–254. https://doi.org/10.1242/dev.123.1.241</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Whitfield, T. T., Riley, B. B., Chiang, M. Y., & Phillips, B. (2002). Development of the zebrafish inner ear. <em>Developmental Dynamics</em>, <em>223</em>(4), 427–458. https://doi.org/10.1002/dvdy.10073</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Xu, P. X., Adams, J., Peters, H., Brown, M. C., Heaney, S., & Maas, R. (1999). Eya1-deficient mice lack ears and kidneys and show abnormal apoptosis of organ primordia. <em>Nature Genetics</em>, <em>23</em>(1), 113–117. https://doi.org/10.1038/12722</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Zhou, J. J., Huang, Y., Zhang, X., Cheng, Y., Tang, L., & Ma, X. (2017). <em>Eyes absent gene (EYA1) is a pathogenic driver and a therapeutic target for melanoma</em> (Vol. 8, Issue 62). www.impactjournals.com/oncotarget</span></span></p>
<p> </p>
</div>
<div>
<h4><a href="/relationships/2468">Relationship: 2468: Altered, inner ear development leads to Reduced, Hearing</a></h4>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Key event relationship is applicable to wide range of vertebrates (Whitfield, 2015).</span></span></p>
<h4>Key Event Relationship Description</h4>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">The inner ear is the vertebrate organ of hearing and balance </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Whitfield, 2002)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">.</span></span></p>
<h4>Evidence Supporting this KER</h4>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Inner ear develops from an ectodermal thickening, the otic placode, visible on either side of the hindbrain from mid-somite stages. In the zebrafish, this placode cavitates to form a hollow ball of epithelium, the otic vesicle, from which all structures of the membranous labyrinth and the neurons of the statoacoustic (VIIIth) ganglion arise </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Haddon and Lewis, 1996; Whitfield <em>et al.</em>, 2002)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">.</span></span></p>
<strong>Biological Plausibility</strong>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Zebrafish serves as a model organism for hearing and deafness. Mutations in several genes connected to development of inner ear affect morphology and patterning of the inner ear epithelium, including formation of the semicircular canals and, in some, development of sensory patches (maculae and cristae). Zebrafish mutant embryos fail to balance correctly, and may swim on their sides, upside down, or in circles (Whitfield <em>et al.</em>, 1996). This is reminiscent of the behavior of deaf mouse mutants, which often display hyperactive circling or head bobbing due to vestibular dysfunction (Whitfield, 2002).</span></span></p>
<ul>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Dog<em>-eared</em> mutants show abnormal development of semicircular canals and lack cristae within the ear (Kozlowski <em>et al.</em>, 2005), while in <em>van gogh</em>, semicircular canals fail to form altogether, resulting in a tiny otic vesicle containing a single sensory patch. Both mutants show irregular swimming pattern (Whitfield <em>et al.</em>, 1996).</span></span></li>
</ul>
<strong>Empirical Evidence</strong>
<p>No Data.</p>
<strong>Uncertainties and Inconsistencies</strong>
<p>No Data.</p>
<h4>Quantitative Understanding of the Linkage</h4>
<p>No Data.</p>
<strong>Response-response relationship</strong>
<p>No Data.</p>
<strong>Time-scale</strong>
<p>No Data.</p>
<strong>Known modulating factors</strong>
<p>No Data.</p>
<strong>Known Feedforward/Feedback loops influencing this KER</strong>
<p>No Data.</p>
<h4>References</h4>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Haddon, C. and Lewis, J. (1996) ‘Early ear development in the embryo of the zebrafish, Danio rerio’, <em>Journal of Comparative Neurology</em>, 365(1), pp. 113–128. doi: 10.1002/(SICI)1096-9861(19960129)365:1<113::AID-CNE9>3.0.CO;2-6.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Kozlowski, D. J. <em>et al.</em> (2005) ‘The zebrafish dog-eared mutation disrupts eya1, a gene required for cell survival and differentiation in the inner ear and lateral line’, <em>Developmental Biology</em>, 277(1), pp. 27–41. doi: 10.1016/j.ydbio.2004.08.033.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Whitfield, T. T. <em>et al.</em> (1996) ‘Mutations affecting development of the zebrafish inner ear and lateral line’, <em>Development</em>, 123, pp. 241–254. doi: 10.1242/dev.123.1.241.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Whitfield, T. T. <em>et al.</em> (2002) ‘Development of the zebrafish inner ear’, <em>Developmental Dynamics</em>, 223(4), pp. 427–458. doi: 10.1002/dvdy.10073.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Whitfield, T. T. (2002) ‘Zebrafish as a Model for Hearing and Deafness’, <em>J Neurobiol</em>, 53, pp. 157–171. doi: 10.1002/neu.10123.</span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Whitfield, T. T. (2015) ‘Development of the inner ear’, <em>Current Opinion in Genetics and Development</em>, 32, pp. 112–118. doi: 10.1016/j.gde.2015.02.006.</span></span></p>
</div>
<div>
<h4><a href="/relationships/2231">Relationship: 2231: Reduced, Hearing leads to Increased Mortality</a></h4>
<p>Impaired hearing could result in an impact on ecologically relevant endpoint, such as predator avoidance and prey capture. Therefore, it can be assumed that an affect on hearing could reduce young of year survival.</p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<div>
<ul>
<li>In birds, acoustic signals play key roles in territory defense and mate attraction (Slabbekoorn and Ripmeester, 2008).</li>
</ul>
<p>Roles of Acoustic signaling in fish (reviewed by Kasumayan 2009):</p>
<ul>
<li>Reproductive isolation - among fish capable of generating sound, sound emission during spawning is the most prominent life stage during which acoustic signaling occurs. Includes mate attraction, courtship, establishment of territory.</li>
<li>Defensive sounds - fright and stress, alert conspecifics to potential threats.</li>
<li>Organization of group/aggregative behaviors</li>
<li>Feeding behaviors - in many fish conditioned reflex to the sounds of conspecifics feeding can be formed and cause orientation or attraction of fish toward their source, particularly in combination with corresponding visual stimuli and odors.</li>
</ul>
</div>
<h4>References</h4>
<div>
<ul>
<li>Kasumayan AO. 2009. Acoustic signaling in fish. J. Ichthyology. 49:963-1020.</li>
<li>SLABBEKOORN, H. and RIPMEESTER, E. A. P. (2008), Birdsong and anthropogenic noise: implications and applications for conservation. Molecular Ecology, 17: 72–83. doi:10.1111/j.1365-294X.2007.03487.x</li>
</ul>
</div>
</div>
<div>
<h4><a href="/relationships/2013">Relationship: 2013: Increased Mortality leads to Decrease, Population growth rate</a></h4>
<td><a href="/aops/312">Acetylcholinesterase Inhibition leading to Acute Mortality via Impaired Coordination & Movement</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/16">Acetylcholinesterase inhibition leading to acute mortality</a></td>
<td>adjacent</td>
<td>Moderate</td>
<td>Moderate</td>
</tr>
<tr>
<td><a href="/aops/155">Deiodinase 2 inhibition leading to increased mortality via reduced posterior swim bladder inflation</a></td>
<td>adjacent</td>
<td>Moderate</td>
<td>Moderate</td>
</tr>
<tr>
<td><a href="/aops/156">Deiodinase 2 inhibition leading to increased mortality via reduced anterior swim bladder inflation</a></td>
<td>adjacent</td>
<td>Moderate</td>
<td>Moderate</td>
</tr>
<tr>
<td><a href="/aops/157">Deiodinase 1 inhibition leading to increased mortality via reduced posterior swim bladder inflation </a></td>
<td>adjacent</td>
<td>Moderate</td>
<td>Moderate</td>
</tr>
<tr>
<td><a href="/aops/158">Deiodinase 1 inhibition leading to increased mortality via reduced anterior swim bladder inflation</a></td>
<td>adjacent</td>
<td>Moderate</td>
<td>Moderate</td>
</tr>
<tr>
<td><a href="/aops/159">Thyroperoxidase inhibition leading to increased mortality via reduced anterior swim bladder inflation</a></td>
<td>adjacent</td>
<td>Moderate</td>
<td>Moderate</td>
</tr>
<tr>
<td><a href="/aops/363">Thyroperoxidase inhibition leading to altered visual function via altered retinal layer structure</a></td>
<td>adjacent</td>
<td>Moderate</td>
<td>Moderate</td>
</tr>
<tr>
<td><a href="/aops/364">Thyroperoxidase inhibition leading to altered visual function via decreased eye size</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/365">Thyroperoxidase inhibition leading to altered visual function via altered photoreceptor patterning</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/399">Inhibition of Fyna leading to increased mortality via decreased eye size (Microphthalmos)</a></td>
<td>adjacent</td>
<td>High</td>
<td>High</td>
</tr>
<tr>
<td><a href="/aops/410">GSK3beta inactivation leading to increased mortality via defects in developing inner ear</a></td>
<td>adjacent</td>
<td>High</td>
<td>High</td>
</tr>
<tr>
<td><a href="/aops/536">Estrogen receptor agonism leading to reduced survival and population growth due to renal failure</a></td>
<td>adjacent</td>
<td>Moderate</td>
<td>Moderate</td>
</tr>
<tr>
<td><a href="/aops/564">DBDPE-induced inhibition of mitochondrial complex Ⅰ leading to population decline via neurotoxicity and metabotoxicity.</a></td>
<td>adjacent</td>
<td>High</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
<h4>Evidence Supporting Applicability of this Relationship</h4>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><strong><span style="color:black">Taxonomic</span></strong><span style="color:black">: All organisms must survive to reproductive age in order to reproduce and sustain populations. The additional considerations related to survival made above are applicable to other fish species in addition to zebrafish and fathead minnows with the same reproductive strategy (r-strategist as described in the theory of MaxArthur and Wilson (1967). The impact of reduced survival on population size is even greater for k-strategists that invest more energy in a lower number of offspring.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><strong><span style="color:black">Life stage</span></strong><span style="color:black">: Density dependent effects start to play a role in the larval stage of fish when free-feeding starts (Hazlerigg et al., 2014).</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><strong><span style="color:black">Sex</span></strong><span style="color:black">: This linkage is independent of sex.</span></span></span></span></p>
<p> </p>
<h4>Key Event Relationship Description</h4>
<p><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Increased mortality in the reproductive population may lead to a declining population. This depends on the excess mortality due to the applied stressor and the environmental parameters such as food availability and predation rate. Most fish species are r-strategist, meaning they produce a lot of offspring instead of investing in parental care. This results in natural high larval mortality causing only a small percentage of the larvae to survive to maturity. If the excess larval mortality due to a stressor is small, the population dynamics might result in constant population size. Should the larval excess be more significant, or last on the long-term, this will affect the population. To calculate the long-term persistence of the population, population dynamic models should be used.</span></span></span></span></p>
<h4>Evidence Supporting this KER</h4>
<p><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Survival rate is an obvious determinant of population size and is therefore included in population modeling (e.g., Miller et al., 2020).</span></span></span></span></p>
<strong>Biological Plausibility</strong>
<ul>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Survival to reproductive maturity is a parameter of demographic significance. Assuming resource availability (i.e., food, habitat, etc.) is not limiting to the extant population, sufficient mortality in the reproductive population may ultimately lead to declining population trajectories.</span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Under some conditions, reduced larval survival may be compensated by reduced predation and increased food availability, and therefore not result in population decline (Stige et al., 2019).</span></span></li>
</ul>
<strong>Empirical Evidence</strong>
<ul>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">According to empirical data, combined with population dynamic models, feeding larvae are the crucial life stage in zebrafish (and other r-strategists) for the regulation of the population. (Schäfers et al., 1993)</span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">In fathead minnow, natural survival of early life stages has been found to be highly variable and influential on population growth (Miller and Ankley, 2004)</span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Rearick et al. (2018) used data from behavioural assays linked to survival trials and applied a modelling approach to quantify changes in antipredator escape performance of larval fathead minnows in order to predict changes in population abundance. This work was done in the context of exposure to an environmental oestrogen. Exposed fish had delayed response times and slower escape speeds, and were more susceptible to predation. Population modelling showed that this can result in population decline.</span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">In the context of fishing and fisheries, ample evidence of a link between increased mortality and a decrease of population size has been given. Important insights can result from the investigation of optimum modes of fishing that allow for maintaining a population (Alekseeva and Rudenko, 2018). Jacobsen and Essington (2018) showed the impact of varying predation mortality on forage fish populations.</span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Boreman (1997) reviewed methods for comparing the population-level effects of mortality in fish populations induced by pollution or fishing.</span></span></li>
</ul>
<strong>Uncertainties and Inconsistencies</strong>
<ul>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">The extent to which larval mortality affects population size could depend on the fraction of surplus mortality compared to a natural situation.</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">There are scenarios in which individual mortality may not lead to declining population size. These include instances where populations are limited by the availability of habitat and food resources, which can be replenished through immigration. Effects of mortality in the larvae can be compensated by reduced competition for resources (Stige et al., 2019).</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">The direct impact of pesticides on migration behavior can be difficult to track in the field, and documentation of mortality during migration is likely underestimated (Eng 2017).</span></span></span></li>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Beaudouin, R., Goussen, B., Piccini, B., Augustine, S., Devillers, J., Brion, F., Pery, A.R., 2015. An individual-based model of zebrafish population dynamics accounting for energy dynamics. PloS one 10, e0125841.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Boreman J. 1997. Methods for comparing the impacts of pollution and fishing on fish populations. Transactions of the American Fisheries Society. 126(3):506-513.</span></span></span></span></p>
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