# The expression of Pax6 and retinal determination genes in the eyeless arachnid A. longisetosus reveals vestigial eye primordia

## TL;DR

This study investigates how Pax6 and related genes are expressed in an eyeless mite, suggesting they may be involved in brain development and vestigial eye structures.

## Contribution

The study provides new insights into Pax6 gene function in an eyeless arachnid, linking it to brain development and vestigial eye primordia.

## Key findings

- Pax6 paralogs in A. longisetosus are expressed in the central nervous system, not the eyes.
- RDGN genes like sine oculis and atonal show expression patterns consistent with vestigial eye primordia.
- Co-expression with orthodenticle suggests Pax6 and RDGN genes contribute to brain patterning in early embryos.

## Abstract

Evidence suggests that Pax6 genes are necessary for the specification of eyes in a variety of metazoans, including mandibulate arthropods. In these arthropods, Pax6 genes usually interact with a conserved set of genes, collectively called the retinal determination gene network (RDGN), to specify eye cells. 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). A genome sequence of the eyeless mite Archegozetes longisetosus revealed that it retains two Pax6 paralogs, as well as singleton orthologs of all RDGN genes. We hypothesized that the retention of these two Pax6 paralogs could be due to their non-eye determining roles, and/or their expression in vestigial eye primordia. We therefore used hybridization chain reactions (HCRs) to follow the embryonic expression of these genes.

To provide a basis for understanding RDGN expression patterns, we developed a staging system for A. longisetosus head development. This showed the presence of structures that in other arachnids form neural components of all eye types. We then showed that two genes in the RDGN of eyed arachnids, i.e., sine oculis and atonal, are expressed in a manner that are suggestive of vestigial eye primordia. We also found that the expression of the Pax6 paralogs was consistent with their roles in the development of the central nervous system. By co-staining for these genes with the conserved head-patterning gene orthodenticle, we observed early expression patterns of these genes in the brains of early A. longisetosus embryos that are comparable to those arachnids with embryonic eyes.

Our data provide support for the hypothesis that the retention of Pax6 genes in A. longisetosus is due to their non-eye patterning roles. Furthermore, our survey of RDGN gene expression also provides support that A. longisetosus patterns vestigial eye primordia. Lastly, our data suggest that the Pax6 genes, with orthodenticle, acts to specify the ancestral arachnid brain. We then discuss our results considering eye loss in other arachnids.

The online version contains supplementary material available at 10.1186/s13227-025-00245-7.