PREreview del Developmental analysis of the cone photoreceptor-less little skate retina

PREreview of “Developmental analysis of the cone photoreceptor-less little skate retina”

Summary:

In this preprint, the authors investigate the developmental mechanisms underlying the uniquely rod-only retina of the little skate, an unusual exception within the otherwise diverse retinal phenotypes of elasmobranchs. Motivated by the open question of how the little skate develops a photoreceptor system lacking cones - despite cones being present in many related sharks and rays - the authors combine transcriptomic, gene-expression, and functional assays to test whether cone-associated regulatory networks, particularly those controlled by Onecut1, are conserved or altered in this species. They identify a novel Onecut1 splice isoform containing a 48–amino acid spacer and examine its regulatory activity using AlphaFold3 modeling and mouse retina electroporation. By linking this isoform to disrupted cone-associated pathways and developmental gene-expression changes, the work provides new insight into regulatory diversification and the evolutionary modification of photoreceptors in elasmobranchs.

Major Points:

•       Cross-species functional assay interpretation: The use of mouse retina electroporation with the mammalian ThrbCRM1::GFP reporter to infer developmental roles of skate Onecut1 is problematic, as the heterologous context limits the biological conclusions that can be drawn. The assay demonstrates that the skate proteins can engage mammalian transcriptional machinery, but it does not necessarily reflect in vivo regulatory function in the skate retina. The authors could explicitly state that this is a heterologous assay testing biochemical capacity rather than developmental function. Ideally, adding a skate-specific validation (even partial) would more directly connect the isoforms to endogenous regulatory pathways and thus significantly strengthen the conclusions.

•       AlphaFold3 modeling: The underlying assumptions for the modeling of a multi-protein–DNA complex that includes a disordered region (the spacer exon) are neither explained nor justified in the manuscript. The authors introduce a specific stoichiometry (two Otx2 molecules and one Onecut1 molecule) without describing why these components were chosen, whether this arrangement is supported by prior literature, or how the parameters were defined during model construction. As a result, the reader cannot evaluate whether the model reflects a biologically plausible scenario or simply an arbitrary configuration. To help, the authors should clearly articulate the rationale for their modeling assumptions, describe the parameters used, and acknowledge the limitations inherent to applying structure prediction to a disordered domain. Experimental validation (e.g., EMSA, pulldown, or binding assays) would provide stronger support; alternatively, the modeling could be explicitly framed as hypothesis-generating rather than as evidence for a specific molecular interaction.

•       Title: The current title, “Developmental analysis of the cone photoreceptor-less little skate retina”, gives the impression that the paper will provide a broader morphological and developmental characterization of the little skate retina. However, the study is primarily focused on Onecut1 isoform expression and characterization. To better reflect the main findings, I would suggest adjusting the title accordingly, as the major contribution of this work is the identification and functional characterization of two distinct Onecut1 isoforms expressed during retinal development.

Minor Points:

•       Introduction: The introduction contains extensive general background before clearly presenting the central question. Condensing this section and sharpening the framing would help guide the reader toward the study’s motivation and significance.

•       Results organization: The results section often shifts between methods, background, and data interpretation without clear transitions (e.g., in the discussion of Fig. 2, the text jumps from panel D to B/C). Reorganizing the results into clear thematic blocks - each ending with a short summary paragraph linking findings back to the central question - would greatly improve readability and logical flow.

•       Discussion: The discussion currently includes extended sections on Onecut1 functions in other organs (e.g. pancreas, liver), which masks reader's understanding of its role in the retina. These could be shortened or refocused to emphasize how the present results advance understanding of Onecut1 evolution and function in the skate retina. The discussion would also benefit from a more explicit summary of the main findings, acknowledgment of limitations, and a clearer statement of how these results contribute to our understanding of photoreceptor evolution.

•       Figure 1: Move extensive electroporation controls (Fig. 1G–K) to Supplement and keep only the essential ThrbCRM1 reporter validation in the main figure, because including the controls in Fig. 1G-K interrupts the flow of the main narrative. Add scale bars, annotate gene names on images, and choose brighter representative images for clarity.

•       Figure 2: Present panels A and B side-by-side; choose a color palette with more variety (not only shades of blue/red) to improve curve distinction. Explain why a human (Fig. 2D) was included in comparative analyses. Validate RNA-seq changes for key genes with qRT-PCR, HCR, or Western blot if possible, as it would strengthen the robustness of the conclusions.

•       Figure 4: For the electroporation data, include arrows (or other markers) clearly in the images if you refer to them in the text. Define abbreviations (e.g., “OC1”) upon first use.

•       Textual adjustments: Move detailed methodological descriptions from results to the methods section, as their current placement in the main text overwhelms the narrative and makes it difficult for readers to clearly follow and understand the results.

Competing interests: None declared.

Competing interests

The author declares that they have no competing interests.

Use of Artificial Intelligence (AI)

The author declares that they did not use generative AI to come up with new ideas for their review.