Relationship: 1027



Decreased, Triiodothyronine (T3) in serum leads to Reduced, Posterior swim bladder inflation

Upstream event


Decreased, Triiodothyronine (T3) in serum

Downstream event


Reduced, Posterior swim bladder inflation

Key Event Relationship Overview


AOPs Referencing Relationship


Taxonomic Applicability


Term Scientific Term Evidence Link
zebrafish Danio rerio High NCBI
fathead minnow Pimephales promelas Moderate NCBI

Sex Applicability


Sex Evidence
Unspecific High

Life Stage Applicability


Term Evidence
Embryo High

Key Event Relationship Description


Reduced T3 levels in serum prohibit local TH action in the target tissues. Since swim bladder development and/or inflation is regulated by thyroid hormones, this results in impaired posterior chamber inflation.

Evidence Supporting this KER


There is convincing evidence that decreased T3 levels result in impaired posterior chamber inflation, but the underlying mechanisms are not completely understood. The quantitative understanding is currently very limited because T3 levels and posterior inflation are seldom measured in the same study. Therefore the evidence supporting this KER can be considered moderate.

Biological Plausibility


Thyroid hormones are known to be involved in development, especially in metamorphosis in amphibians and in embryonic-to-larval transition (Liu and Chan, 2002) and larval-to-juvenile transition (Brown et al., 1997) in fish. Inflation of the posterior chamber is part of the embryonic-to-larval transition in fish, together with structural and functional maturation of the mouth and gastrointestinal tract, and resorption of the yolk sac (Liu and Chan, 2002). Marelli et al. (2016) showed that thyroid hormone receptor alpha and beta are both expressed in swim bladder tissue of zebrafish at 5 days post fertilization, corresponding to the timing of posterior inflation. this time point has additionally been shown to coincide with increased T3 and T4 levels (Chang et al., 2012), suggesting that posterior inflation is under thyroid hormone regulation.

Empirical Evidence


  • Maternal injection of T3, resulting in increased T3 concentrations in the eggs of striped bass (Morone saxatilis) lead to significant increases in both swim bladder inflation and survival (Brown et al., 1988).
  • Dong et al. (2013) and Thisse et al. (2003) showed localized expression of DIO1 and DIO2 in the swim bladder tissue of 96 and 120 hpf zebrafish larvae, suggesting that local activation of thryoid hormones (i.e. conversion of T4 to T3) is requried in swim bladder tissue around that time period.
  • Marelli et al. (2016) used morpholinos to block translation of thryoid hormone receptor alpha or beta in zebrafish. They found that thyroid hormone receptor alpha and beta knockdowns failed to inflate the posterior chamber of the swim bladder by 120 hpf, indicating that the action of T3 is needed for proper inflation of the posterior chamber. High T3 doses partially rescued the negative impact in partially resistant mutants, further confirming the importance of T3 in this process.
  • Stinckens et al. (2018) showed that effects on posterior chamber inflation in zebrafish could be predicted based on in chemico DIO2 inhibition potential with only few false positives and false negatives. While T3 levels were not determined in this study, DIO2 inhibition is expected to result in decreased T3 levels.
  • Bagci et al. (2015) and Heijlen et al. (2013, 2014) reported that knockdown of DIO1+2 in zebrafish resulted in impairment of the inflation of the posterior chamber of the swim bladder. DIO1 and 2 knockdown is expected to result in reduced T3 levels. Indeed, Walpita et al. (2009, 2010) showed that T3 supplementation effectively rescued the effects of DIO1 and 2 knockdown, while T4 supplementation did not.
  • de Vrieze et al. (2014) found that knockdown of monocarboxylate transporter 8 (mct8) in zebrafish resulted in a dose-dependent impairment of posterior chamber inflation. Since this transporter is known to transport thyroid hormones across cell membranes, this supports the importance of thyroid hormones in regulating posterior chamber inflation.
  • Shi et al. (2019) found that exposure of adult zebrafish to 6:2 chlorinated polyfluorinated ether sulfonate (F-53B), an alternative to perfluorooctanesulfonate (PFOS), decreased T3 levels in both male and female zebrafish. Additionally,  F-53B was maternally transferred to the offspring. Decreased T3 levels together with impaired posterior chamber inflation was observed in the F1 offspring.
  • Wang et al. (2020) observed a decrease of whole-body T3 as well as impaired posterior chamber inflation in zebrafish exposed to perfluorooctanoic acid and perfluoropolyether carboxylic acids from fertilization until the age of 5 days. Exogeneous T3 or T4 supplementation partly rescued PFECA-induced posterior swim bladder malformation, confirming the causal relationship between reduced T3 levels and reduced posterior chamber inflation.
  • Molla et al. (2019) showed that T3 supplementation increased posterior chamber diameter in zebrafish larvae. This confirms that T3 plays an important role in posterior swim bladder inflation.

Uncertainties and Inconsistencies


The mechanism through which altered TH levels result in impaired posterior chamber inflation still needs to be elucidated. It is currently unclear which aspect of swim bladder development and inflation is affected by TH disruption. Based on the developmental stages of the posterior chamber, several hypotheses could explain effects on posterior chamber inflation due to disrupted TH levels. A first hypothesis includes effects on the budding of the posterior chamber inflation. Secondly, the effect on posterior chamber inflation could also be caused by disturbing the formation and growth of the three tissue layers of this organ. It has been reported that the Hedgehog signalling pathway plays an essential role in swim bladder development and is required for growth and differentiation of cells of the swim bladder. The Wnt/β-catenin signalling pathway is required for the organization and growth of all three tissue layers (Yin et al., 2011, 2012, Winata 2009, Kress et al., 2009). Both signalling pathways have been related to THs in amphibian and rodent species (Kress et al., 2009; Plateroti et al., 2006; Stolow and Shi, 1995). Molla et al. (2019) showed that insulin-like growth factor (IGF‐1) plays a role in swim bladder inflation/maturation in zebrafish. Several other hypotheses include effects on the successful initial inflation of the posterior chamber, effects on lactic acid production that is required for the maintenance of the swim bladder volume, or effects on the production of surfactant that is crucial to maintain the surface tension necessary for swim bladder inflation.

Another uncertainty lies in the relative importance of the different T4 actvating iodothyronine deiodinases (DIO1, DIO2) in regulating swim bladder inflation. Stinckens et al. (2018) showed that exposure of zebrafish embryos to seven strong DIO1 inhibitors (measured using in chemico enzyme inhibition assays), six out of seven compounds impaired posterior chamber inflation. Exposure to strong DIO2 inhibitors on the other hand affected posterior chamber inflation and/or surface area in all cases. These results suggest that DIO2 enzymes may play a more important role in swim bladder inflation compared to DIO1 enzymes. it has been previously suggested that DIO2 is the major contributor to TH activation in developing zebrafish embryos (Darras et al., 2015; Walpita et al., 2010). It has been shown that a morpholino knockdown targeting DIO1 mRNA alone did not affect embryonic development in zebrafish, while knockdown of DIO2 delayed progression of otic vesicle length, head-trunk angle and pigmentation index (Houbrechts et al., 2016; Walpita et al., 2010, 2009). DIO1 inhibition may only become essential in hypothyroidal circumstances, for example when DIO2 is inhibited or in case of iodine deficiency, in zebrafish (Walpita et al., 2010) and mice (Galton et al., 2009; Schneider et al., 2006).

As reported by Bagci et al. (2015) and Heijlen et al. (2014), posterior chamber inflation was impaired in DIO3 knockdown zebrafish. Heijlen et al. (2014) additionally reported histologically abnormal tissue layers in the swim bladder of DIO3 knockdown zebrafish. DIO3 is a thyroid hormone inactivating enzyme, which would result in higher levels of T3 in serum. Wei et al. (2018) showed that exposure to bisphenol S in adult zebrafish decreased T4 levels and increased T3 levels, and these changes in thyroid hormone levels were transferred to the offspring, in which impaired swim bladder inflation was observed. This indicates that not only too low, but also too high T3 levels, impact posterior chamber inflation. The underlying mechanism is currently unknown.

In the study of Cavallin et al. (2017) fathead minnow embryos were exposed to IOP, a model iodothyronine deiodinase inhibitor that is assumed to inhibit all three deiodinase enzymes (DIO1,2,3). The authors observed increased whole body T3 concentrations in 4 and 6 day old embryos, together with impaired posterior chamber inflation. Transcript levels of DIO1, 2 and 3 remained unaltered and thus offered no proof of a compensatory mechanism that could explain these results.

The earliest life stages of teleost fish rely on maternally transferred THs to regulate certain developmental processes until embryonic TH synthesis is active (Power et al., 2001). As a result, posterior swim bladder chamber inflation, which occurs early during development, appears to be less sensitive to inhibition of TH synthesis than to inhibition of the conversion of T4 to T3 (Stinckens et al., 2016, 2018; Nelson et al., 2016). There have however been a few reports of reduced posterior inflation upon inhibition of TH synthesis (Liu and Chan, 2002). It must however be noted that these observations could reflect delayed inflation due to a general delay in development rather than a direct effect on the swim bladder. Longer observations would have to clarify this.

Quantitative Understanding of the Linkage


Studies measuring both T3 levels and posterior chamber inflation are scarce and a quantitative relationshiop has not been established yet.

Response-response Relationship




Known modulating factors


Known Feedforward/Feedback loops influencing this KER


Domain of Applicability


Taxonomic: The indirect relationship between deiodinase inhibition, expected to lead to reduced T3 levels, and reduced posterior chamber inflation has been confirmed in early zebrafish larvae around the time of posterior chamber inflation, i.e. around 5 days post fertilization in zebrafish (Stinckens et al., 2018) and around 6 days post fertilization in fathead minnows (Cavallin et al., 2017).

Life stage: This KER is only applicable to early embryonic development, which is the period where the posterior swim bladder chamber inflates. The relationship between reduced T3 levels and reduced posterior chamber inflation is not applicable to older larvae that successfully inflated the posterior chamber but show impaired anterior chamber inflation after chronic exposure to low concentrations of thyroid hormone system disruptors. In 32 day old zebrafish exposed to methimazole, propylthiouracil, 2-mercaptobenzothiazole or iopaonic acid (Stinckens et al., 2016, 2020) as well as in 14-21 day old fathead minnows exposed to iopaonic acid (Cavallin et al., 2017), a clear inverse relationship was found. With decreasing whole body T3 concentrations, posterior chamber volume increased, suggesting a possible compensatory mechanism for the observed decrease in anterior chamber volume. As a result, the sum of both chamber surfaces, reflecting the total amount of gas, was equal to controls for most treatments (Stinckens et al., 2016; Stinckens et al., 2020).

Sex: Zebrafish are undifferentiated gonochorists since both sexes initially develop an immature ovary (Maack and Segner, 2003). Immature ovary development progresses until approximately the onset of the third week. Later, in female fish immature ovaries continue to develop further, while male fish undergo transformation of ovaries into testes. Final transformation into testes varies among male individuals, however finishes usually around 6 weeks post fertilization. Since the posterior chamber inflates around 5 days post fertilization, when sex differentiation has not started yet, sex differences are expected to play a minor role.



Bagci, E., Heijlen, M., Vergauwen, L., Hagenaars, A., Houbrechts, A.M., Esguerra, C.V., Blust, R., Darras, V.M., Knapen, D., 2015. Deiodinase knockdown during early zebrafish development affects growth, development, energy metabolism, motility and phototransduction. PLOS One 10, e0123285.

Brown, C.L., Doroshov, S.I., Nunez, J.M., Hadley, C., Vaneenennaam, J., Nishioka, R.S., Bern, H.A., 1988. MATERNAL TRIIODOTHYRONINE INJECTIONS CAUSE INCREASES IN SWIMBLADDER INFLATION AND SURVIVAL RATES IN LARVAL STRIPED BASS, MORONE-SAXATILIS. Journal of Experimental Zoology 248, 168-176.

Brown, D.D., 1997. The role of thyroid hormone in zebrafish and axolotl development. Proceedings of the National Academy of Sciences of the United States of America 94, 13011-13016.

Cavallin, J.E., Ankley, G.T., Blackwell, B.R., Blanksma, C.A., Fay, K.A., Jensen, K.M., Kahl, M.D., Knapen, D., Kosian, P.A., Poole, S.T., Randolph, E.C., Schroeder, A.L., Vergauwen, L., Villeneuve, D.L., 2017. Impaired swim bladder inflation in early life stage fathead minnows exposed to a deiodinase inhibitor, iopanoic acid. Environmental Toxicology and Chemistry 36, 2942-2952.

Chang, J., Wang, M., Gui, W., Zhao, Y., Yu, L., Zhu, G., 2012. Changes in Thyroid Hormone Levels during Zebrafish Development. Zoological Science 29, 181-184.

Darras, V.M., Houbrechts, A.M., Van Herck, S.L.J., 2015. Intracellular thyroid hormone metabolism as a local regulator of nuclear thyroid hormone receptor-mediated impact on vertebrate development. Biochimica Et Biophysica Acta-Gene Regulatory Mechanisms 1849, 130-141.

de Vrieze, E., van de Wiel, S.M.W., Zethof, J., Flik, G., Klaren, P.H.M., Arjona, F.J., 2014. Knockdown of Monocarboxylate Transporter 8 (mct8) Disturbs Brain Development and Locomotion in Zebrafish. Endocrinology 155, 2320-2330.

Dong, W., Macaulay, L.J., Kwok, K.W., Hinton, D.E., Stapleton, H.M., 2013. Using whole mount in situ hybridization to examine thyroid hormone deiodinase expression in embryonic and larval zebrafish: a tool for examining OH-BDE toxicity to early life stages. Aquat Toxicol 132-133, 190-199.

Galton, V.A., Schneider, M.J., Clark, A.S., St Germain, D.L., 2009. Life without thyroxine to 3,5,3'-triiodothyronine conversion: studies in mice devoid of the 5'-deiodinases. Endocrinology 150, 2957-2963.

Heijlen, M., Houbrechts, A., Bagci, E., Van Herck, S., Kersseboom, S., Esguerra, C., Blust, R., Visser, T., Knapen, D., Darras, V., 2014. Knockdown of type 3 iodothyronine deiodinase severely perturbs both embryonic and  early larval development in zebrafish. Endocrinology 155, 1547-1559.

Heijlen, M., Houbrechts, A.M., Darras, V.M., 2013. Zebrafish as a model to study peripheral thyroid hormone metabolism in vertebrate development. Gen.Comp. Endocrinol. 188, 289–296, http://dx.doi.org/10.1016/j.ygcen.2013.04.004.

Kress, E., Rezza, A., Nadjar, J., Samarut, J., Plateroti, M., 2009. The frizzled-related sFRP2 gene is a target of thyroid hormone receptor alpha1 and activates beta-catenin signaling in mouse intestine. J Biol Chem 284, 1234-1241.

Liu, Y.W., Chan, W.K., 2002. Thyroid hormones are important for embryonic to larval transitory phase in zebrafish. Differentiation 70, 36-45.

Maack, G., Segner, H., 2003. Morphological development of the gonads in zebrafish. Journal of Fish Biology 62, 895-906.

Marelli, F., Carra, S., Agostini, M., Cotelli, F., Peeters, R., Chatterjee, K., Persani, L., 2016. Patterns of thyroid hormone receptor expression in zebrafish and generation of a novel model of resistance to thyroid hormone action. Molecular and Cellular Endocrinology 424, 102-117.

Molla, M.H.R., Hasan, M.T., Jang, W.J., Diaz, C.D.S., Appenteng, P., Marufchoni, H., Jahan, B., Brown, C.L., 2019. Thyroid hormone-induced swim bladder and eye maturation are transduced by IGF-1 in zebrafish embryos. Aquaculture Research 50, 3462-3470.

Nelson, K., Schroeder, A., Ankley, G., Blackwell, B., Blanksma, C., Degitz, S., Flynn, K., Jensen, K., Johnson, R., Kahl, M., Knapen, D., Kosian, P., Milsk, R., Randolph, E., Saari, T., Stinckens, E., Vergauwen, L., Villeneuve, D., 2016. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part I: Fathead minnow. Aquatic Toxicology 173, 192-203.

Plateroti, M., Kress, E., Mori, J.I., Samarut, J., 2006. Thyroid hormone receptor alpha1 directly controls transcription of the beta-catenin gene in intestinal epithelial cells. Mol Cell Biol 26, 3204-3214.

Schneider, M.J., Fiering, S.N., Thai, B., Wu, S.Y., St Germain, E., Parlow, A.F., St Germain, D.L., Galton, V.A., 2006. Targeted disruption of the type 1 selenodeiodinase gene (Dio1) results in marked changes in thyroid hormone economy in mice. Endocrinology 147, 580-589.

Shi, G., Wang, J., Guoa, H., Shenga, N., Cui, Q., Pan, Y., Guob, Y., Sun, Y., Dai, J., 2019. Parental exposure to 6:2 chlorinated polyfluorinated ether sulfonate (F-53B) induced transgenerational thyroid hormone disruption in zebrafish. Science of the Total Environment 665, 855-863.

Stinckens, E., Vergauwen, L., Schroeder, A., Maho, W., Blackwell, B., Witters, H., Blust, R., Ankley, G., Covaci, A., Villeneuve, D., Knapen, D., 2016. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part II: Zebrafish. Aquatic Toxicology 173, 204-217.

Stinckens, E., Vergauwen, L., Ankley, G.T., Blust, R., Darras, V.M., Villeneuve, D.L., Witters, H., Volz, D.C., Knapen, D., 2018. An AOP-based alternative testing strategy to predict the impact of thyroid hormone disruption on swim bladder inflation in zebrafish. Aquatic Toxicology 200, 1-12.

Stinckens, E., Vergauwen, L., Blackwell, B.R., Anldey, G.T., Villeneuve, D.L., Knapen, D., 2020. Effect of Thyroperoxidase and Deiodinase Inhibition on Anterior Swim Bladder Inflation in the Zebrafish. Environmental Science & Technology 54, 6213-6223.


Thisse, C., Degrave, A., Kryukov, G.V., Gladyshev, V.N., Obrecht-Pflumio, S., Krol, A., Thisse, B., Lescure, A., 2003. Spatial and temporal expression patterns of selenoprotein genes during embryogenesis in zebrafish. Gene Expr Patterns 3, 525-532.

Walpita, C.N., Crawford, A.D., Darras, V.M., 2010. Combined antisense knockdown of type 1 and type 2 iodothyronine deiodinases disrupts embryonic development in zebrafish (Danio rerio). General and Comparative Endocrinology 166, 134-141.

Walpita, C.N., Crawford, A.D., Janssens, E.D., Van der Geyten, S., Darras, V.M., 2009. Type 2 iodothyronine deiodinase is essential for thyroid hormone-dependent embryonic development and pigmentation in zebrafish. Endocrinology 150, 530-539.

Wang, J.X., Shi, G.H., Yao, J.Z., Sheng, N., Cui, R.N., Su, Z.B., Guo, Y., Dai, J.Y., 2020. Perfluoropolyether carboxylic acids (novel alternatives to PFOA) impair zebrafish posterior swim bladder development via thyroid hormone disruption. Environment International 134.

Wei, P.H., Zhao, F., Zhang, X.N., Liu, W.M., Jiang, G.B., Wang, H.F., Ru, S.G., 2018. Transgenerational thyroid endocrine disruption induced by bisphenol S affects the early development of zebrafish offspring. Environmental Pollution 243, 800-808.

Winata, C.L., Korzh, S., Kondrychyn, I., Zheng, W., Korzh, V., Gong, Z., 2009. Development of zebrafish swimbladder: The requirement of Hedgehog signaling in specification and organization of the three tissue layers. Developmental Biology 331, 222-236.

Yin, A., Korzh, S., Winata, C.L., Korzh, V., Gong, Z., 2011. Wnt signaling is required for early development of zebrafish swimbladder. PLoS One 6, e18431.

Yin, A., Korzh, V., Gong, Z., 2012. Perturbation of zebrafish swimbladder development by enhancing Wnt signaling in Wif1 morphants. Biochim Biophys Acta 1823, 236-244.