Subterranean Fishes of the World

3208

2013

AIM 2013

Lens apoptosis plays a critical role in eye degeneration in Astyanax cavefish but the underlying molecular mechanisms are poorly understood. Developmental and genetic studies suggest that multiple genes are involved in cavefish eye degeneration. One of these genes is αA-crystallin (αA-crys), which is downregulated during cavefish lens development and maps near an eye QTL on relevant Astyanax genetic maps. Here we investigate the role of αA-crys in cavefish lens degeneration. Knockdown of αA-crys expression by injection of translation blocking and premRNA splice-blocking morpholinos (MO) into surface fish eggs resulted in the inhibition of lens development and induction of apoptosis. Thus far, overexpression of αA-crys by injection of synthetic capped mRNA into cavefish embryos has not rescued lens apoptosis or eye development. Together, these results suggest that αA-crys is required but insufficient for normal lens development. Experiments were conducted to determine whether αA-crys downregulation is caused by cis- or trans-acting regulatory mutations in cavefish. First, the levels of αA-crys expression in F1 hybrids, containing both the surface fish and cavefish αA-crys alleles in a transacting background, were compared to the levels in surface fish by in situ hybridization and semiquantitative PCR, and were found to be the same. Second, about 10 kb of DNA sequence including the αA-crys locus (through the upstream adjacent cystathionine β-synthase gene) was sequenced in surface fish and cavefish. There are two conserved non-coding regions with minor sequence differences in cavefish: one is in the 5’upstream deleting a “CA” repeat and another is in the second intron, deleting a “CT”. These two sequence differences could be part of enhancers responsible for cis-acting regulation. In addition to cis-acting regulation, aA-crys downregulation in cavefish may also be caused by changes in trans-acting factors. The pax6 and sox2 genes encode transcription factors responsible for the activation of crystallin gene expression. Of the two candidate genes, in situ hybridization showed that sox2 is downregulated during cavefish lens development. Furthermore, most surface fish embryos lost αA-crys expression in the lens after MO-based knockdown of the sox2 gene, while αA-crys expression was not affected after MO knockdown of the pax6 gene. The results suggest that a genetic pathway leading from sox2 to αA-crys is involved in normal lens development in Astyanax surface fish and that defects in this pathway result in lens apoptosis and eye degeneration in cavefish.

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