Altered, Photoreceptor patterning

Photoreceptors in the retina of vertebrates and invertebrates are the cells that are responsible for phototransduction. The main groups of photoreceptor cells are rods, active at very low light levels, and cones, active at higher light levels and responsible for color vision. Photoreceptor subtypes are characterized by different opsins (light-sensitive proteins) that respond to light with different wavelengths.

The opsin characterizing rods is rhodopsin. Cones are further divided in several subtypes. The opsins characterizing these subtypes are generally grouped in S-opsins (short wavelength-sensitive), M-opsins (medium wavelength-sensitive) and L-opsins (long wavelength-sensitive). The occurrence of different opsins is species-specific (see Taxonomic applicability). 

The distribution of photoreceptor subtypes within the retina is also referred to as photoreceptor patterning and has a quantitative component (typical ratios of photoreceptor subtypes) as well as a spatial component (organization of photoreceptor subtypes). Depending on the species, the spatial organization is stochastic/regionalized (human, fruit fly), regionalized (mouse), or ordered (zebrafish).

During early development, photoreceptor subtypes differentiate from retinal progenitor cells. In a later stage of embryo or juvenile development, already differentiated cone photoreceptors can also switch opsin expression to a different opsin type. In general, this opsin switch is characterized by a switch in opsin expression from short to longer wavelength-sensitive opsins. An opsin switch is part of normal eye development and has been documented mostly in fish species (Shand et al., 2002; Cheng et al., 2006; Cheng and Flamarique, 2007; Matsumoto and Ishibashi, 2016; Mackin et al., 2019), and also in rodents (Lukats et al., 2005). 

Under some circumstances, photoreceptor patterning and opsin switching can be altered. This can manifest itself as altered numbers of photoreceptor subtypes leading to an altered ratio of photoreceptor subtypes and/or altered spatial organization (Raymond et al., 2014).

In general, photoreceptor cell types are quantified and/or localized based on their opsin expression. The target for measurement therefore are the opsins. They can be measured either on the mRNA or on the protein level.

Altered opsin expression patterns indicative of altered ratios of photoreceptor subtypes are often detected by relative quantification of mRNA coding for the specific opsins expressed in the photoreceptor subtypes using qPCR (quantitative polymerase chain reaction) (Allison et al., 2006; Mackin et al., 2019). This is a straightforward technique that many laboratories have available.

Several methods can be used to obtain information on spatial patterning and to count photoreceptor types. 

• Immunohistochemistry allows for labelling specific opsins (protein level) with antibodies, mostly through the use of primary antibodies specific to the target and secondary antibodies that bind to the primary antibodies and are conjugated to e.g., a fluorescent label or alkaline phosphatase that produces a measurable product (Allison et al., 2006; DuVal et al., 2014; Houbrechts et al., 2016; DuVal and Allison, 2018). This is typically performed on retinal wholemounts or cryosections. 

• In situ hybridization is used to label opsin mRNA using complementary oligonucleotide probes on retinal sections (Allison et al., 2006; Gan and Flamarique, 2010; Glaschke et al., 2010; DuVal et al., 2014; Karagic et al., 2018; Mackin et al., 2019).

• Zebrafish transgenic lines expressing fluorescent reporters in specific photoreceptor types (again associated with opsin expression) have also been successfully used to analyse photoreceptor counts, spatial patterning and opsin switches (Raymond et al., 2014; Mackin et al., 2019). Examples include lines reporting rhodopsin, blue  cone or UV cone expression (Raymond et al., 2014).

Based on available evidence, it seems plausible that this key event is applicable across all life stages and for a wide variety of taxa including vertebrates and invertebrates.

1. Taxonomic applicability

Rod and cone pigments all diverged from a common ancestor through a series of duplication events (Nathans et al., 1986). These duplication events gave rise to important taxonomic differences in opsin genes. As an example, humans have three cone photoreceptor types expressing long (L, red), medium (M, green), or short (S, blue) wavelength‐specific opsins (Nathans et al., 1986), while the zebrafish genome has two red (LWS-1 and LWS-2), four green (RH2-1, RH2-2, RH2-3, and RH2-4), and single blue (SWS2) and ultraviolet (SWS1) opsin genes (Chinen et al., 2003). Suzuki et al. (2013) further discuss that some species have pure cone types that express a single opsin, while others have mixed cone types expressing different opsins simultaneously. The authors suggested that expression of thrbeta2 in progenitor cells results in pure L-opsin cones in zebrafish. This is opposed to expression of thrbeta2 in later in postmitotic cells resulting in mixed cones in mice.

The importance of normal ratios of photoreceptor types and the concept of photoreceptor patterning however seems to be applicable to a wide range of species, including vertebrates and invertebrates. Retinal patterning of different taxa can be stochastic/regionalized (human, fruit fly), regionalized (mouse), or ordered (zebrafish) and has evolved to suit different environments and behaviors (Raymond et al., 2014; Viets et al., 2016). Since normal patterning differs among taxa, changes in patterning should be considered within species.

During early development, photoreceptor subtypes differentiate from retinal progenitor cells. In a later stage of embryo or juvenile development, already differentiated cone photoreceptors can also switch opsin expression to a different opsin type. Such opsin switch is part of normal eye development and has been documented mostly in fish (Shand et al., 2002; Cheng et al., 2006; Cheng and Flamarique, 2007; Matsumoto and Ishibashi, 2016), and also in rodents (Lukats et al., 2005) and humans (Cornish et al., 2004). This opsin switch is characterized by a switch in opsin expression from short to longer wavelength-sensitive opsins. For example, in salmonids, single cones express ultraviolet (SWS-1) opsin during embryonic development and switch to blue (SWS-2) opsin as the fish grow (Gan and Flamarique, 2010). In zebrafish a switch occurs from LWS-2 to the longer wavelength LWS-1 opsin (Tsujimura et al., 2010; Mitchell et al., 2015; Mackin et al., 2019). Mitchell et al. (2015) and Mackin et al. (2019) even confirmed opsin switching in real time using developing transgenic zebrafish. In rodents and humans, the opsin switch involves a switch from S to M opsins.

Teleost fish and salamanders are capable of regenerating the retina following injury, while mammals do not have this innate capacity to regenerate the retina (Mader and Cameron, 2004; Lamba et al., 2008; Van Gelder and Kaur, 2015). Studies have shown however that opsin expression in terminally differentiated mammalian cones also remains subject to alterations (Glaschke et al., 2011). Therefore, in addition to alterations during development, alterations of photoreceptor patterning in the adult retina are also expected to be relevant across taxa.

2. Life-stage applicability

Normal photoreceptor patterning is established during development and this process can be altered by various circumstances (Mackin et al., 2019). Therefore, this key event is applicable to early life stages in which the retina is under development.

Juvenile zebrafish show some plasticity in opsin expression (Mackin et al., 2019). This type of phenotypic plasticity appears common among fish as a result of changes in habitats. 

Since teleosts and salamanders can regenerate the retina after injury, the process of restoring photoreceptor patterning can also be affected in the adult life stage (Mader and Cameron, 2006). 

In adult mice and rats, the normal pattern of opsin expression and distribution can be reversibly altered, suggesting that opsin expression in terminally differentiated mammalian cones also remains subject to alterations (Glaschke et al., 2011). 

Taken together, there is good evidence that this key event is applicable across all life stages.

3. Sex applicability

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. Establishment of photoreceptor patterning during early development is therefore expected to be independent of sex.

A few studies have shown that sex hormones can regulate spectral sensitivity, and this is probably related to the importance of perceiving breeding coloration. In sexually mature male sticklebacks, androgen is a key factor in enhancing sensitivity to red light via regulation of opsin gene expression (Shao et al., 2014). This is in line with the need to detect the red breeding color of males during the breeding season. Lizards also regulate opsin expression seasonally, and this appears to be related to evaluation of the coloration of potential mates. Tseng et al. (2018) showed that testosterone regulates opsin expression in a sexually dimorphic lizard and that males and females show opposite shifts in opsin expression during the breeding season.

In mammals, medium (green) and long (red) wavelength-sensitive opsin genes are located on the X chromosome, leading to sex-linked color vision deficiencies where male individuals are more susceptible (Jacobs, 2009). 

Studies have shown sexual dimorphism of photoreceptor patterning in Arthropoda such as the fruitfly and the small white butterfly (Arikawa et al., 2005; Hilbrant et al., 2014). The crustacean Euphilomedes carcharodonta exhibits radical sexual dimorphism of the lateral eyes. Females have only a tiny, simple lateral eye while males have elaborate ommatidial eyes. This coincides with differences in the expression of genes related to eye development and phototransduction (Sajuthi et al., 2015). 

Alterations in normal photoreceptor patterning can be expected to occur across sexes. Based on the general evidence of sexual dimorphism in terms of spectral sensitivity, sex specific alterations may occur in sexually mature organisms.

Allison, W.T., Dann, S.G., Veldhoen, K.M., Hawryshyn, C.W., 2006. Degeneration and regeneration of ultraviolet cone photoreceptors during development in rainbow trout. Journal of Comparative Neurology 499, 702-715.

Arikawa, K., Wakakuwa, M., Qiu, X.D., Kurasawa, M., Stavenga, D.G., 2005. Sexual dimorphism of short-wavelength photoreceptors in the small white butterfly, Pieris rapae crucivora. Journal of Neuroscience 25, 5935-5942.

Cheng, C.L., Flamarique, I.N., 2007. Chromatic organization of cone photoreceptors in the retina of rainbow trout: single cones irreversibly switch from UV (SWS1) to blue (SWS2) light sensitive opsin during natural development. Journal of Experimental Biology 210, 4123-4135.

Cheng, C.L., Flamarique, I.N., Harosi, F.I., Rickers-Haunerland, J., Haunerland, N.H., 2006. Photoreceptor layer of salmonid fishes: Transformation and loss of single cones in juvenile fish. Journal of Comparative Neurology 495, 213-235.

Chinen, A., Hamaoka, T., Yamada, Y., Kawamura, S., 2003. Gene duplication and spectral diversification of cone visual pigments of zebrafish. Genetics 163, 663-675.

Cornish, E.E., Xiao, M., Yang, Z.T., Provis, J.M., Hendrickson, A.E., 2004. The role of opsin expression and apoptosis in determination of cone types in human retina. Experimental Eye Research 78, 1143-1154.

DuVal, M.G., Allison, W.T., 2018. Photoreceptor Progenitors Depend Upon Coordination of gdf6a, thr beta, and tbx2b to Generate Precise Populations of Cone Photoreceptor Subtypes. Investigative Ophthalmology & Visual Science 59, 6089-6101.

DuVal, M.G., Oel, A.P., Allison, W.T., 2014. gdf6a Is Required for Cone Photoreceptor Subtype Differentiation and for the Actions of tbx2b in Determining Rod Versus Cone Photoreceptor Fate. Plos One 9.

Gan, K.J., Flamarique, I.N., 2010. Thyroid Hormone Accelerates Opsin Expression During Early Photoreceptor Differentiation and Induces Opsin Switching in Differentiated TR alpha-Expressing Cones of the Salmonid Retina. Developmental Dynamics 239, 2700-2713.

Glaschke, A., Glosmann, M., Peichl, L., 2010. Developmental Changes of Cone Opsin Expression but Not Retinal Morphology in the Hypothyroid Pax8 Knockout Mouse. Investigative Ophthalmology & Visual Science 51, 1719-1727.

Glaschke, A., Weiland, J., Del Turco, D., Steiner, M., Peichl, L., Glosmann, M., 2011. Thyroid Hormone Controls Cone Opsin Expression in the Retina of Adult Rodents. Journal of Neuroscience 31, 4844-4851.

Hilbrant, M., Almudi, I., Leite, D.J., Kuncheria, L., Posnien, N., Nunes, M.D.S., McGregor, A.P., 2014. Sexual dimorphism and natural variation within and among species in the Drosophila retinal mosaic. Bmc Evolutionary Biology 14.

Houbrechts, A.M., Vergauwen, L., Bagci, E., Van Houcke, J., Heijlen, M., Kulemeka, B., Hyde, D.R., Knapen, D., Darras, V.M., 2016. Deiodinase knockdown affects zebrafish eye development at the level of gene expression, morphology and function. Molecular and Cellular Endocrinology 424, 81-93.

Jacobs, G.H., 2009. Evolution of colour vision in mammals. Philosophical Transactions of the Royal Society B-Biological Sciences 364, 2957-2967.

Karagic, N., Harer, A., Meyer, A., Torres-Dowdall, J., 2018. Heterochronic opsin expression due to early light deprivation results in drastically shifted visual sensitivity in a cichlid fish: Possible role of thyroid hormone signaling. Journal of Experimental Zoology Part B-Molecular and Developmental Evolution 330, 202-214.

Lamba, D., Karl, M., Rehl, T., 2008. Neural regeneration and cell replacement: A view from the eye. Cell Stem Cell 2, 538-549.

Lukats, A., Szabo, A., Rohlich, P., Vigh, B., Szel, A., 2005. Photopigment coexpression in mammals: comparative and developmental aspects. Histology and Histopathology 20, 551-574.

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

Mackin, R.D., Frey, R.A., Gutierrez, C., Farre, A.A., Kawamura, S., Mitchell, D.M., Stenkamp, D.L., 2019. Endocrine regulation of multichromatic color vision. Proceedings of the National Academy of Sciences of the United States of America 116, 16882-16891.

Mader, M., Cameron, D., 2006. Effects of induced systemic hypothyroidism upon the retina: Regulation of thyroid hormone receptor alpha and photoreceptor production. Molecular Vision 12, 915-930.

Mader, M.M., Cameron, D.A., 2004. Photoreceptor differentiation during retinal development, growth, and regeneration in a metamorphic vertebrate. Journal of Neuroscience 24, 11463-11472.

Matsumoto, T., Ishibashi, Y., 2016. Sequence analysis and expression patterns of opsin genes in the longtooth grouper Epinephelus bruneus. Fisheries Science 82, 17-27.

Mitchell, D.M., Stevens, C.B., Frey, R.A., Hunter, S.S., Ashino, R., Kawamura, S., Stenkamp, D.L., 2015. Retinoic Acid Signaling Regulates Differential Expression of the Tandemly-Duplicated Long Wavelength-Sensitive Cone Opsin Genes in Zebrafish. Plos Genetics 11.

Nathans, J., Thomas, D., Hogness, D.S., 1986. MOLECULAR-GENETICS OF HUMAN COLOR-VISION - THE GENES ENCODING BLUE, GREEN, AND RED PIGMENTS. Science 232, 193-202.

Raymond, P.A., Colvin, S.M., Jabeen, Z., Nagashima, M., Barthel, L.K., Hadidjojo, J., Popova, L., Pejaver, V.R., Lubensky, D.K., 2014. Patterning the Cone Mosaic Array in Zebrafish Retina Requires Specification of Ultraviolet-Sensitive Cones. Plos One 9.

Sajuthi, A., Carrillo-Zazueta, B., Hu, B., Wang, A., Brodnansky, L., Mayberry, J., Rivera, A.S., 2015. Sexually dimorphic gene expression in the lateral eyes of Euphilomedes carcharodonta (Ostracoda, Pancrustacea). Evodevo 6.

Shand, J., Hart, N.S., Thomas, N., Partridge, J.C., 2002. Developmental changes in the cone visual pigments of black bream Acanthopagrus butcheri. Journal of Experimental Biology 205, 3661-3667.

Shao, Y.T., Wang, F.Y., Fu, W.C., Yan, H.Y., Anraku, K., Chen, I.S., Borg, B., 2014. Androgens Increase Iws Opsin Expression and Red Sensitivity in Male Three-Spined Sticklebacks. Plos One 9.

Tseng, W.H., Lin, J.W., Lou, C.H., Lee, K.H., Wu, L.S., Wang, T.Y., Wang, F.Y., Irschick, D.J., Lin, S.M., 2018. Opsin gene expression regulated by testosterone level in a sexually dimorphic lizard. Scientific Reports 8.

Tsujimura, T., Hosoya, T., Kawamura, S., 2010. A Single Enhancer Regulating the Differential Expression of Duplicated Red-Sensitive Opsin Genes in Zebrafish. Plos Genetics 6.

Van Gelder, R.N., Kaur, K., 2015. Vision Science: Can Rhodopsin Cure Blindness? Current Biology 25, R713-R715.

Viets, K., Eldred, K.C., Johnston, R.J., 2016. Mechanisms of Photoreceptor Patterning in Vertebrates and Invertebrates. Trends in Genetics 32, 638-659.