Albinism, the loss of melanin pigmentation, has evolved in a diverse variety of cave animals but the responsible evolutionary mechanisms are unknown. In Astyanax mexicanus, which has a pigmented surface dwelling form (surface fish) and several albino cave-dwelling forms (cavefish), albinism is caused by loss of function mutations in the oca2 gene, which operates during the first step of the melanin synthesis pathway. In addition to albinism, cavefish have evolved differences in behavior, including feeding and sleep, which are under the control of the catecholamine system. The catecholamine and melanin synthesis pathways diverge after beginning with the same substrate, L-tyrosine. Here we describe a novel relationship between the catecholamine and melanin synthesis pathways in Astyanax. Our results show significant increases in L-tyrosine, dopamine, and norepinephrine in pre-feeding larvae and adult brains of Pachón cavefish relative to surface fish. In addition, norepinephrine is elevated in cavefish adult kidneys, which contain the teleost homologs of catecholamine synthesizing adrenal cells. We further show that the oca2 gene is expressed during surface fish development but is downregulated in cavefish embryos. A key finding is that knockdown of oca2 expression in surface fish embryos delays the development of pigmented melanophores and simultaneously increases L-tyrosine and dopamine. We conclude that a potential evolutionary benefit of albinism in Astyanax cavefish may be to provide surplus L-tyrosine as a precursor for the elevated catecholamine synthesis pathway, which could be important for adaptation to the challenging cave environment.

The role of maternal factors in the evolution of development is poorly understood. Here we describe the use of reciprocal hybridization between the surface dwelling (surface fish, SF) and cave dwelling (cavefish, CF) morphs of the teleost Astyanax mexicanus to investigate the roles of maternal genetic effects in cavefish development. Reciprocal hybridization, a procedure in which F1 hybrids are generated by fertilizing SF eggs with CF sperm (SF × CF hybrids) and CF eggs with SF sperm (CF × SF hybrids), revealed that the CF degenerative eye phenotype showed maternal genetic effects. The eyes of CF × SF hybrids resembled the degenerate eyes of CF in showing ventral reduction of the retina and corresponding displacement of the lens within the optic cup, a smaller lens and eyeball, more lens apoptosis, a smaller cartilaginous sclera, and lens-specific gene expression characteristics compared to SF × CF hybrids, which showed eye and lens gene expression phenotypes resembling SF. In contrast, reciprocal hybridization failed to support roles for maternal genetic effects in the CF regressive pigmentation phenotype or in CF constructive changes related to enhanced jaw development. Maternal transcripts encoded by the pou2f1b, runx2b, and axin1 genes, which are involved in determining ventral embryonic fates, were increased in unfertilized CF eggs. In contrast, maternal mRNAs encoded by the ß-catenin and syntabulin genes, which control dorsal embryonic fates, showed similar expression levels in unfertilized SF and CF eggs. Furthermore, maternal transcripts of a sonic hedgehog gene were detected in SF and CF eggs and early cleaving embryos. This study reveals that CF eye degeneration is controlled by changes in maternal factors produced during oogenesis and introduces A. mexicanus as a model system for studying the role of maternal changes in the evolution of development.

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.

BackgroundThe teleost Astyanax mexicanus is a single species consisting of eyed surface-dwelling (surface fish) and blind cave-dwelling (cavefish) morphs. Cavefish eyes are lost through apoptosis of the lens, which in turn promotes the degeneration of other optic tissues. The αA-crystallin (αA-crys) gene is strongly downregulated in the cavefish lens and is located in a genomic region (QTL) responsible for eye loss. Therefore, αA-crys has been proposed as a candidate for regulating cavefish eye degeneration. The purpose of this study was to determine the mechanism of αA-crys downregulation and its role in cavefish eye degeneration.ResultsThe involvement of αA-crys in eye degeneration was confirmed by knocking down its expression in surface fish, which led to apoptosis of the lens. The underlying reason for αA-crys downregulation in cavefish was investigated by comparing genomic αA-crys DNA sequences in surface fish and cavefish, however, no obvious cis-regulatory factors were discovered. Furthermore, the cavefish αA-crys allele is expressed in surface fish x cavefish F1 hybrids, indicating that evolutionary changes in upstream genes are most likely responsible for αA-crys downregulation. In other species, Sox2 is one of the transcription factors that regulate lens crystallin genes during eye development. Determination of sox2 expression patterns during surface fish and cavefish development showed that sox2 is specifically downregulated in the cavefish lens. The upstream regulatory function of Sox2 was demonstrated by knockdown in surface fish, which abolished αA-crys expression and induced lens apoptosis.ConclusionsThe results suggest that αA-crys is required for normal eye development in cavefish via suppression of lens apoptosis. The regulatory changes involved in αA-crys downregulation in cavefish are in trans-acting factors rather than cis-acting mutations in the αA-crys gene. Therefore, αA-crys is unlikely to be the mutated gene(s) associated with an Astyanax eye QTL. The results reveal a genetic pathway leading from sox2 to αA-crys that is required for survival of the lens in Astyanax surface fish. Defects in this pathway may be involved in lens apoptosis and thus a cause of cavefish eye degeneration.

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...