Developmental analysis of the cone photoreceptor-less little skate retina

The retinal development of elasmobranchs--the superclass comprising sharks, skates and rays--is a poorly understood phenomenon. The clade is diverse in retinal phenotypes, with many sharks and rays possessing rods and multiple cone types. In contrast, the little skate ( Leucoraja erinacea ) has only a single type of rod photoreceptor, which is reported to have taken on some physiological and anatomical properties of cones. How does the little skate develop this photoreceptor system? To investigate this question, we identified an early stage of embryonic photoreceptor formation based on Otx2 expression. To assess whether cone photoreceptor gene regulatory networks regulated by the Onecut1 transcription factor were intact we developed an electroporation approach. Activation of a Onecut1-dependent reporter was not detected supporting a change in early Onecut1-transcriptional networks associated with cone photoreceptor formation. To assess developmental changes in gene expression, bulk RNA-Seq samples of embryos, juvenile hatchlings and adult retinas were generated. As expected, gene expression changes associated with cone photoreceptors of other species were not identified in the little skate and several cone-enriched genes were found to be pseudogenes. A splice isoform of Onecut1 with an additional 48 amino acid sequence located between the CUT and Homeodomain DNA-binding domains – referred to as “spacer isoform” or LSOC1X2 was identified and exhibited high expression in the embryo compared to juvenile and adult stages. To test if this LSOC1X2 isoform retained its regulatory potential compared to the isoform without the additional sequence, we tested it in a mouse retina reporter assay. Both skate Onecut1 isoforms were able to activate the Onecut1-dependent transcriptional reporter. Thus, the spacer isoform represents a developmentally regulated, novel retinal Onecut1 isoform with regulatory potential. This identifies it as a target for further analysis in the retinal development of the little skate and its elasmobranch relatives.