Surface Fish Research Articles

Environmental Hypoxia is a Driver of Sonic Hedgehog-dependent Troglomorphic Evolution in Astyanax Cavefish

Astyanax mexicanus cavefish (CF) have evolved numerous troglomorphic traits since their divergence from surface fish (SF) ancestors about 20,000 years ago. A large number of these traits are under the control of Sonic Hedgehog (Shh), a signaling morphogen that is overexpressed along the midline in CF relative to SF embryos. The Shh-dependent traits include eye degeneration and increases in the olfactory lobes, hypothalamus, jaws, and oral taste buds. Eye degeneration and tastebud enhancement are antagonistic tradeoffs governed by the pleiotropic shh gene. CF have also evolved multiple changes in circulatory system development, highlighted by larger hematopoietic domains, more erythrocytes, and reversals in normal heart asymmetry, which are also controlled by Shh signaling. Although Shh signaling is an important factor in CF troglomorphism, the shh gene is not associated with any of the many QTL identified by genetic analysis, suggesting that upstream processes, rather than mutation of the shh locus itself, may be important in Shh regulation. Due to the absence of primary productivity, cave pools harboring Astyanax CF are deficient in dissolved oxygen. Accordingly, the Hypoxia Inducing Factor (HIF) transcription factors and their downstream targets are constitutively active during CF development. To determine if oxygen deficiency is an upstream regulator of Shh signaling, we subjected early SF embryos to 8 or 16 hours of hypoxia in the laboratory and assayed the effects on shh expression, erythrocyte formation, heart asymmetry, and eye development. The results indicated that hypoxia can cause shh overexpression, increase erythropoiesis, reverse heart asymmetry, and induce eye degeneration in SF embryos. We conclude that environmental hypoxia may be responsible for the evolution of troglomorphic traits in Astyanax CF through hyperactivation of the Shh signaling pathway.

Lens defects in Astyanax mexicanus Cavefish: evolution of crystallins and a role for alphaA-crystallin.

The fish Astyanax mexicanus presents, within the same species, populations of river-dwelling surface fish (SF) and blind cave-living fish. In cavefish (CF), the eyes develop almost normally during embryogenesis. But 40 h after fertilization, the lens enters apoptosis, triggering the progressive degeneration of the entire eye. Before apoptosis, the CF lens expresses early differentiation factors correctly. Here, we searched for possible late differentiation defects that would be causal in CF lens degeneration. We reasoned that crystallins, the major lens structural proteins, could be defective or misregulated. We surveyed the CF and SF transcriptomes and uncovered 14 Astyanax crystallins from the beta, gamma, lambda, mu, and zeta families. These proteins are less polymorphic and accumulate more fixed mutations, some at highly conserved positions, in CF than in SF, suggesting relaxed selection at these loci in CF. In situ hybridizations and qPCR show that crybb1c, crybgx, crygm5 are expressed at much lower levels or are not expressed in the CF lens. For the best crystallin candidates, we tested a potential causal role in CF lens apoptosis. Crybgx, crybb1c (not expressed in CF from very early on), and cryaa (previously shown to be faintly expressed in CF) failed to induce any defect when knocked-down in zebrafish embryos. However, the anti-apoptotic cryaa protected lens cells from apoptosis when reexpressed by transgenesis in CF, suggesting a cell-autonomous effect of cryaa on lens cell survival. Altogether, these data suggest that crystallin sequence evolution and expression defects may contribute to the loss of eyes in CF.