Developmental analysis of the cone photoreceptor-less little skate retina

  1. Chetan C. Rangachar
  2. Denice D. Moran
  3. Mark M. Emerson

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Abstract

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.

Article activity feed

  1. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/17933328.

    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.

  2. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/17942131.

    Summary:

    This manuscript investigates retinal development in the cone-less little skate, focusing on how Onecut1 (OC1) and cone phototransduction genes might underlie the absence of cones. The central question, as I interpret it, is: Does the loss of cones in little skate arise from changes in OC1 function/regulation and/or absence of cone-specific genes, and what does this reveal about photoreceptor evolution? 

    The authors combine embryonic histology, electroporation, bulk RNA-seq, and molecular assays to explore the regulation of OC1 - a transcription factor central to cone specification in other vertebrates. They identify a novel OC1 splice isoform (LSOC1X2) containing a 48-amino-acid "spacer" sequence between its CUT and homeodomain regions, which is developmentally regulated and capable of activating a ThrbCRM1 reporter in a mouse retina assay. The study suggests that alternative OC1 splicing may underlie the skate's cone-loss phenotype while preserving partial cone-like functional traits in rods.

    Overall, the paper presents a creative molecular characterization of an evolutionarily informative model. The combination of comparative genomics, functional assays, and careful developmental staging adds novelty to our understanding of retinal evolution. However, some of the core conclusions, especially those connecting the OC1 isoform and the developmental data to cone loss - remain indirect. In several cases, the methods generate rich descriptive data but do not yet fully answer the central mechanistic question. With additional functional controls in skate, cell-type–resolved expression data, and clarified narrative structure, the manuscript could make a much stronger and more coherent contribution to developmental and evolutionary biology.

    Major Comments

    1. Functional significance of LSOC1X2 would benefit from a deeper interpretation - Both Onecut1 isoforms activate ThrbCRM1 in the mouse retina (Fig. 4), suggesting that the spacer insertion does not overtly disrupt canonical Onecut1 function. However, to improve reader understanding, the authors could consider clarifying what mechanistic or evolutionary advantage the spacer might provide, and how its developmental regulation could influence OC1 activity - for example, by altering cofactor-binding preferences, protein stability, or DNA-binding kinetics. A concise discussion outlining these plausible mechanisms would significantly strengthen the interpretation, even if the authors are not able to perform additional experiments. That said, functional tests (such as assessing whether the spacer modulates cofactor interactions or affects OC1 kinetics) would further support the proposed roles, should the authors choose to pursue them. Without such contextualization, it remains difficult to link this isoform to potential developmental or evolutionary roles.

    2. It may help readers if the interpretation of the ThrbCRM1 reporter results were supported by some validation within the skate system. Showing that the reporter can be activated in skate by introducing mouse OC1 and OTX2, followed by skate OC1 with or without the spacer together with skate OTX2, would confirm that the assay context is permissive. Including a second OC1/OTX2-responsive enhancer as an orthogonal control would further ensure that any observed differences are biological rather than reporter-specific.

    3. The RNA-seq data are bulk, so cell-specific differences in Onecut1 expression are inferred but not demonstrated. The Discussion notes this limitation, but it would strengthen the paper to add either in situ hybridization, single-cell validation, or at least computational deconvolution to identify likely expressing cell types for OC1, OTX2, Thrb/TRβ2, RXRγ, CRX, and NRL.

    4. The alphafold models are visually appealing but lack quantitative metrics (e.g., confidence scores). Including these and clarifying whether differences between isoforms are within confidence thresholds would make this analysis more convincing.

    Minor Comments

    1. Figure 1 labeling: Fig. 1A–E currently lacks clear labels; adding channel labels, and staging info on the figure would help readers interpret the developmental characterization.

    2. Introduction context: A brief paragraph summarizing the established roles of OC1 and OTX2 in cone and horizontal cell specification in chick/mouse would orient readers who are less familiar with this literature and clarify why these factors were chosen as candidates in skate.

    3. The manuscript would benefit from clearer signposting of the question ("We ask whether…"), a short explanation of the OC1–OTX2–ThrbCRM1 framework for readers outside this niche, and a more linear connection between "question → experiment → result → interpretation." Tightening paragraphs, reducing method details in the Results (with cross-references to Methods), and explicitly indicating why each experiment was done would significantly improve readability without changing the underlying science.

    4. Nomenclature and formatting: Consistent formatting for genes vs. proteins, italicization of species names, and uniform use of "ThrbCRM1" will give the manuscript a more polished presentation.

    5. Supplementary figures: Short, explicit captions describing how each supplemental figure supports specific main-text claims (e.g., "supports Fig. 2 by…") would guide readers through the additional analyses.

    Competing interests

    The authors declare that they have no competing interests.

    Use of Artificial Intelligence (AI)

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

  3. Version published to 10.1101/2025.07.30.667746 on bioRxiv