Coding and non-coding mutations in DNA contribute significantly to phenotypic variability during evolution. However, less is known about the role of epigenetics in this process. Although previous studies have identified eye development genes associated with the loss of eyes phenotype in the Pachón blind cave morph of the Mexican tetra Astyanax mexicanus, no inactivating mutations have been found in any of these genes. Here we show that excess DNA methylation-based epigenetic silencing promotes eye degeneration in blind cave Astyanax mexicanus. By performing parallel analyses in Astyanax mexicanus cave and surface morphs and in the zebrafish Danio rerio, we have discovered that DNA methylation mediates eye-specific gene repression and globally regulates early eye development. The most significantly hypermethylated and down-regulated genes in the cave morph are also linked to human eye disorders, suggesting the function of these genes is conserved across the vertebrates. Our results show that changes in DNA methylation-based gene repression can serve as an important molecular mechanism generating phenotypic diversity during development and evolution.

DNA methylation in cavefishGore et al 2 Coding and non-coding mutations in DNA contribute significantly to phenotypic variability during evolution. However, less is known about the role of epigenetics in this process. Although previous studies have identified eye development genes associated with the loss of eyes phenotype in the Pachón blind cave morph of the Mexican tetra Astyanax mexicanus 1-6 , no inactivating mutations have been found in any of these genes 2,3,7-10 . Here we show that excess DNA methylation-based epigenetic silencing promotes eye degeneration in blind cave Astyanax mexicanus.By performing parallel analyses in Astyanax mexicanus cave and surface morphs and in the zebrafish Danio rerio, we have discovered that DNA methylation mediates eye-specific gene repression and globally regulates early eye development.The most significantly hypermethylated and down-regulated genes in the cave morph are also linked to human eye disorders, suggesting the function of these genes is conserved across the vertebrates. Our results show that changes in DNA methylation-based gene repression can serve as an important molecular mechanism generating phenotypic diversity during development and evolution.Subterranean animals offer an excellent opportunity to study morphological, molecular and physiological changes that allow organisms to adapt to unique environments. Loss of eyes is one of the most common morphological features of cave-adapted animals, including many fish species. Blind cave fish (CF) morphs of Astyanax mexicanus evolved from surface fish (SF) during a few million years of isolation in dark Mexican caves 11 , with recent studies suggesting that regression of eyes evolved as part of a strategy to conserve energy in fish adapted to dark and nutrient deficient caves 12 .Although a number of studies have examined molecular mechanisms underlying eye loss DNA methylation in cavefish Gore et al 3 in Pachón cave-derived Astyanax mexicanus CF, recent sequencing of the Pachón CF genome and other studies revealed no inactivating null mutations in essential eye development genes 2,3,7-9 . In contrast, genome sequencing of another subterranean animal, the naked mole rat Heterocephalus glaber, showed combined functional loss of more than a dozen key eye genes due to inactivating mutations 13 . These findings suggest the possibility that epigenetic rather than genetic changes may mediate eye loss in Pachón cave fish. To test this possibility, we used CF and SF morphs of Astyanax mexicanus as well as wild type and DNA methylation and demethylation-deficient zebrafish Danio rerio to examine whether DNA methylation regulates eye formation, and whether eye loss in Pachón cave fish evolved at least in part through hypermethylation of key eye genes.At 36 hpf Astyanax mexicanus CF and SF embryos are superficially indistinguishable with properly formed lenses and optic cups in both morphs (Fig. 1a,b). By five days of development, however, degeneration of eye tissue is clearly evident (Fig. 1c,d), and by adulthood CF eyes ar...

Vestigial structures are key indicators of evolutionary descent, but the mechanisms underlying their development are poorly understood. This study examines vestigial eye formation in the teleost Astyanax mexicanus, which consists of a sighted surface-dwelling morph and multiple populations of blind cave morphs. Cavefish embryos initially develop eyes, but they subsequently degenerate and become vestigial structures embedded in the head. The mutated genes involved in cavefish vestigial eye formation have not been characterized. Here we identify cystathionine ß-synthase a (cbsa), which encodes the key enzyme of the transsulfuration pathway, as one of the mutated genes responsible for eye degeneration in multiple cavefish populations. The inactivation of cbsa affects eye development by increasing the transsulfuration intermediate homocysteine and inducing defects in optic vasculature, which result in aneurysms and eye hemorrhages. Our findings suggest that localized modifications in the circulatory system may have contributed to the evolution of vestigial eyes in cavefish.

17Vestigial structures are key indicators of evolutionary descent but the 18 mechanisms underlying their development are poorly understood. This study examines 19 vestigial eye formation in the teleost Astyanax mexicanus, which consists of a sighted 20 surface-dwelling morph and different populations of blind cave morphs. Cavefish 21 embryos initially develop optic primordia but vestigial eyes are formed during larval 22 development. Multiple genetic factors are involved in cavefish eye loss but none of the 23 mutated genes have been identified. Here we identify cystathionine ß-synthase (cbsa), 24 which encodes the key enzyme of the transsulfuration pathway, as a mutated gene 25 responsible for eye degeneration in multiple cavefish populations. The inactivation of 26 cbsa affects eye development by inducing accumulation of the transsulfuration 27 intermediate homocysteine and defects in optic vasculature, including aneurysms and 28 eye hemorrhages, leading to oxygen deficiency. Our findings suggest that localized 29 modifications in the circulatory system and hypoxia had important roles in the evolution 30 of vestigial eyes in blind cavefish. 31In the teleost Astyanax mexicanus, blind cavefish (CF) have been derived 39 repeatedly from sighted surface fish (SF) ancestors 1-3 . The loss of eyes in A. 40 mexicanus CF involves initial optic development followed by subsequent degeneration 1, 41 2 . Eye primordia with a lens and retina are formed in CF embryos, but the lens 42 undergoes massive apoptosis, the retina becomes apoptotic and disorganized, and eye 43 growth is eventually arrested during larval development [3][4][5] . Accordingly, the rate of 44 optic growth fails to keep pace with the increase in overall body size as CF larvae 45 develop into adults, and the small non-functional eyes are overgrown by skin and 46 connective tissue. About 30 distinct CF populations with various degrees of eye 47 reduction have evolved in Mexican caves after colonization by surface fish ancestors 48 during the mid-to late Pleistocene, and several of these CF populations have evolved 49 vestigial eye phenotypes independently 6,7 . Similar eye reduction or loss occurs in other 50 cave dwelling species 8 and animals adapted to other perpetually dark habitats 9 . 51The existence of hybridizable surface dwelling and cave adapted morphs in A. 52 mexicanus has allowed eye degeneration to be studied by genetic methods 2 . These 53 studies have shown that eye loss in the Pachón CF (PA-CF) population is controlled by 54 multiple genetic factors, each controlling a part of the complex vestigial eye 55 phenotype 10-12 . Furthermore, complementation crosses between different CF 56 populations have demonstrated that some of the genetic factors involved in eye loss are 57 the same whereas others are unique 13 . In addition to genetic changes, epigenetic 58 events may also have roles in the evolution of CF eye loss 14 . Quantitative trait locus 59 (QTL) analysis revealed about 15 non-overlapping genomic regions that are responsible 60 for len...