Subterranean Fishes of the World

5612

2022

AIM 2022

19

The direction of visceral asymmetry is conserved during vertebrate evolution. As in other vertebrates, heart development is biased to the left in Astyanax surface fish (SF), but development is reversed to the right in a significant number of cavefish (CF). The heritable change in CF left-right (L-R) asymmetry is reflected by right-sided or bilateral expression of the Nodal/Lefty-Pitx2 signaling system, which is normally restricted to the left lateral mesoderm in SF. Critical changes in key Nodal/Lefty-Pitx2 system genes are not evident in CF, thus focusing our attention to differences in upstream processes. Accordingly, reciprocal hybridizations between SF and CF revealed a maternal basis for CF heart asymmetry. Here we probe zygotic events upstream of the lateralized Nodal/Lefty-Pitx2 signal as potential targets of maternal effects in Pachón CF embryos. We found that Kupffer’s Vesicle (KV), the ciliated L-R organizer, is shaped differently, larger, and has significantly more cilia in CF than SF. In addition, dorsal forerunner cells (DFC), the precursors of the KV, are more abundant in CF compared to SF. In zebrafish, KV ciliation is regulated by the Foxj1a transcription factor. In situ hybridization indicated that foxj1a is expressed in DFC and the KV of Astyanax, and qPCR quantification showed a 10-fold foxj1a transcript increase in CF relative to SF. Previous studies showed that shh expression, an upstream regulator of foxj1a in zebrafish, is increased and expanded along the anterior midline of CF embryos. In situ hybridization indicated that shh expression is also expanded posteriorly in the KV region of CF embryos. Treatment of SF embryos with Smoothened Agonist, a Hedgehog pathway activator, induced the reversal of heart asymmetry. We conclude that maternal factors responsible for L-R asymmetry may target the Shh-Foxj1a pathway and that visceral reversal could be linked to enhancement of Shh-dependent constructive traits during CF evolution.

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