A HypomorphicCystathionine ß-SynthaseGene Contributes to Cavefish Eye Loss by Disrupting Optic Vasculature

Abstract

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...

Evolutionary Changes in Left-Right Visceral Asymmetry inAstyanaxCavefish

“…The positions of liver and pancreas development were compared in surface fish, PA-CF, and TI-CF by in situ hybridization using the cbsa gene as a marker [13]. Consistent with the conventional L-R laterality in zebrafish [14], at 3 dpf the liver developed on the left and the pancreas on the right in more than 98% of surface fish larvae ( Fig. 1D, E), and TI-CF larvae showed the similar liver-pancreas asymmetry (Fig.…”

“…The two sgRNA were synthesized according to lft2 sequence information (Ensembl ENSAMXT00000000477): the sequence of the first sgRNA was 5'-GCUUGCAGCACUGUCAAACA-3', and the sequence of the second sgRNA was 5'-CAAACAGAAACAUGUGCAUG-3'. Microinjection was carried out as described previously [14,38]. CRISPR-Cas9 injected and control un-injected embryos from the same clutch were cultured at 25 °C.…”