A hypomorphic cystathionine ß-synthase gene contributes to cavefish eye loss by disrupting optic vasculature

Abstract

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.

Astyanax surface and cave fish morphs

“…These studies have shown that eye loss is an additive trait controlled by more than a dozen QTL scattered across the genome [20,21]. Thus far, cbsa, which affects eye growth by interfering with optic vasculature function, is the only mutated gene that has been positively identified under an eye QTL [22]. The loss of melanin pigmentation is caused by differentiation of fewer melanophores, reduced melanin synthesis, which leads to brown coloration in some cavefish populations (e.g., Tinaja), and the complete loss of melanin, which results in albinism in other cavefish populations (e.g., Pachón and Molino) [23].…”

“…Genetic complementation between CRISPR-Cas9 surface fish mutants and cavefish can be used to confirm the relationship of edited genes to the genes responsible for naturally evolved cavefish traits and to evaluate off-target effects [55]. Gene overexpression has been carried out by injection of synthetic mRNAs or a gene fusion construct containing the zebrafish temperature-inducible hsp70 promoter into eggs [16,22].…”

“…First the lens (arrowhead) and then parts of the retina (arrows) undergo apoptosis detected by TUNEL. Center: steps of eye development in surface fish (right) and eye degeneration in cavefish (left) (modified from [ 22 ]) …”

“…4 b) have shown that genes controlling eye loss can be the same or unique in these populations [ 31 ]. The cbsa gene is mutated at the same cis -regulatory position in six different cavefish populations [ 22 ], suggesting that it functions repeatedly in eye loss. In some cavefish populations (e.g., Pachón, Tinaja) cbsa shows the same mutation, while in others (e.g., Molino) a different mutation occurs at the same site, revealing a possible DNA hotspot for mutational change.…”

“…Gene expression can be knocked down by injection of eggs with morpholinos and the combined use of judicious controls [ 16 , 22 , 53 ]. Gene knockouts at efficiencies of 50–75% are possible by microinjection of TALEN [ 54 ] or CRISPR–Cas9 [ 22 , 55 ] into fertilized eggs, and mutant lines can be maintained by standard husbandry. Genetic complementation between CRISPR–Cas9 surface fish mutants and cavefish can be used to confirm the relationship of edited genes to the genes responsible for naturally evolved cavefish traits and to evaluate off-target effects [ 55 ].…”