The Expression of Pax6 Genes in an Eyeless Arachnid Suggests Their Ancestral Role in Arachnid Head Development

Background: Many animal lineages utilize Pax6 transcription factors during eye development. Within Arthropoda, evidence suggests that Pax6 genes are necessary for the specification of eyes in myriapods, crustaceans, and insects. However, recent data have argued that Pax6 genes lack a role in the development of the eyes in Chelicerata (=arachnids, horseshoe crabs, and sea spiders). An alternative hypothesis argues that the absence of Pax6 expression in developing chelicerate eyes could be explained by an earlier role for these genes in patterning eye precursor cells. The arachnid mite Archegozetes longisetosus lacks eyes, however it retains two Pax6 paralogs in its genome. By leveraging these aspects of A. longisetosus , we tested the hypothesis that ancestrally chelicerates did not use Pax6 genes to pattern their eyes but rather used them to pattern the central nervous system. We reasoned that if we observed comparable expression patterns of Pax6 genes in A. longisetosus in comparison to those in arachnids that have retained eyes, then this would support the hypothesis that Pax6 genes were not ancestrally used for eye specification in chelicerates. Results: We followed the expression of canonical arthropod retinal determination genes to confirm that A. longisetosus does not develop vestigial eyes. We found that the expression of the Pax6 paralogs was consistent with their roles in the development of the ocular region and central nervous system. By co-staining for these genes simultaneously with the conserved head patterning gene orthodenticle , we also observed early expression patterns of these genes in the protocerebrum of early A. longisetosus embryos that are comparable to those arachnids with embryonic eyes. Conclusions: Our data provide support for the hypothesis that Pax6 genes were not ancestrally used to pattern chelicerate eyes. The expression patterns of Pax6 genes in A. longisetosus were comparable to those of other arachnids that have eyes. This suggests that the retention of Pax6 genes in A. longisetosus is due to their ancestral, non-eye patterning roles. Further supporting this hypothesis is our observation that A. longisetosus does not pattern vestigial eyes. Lastly, our data suggests that the Pax6 genes, with orthodenticle , acted to specify the ancestral arachnid protocerebrum.