An RNA ligase shapes transcriptional profiles, neural function, and behaviour in the developing larval zebrafish

  1. Fiona S. Klusmann
  2. Anna C. Kögler
  3. Katja Slangewal
  4. Onur Önder
  5. Heike Naumann
  6. Andreas Marx
  7. Armin Bahl
  8. Patrick Müller

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Abstract

RNA ligases are essential for the repair, splicing, and editing of RNA across various biological systems. Recently, a new enzyme that catalyses 5’-3’ RNA ligation – RNA ligase 1 (Rlig1) – was identified in vitro . However, the in vivo biological functions of Rlig1 have remained elusive. Here, we reveal the role of Rlig1 during vertebrate development using embryonic and larval zebrafish as a model system. We found that rlig1 mRNA is maternally deposited and present ubiquitously during early embryogenesis, whereas at larval stages it localises to the brain and eyes. Interestingly, CRISPR/Cas9-generated rlig1 knockout zebrafish exhibited no overt morphological abnormalities, but showed reduced behavioural responsiveness to visual stimuli along with massively perturbed transcriptomes and widespread dysregulation of core metabolic and translational pathways. Brain-wide calcium imaging in rlig1 knockout larvae revealed decreased neuronal activity in key regions for visual processing, consistent with the observed behavioural defects. Together, our findings identify a role for Rlig1 in maintaining the integrity and function of the nervous system and uncover a new link between neuronal RNA processing, development, and sensory-motor computation.

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    Response to Reviewer's Comments

    We thank the reviewers for their careful, constructive, and encouraging assessment of our manuscript. As described in detail in the point-by-point response below, we have extensively revised the manuscript and Supplementary Information. Together, these changes provide further support for the role of Rlig1 in neural function and visually guided behaviour during zebrafish development.

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    Summary: Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate).

    This study characterizes the function of RNA ligase 1 (Rlig1) in the vertebrate model zebrafish. Rlig1 is one of only two known RNA ligases in vertebrates, and its biological roles remain poorly understood. The authors combine gene expression analysis, loss-of-function approaches, transcriptomic profiling, calcium imaging, and behavioral assays to investigate its function during development. They show that loss of rlig1 (including maternal-zygotic loss) has no major effects on development or morphology, but that it leads to impairments in visually-guided behavior and altered neuronal activity in response to visual stimuli. Transcriptomic analyses reveal widespread dysregulation across multiple developmental stages, nominating genes that may underly the observed neural phenotypes. Together, the findings support a role for Rlig1 in neural development and function in vertebrates.

    We thank the reviewer for this accurate and positive summary of our study and for recognising the complementary, multi-level approaches used to examine the in vivo role of Rlig1.

    Major comments:

    • Are the key conclusions convincing?

    The key conclusion of this study is that Rlig1 plays an important role in the development and function of vertebrate neural circuits. Overall, this overarching conclusion, as well as the individual conclusions from each set of experiments, are well supported by the data presented. The combination of tissue-specific expression of rlig1, robust behavioral phenotypes in mutants, transcriptomic changes across multiple developmental stages, and circuit differences observed through calcium imaging provides a coherent, multi-faceted argument for the importance of this enzyme in brain development and function. While the precise RNA substrates of Rlig1 and the mechanistic link between transcriptomic changes and neural phenotypes remain to be defined, the authors clearly acknowledge these next steps and limitations. This study is a critical foundation for those future experiments.

    We appreciate the reviewer’s positive assessment of the strength and coherence of the evidence.

    • Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

    The claims in the manuscript are generally well-supported. The authors clearly acknowledge limitations and future experiments to further dissect mechanism in the Discussion section.

    • Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

    No major additional experiments appear essential for supporting the current claims.

    • Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

    No experiments are required for the current claims of the manuscript.

    We thank the reviewer for this assessment.

    • Are the data and the methods presented in such a way that they can be reproduced?

    The methods are generally well described. I would suggest that the "raw images, data, and source code for custom scripts used in this work" be made accessible without having to request from the authors. Zenodo provides up to 50 GB of storage, which is likely sufficient for the data presented in this manuscript. In particular, I think it is important to share the behavior analysis, calcium imaging pipeline, and transcriptomics analysis. Even if all the data is too large, a sample dataset and analysis scripts should be publicly available.

    We agree and thank the reviewer for this important suggestion. To ensure that the study can be reproduced without the need to contact the authors, we have made the underlying data and custom analysis code publicly accessible. The RNA-seq data have been deposited in the GEO repository under accession number GSE308510 and are available at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE308510.

    In addition, the raw imaging data, behavioural and calcium-imaging datasets, processed data, and custom scripts used for the behavioural, calcium-imaging, as well as the tRNA and rRNA sequencing data have been deposited on KonDATA (DOI: 10.48606/vpwgm69277srrgaj) – together more than 190 GB – and can be accessed using this link: https://kondata.uni-konstanz.de/radar/en/dataset/vpwgm69277srrgaj?token=gLEaYEENHjmHBhjhHUHK.

    We have revised the Data and code availability statement in the manuscript accordingly.

    • Are the experiments adequately replicated and statistical analysis adequate?

    The experiments appear adequately replicated, and statistical analyses are appropriate for the types of data presented.

    We thank the reviewer for this positive assessment. To further improve transparency, we have revised the figure legends and Methods to define sample-size notation consistently throughout the manuscript. As suggested by Reviewer 3, we now distinguish biological replicates or independent experiments (N) from individual embryos, larvae, cells, imaging planes, or trials (n), as appropriate.

    Minor comments:

    • Specific experimental issues that are easily addressable.
    • Are prior studies referenced appropriately?
    • Are the text and figures clear and accurate?
    • Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

    Throughout the manuscript: use the prime symbol for 5/3 DNA/RNA instead of an apostrophe. The prime symbol is present in a small number of sentences, but mostly the apostrophe is used.

    We thank the reviewer for noting this. We have replaced apostrophes with prime symbols throughout the manuscript to ensure consistent notation of 5′ and 3′ RNA/DNA termini.

    Line 227: "Next, we compared the total number of neurons". The elavl3 driver labels brain cells in addition to neurons.

    • The authors compared to the total number of brain cells, but can they make any comments on the size of the brain across the various areas? I imagine this data is also accessible by analyzing the imaging already collected.

    The elavl3 promoter is widely used as a pan-neuronal driver in zebrafish. Our calcium-imaging experiments used the Tg(elavl3:H2B-GCaMP8s) line, in which nuclear-localised GCaMP8s is expressed under the control of the elavl3 regulatory region. This established configuration enables brain-wide functional imaging of neuronal activity in larval zebrafish.

    To assess whether differences in regional brain size might contribute to the observed phenotype, we quantified brain dimensions in 5 dpf larvae using the existing imaging data. Measurements were performed manually in Fiji in a blinded manner, with genotypes assigned only after completion of the analysis. We quantified tectum width, hindbrain width, and tectum length, as illustrated in the new Supplementary Figure 6.

    MZrlig1 larvae showed a modest reduction in tectum width (MZrlig1: 299 ± 14 µm; WT: 312 ± 10 µm; one-sided t-test, p = 0.00125) and tectum length (MZrlig1: 122 ± 5 µm; WT: 134 ± 9 µm; one-sided t-test, p = 1.03 × 10⁻⁵). In contrast, hindbrain width did not differ between genotypes (MZrlig1: 164 ± 10 µm; WT: 164 ± 10 µm; one-sided t-test, p = 0.52). Following assessment of data distribution, statistical significance was evaluated using one-sided t-tests with Bonferroni correction for three comparisons (n = 18 MZrlig1 and n = 20 WT larvae).

    Importantly, the unchanged hindbrain width indicates that the reduced number of motion-responsive hindbrain neurons in MZrlig1 larvae is unlikely to be explained by a gross difference in hindbrain size. These findings therefore support our interpretation that Rlig1 loss is associated with reduced neuronal responsiveness in the hindbrain.

    Given that there is already a mouse mutant for this gene and transcriptomics, can the authors do a more thorough job comparing the transcriptomics from that study with their own?

    We thank the reviewer for this helpful suggestion. When we applied the differential-expression thresholds used in our zebrafish analysis (absolute log₂ fold change ≥ 1.5 and adjusted p value ≤ 0.05) to the genes reported in the mouse study, only flg2 met these criteria. Thus, the available mouse dataset provides limited scope for a direct gene-by-gene comparison with our data.

    To extend our analysis beyond poly(A)-enriched mRNA sequencing, we additionally performed tRNA and rRNA sequencing using total RNA from 5 dpf WT and MZrlig1 larvae. The tRNA analysis identified 17 significantly altered tRNAs in MZrlig1 larvae, including seven upregulated and ten downregulated species (Figure 5i; Supplementary Tables 8–9). Notably, the affected tRNAs include tRNA-Lys-CTT, which was previously identified among RNAs enriched in human Rlig1 immunoprecipitates, and tRNA-Thr-CGT, which was reported to be increased in female rlig1 knockout mouse brains. Although the direction of change is not fully conserved across these studies, these overlaps further support the possibility that Rlig1 influences tRNA homeostasis.

    In parallel, rRNA sequencing revealed differential abundance of 122 5S rRNA transcripts, with 86 upregulated and 36 downregulated in MZrlig1 larvae (Figure 5h; Supplementary Tables 10–11). Together, these new analyses show that loss of Rlig1 is associated with altered abundance of both tRNA and rRNA species, consistent with previous evidence linking Rlig1 to RNA homeostasis. At the same time, we explicitly state that these data do not identify direct enzymatic substrates of Rlig1, but provide a resource and rationale for future mechanistic studies.

    A clearer statement on the similarities and differences of Rlig1 and RtcB would be helpful. Is it possible RtcB is compensating at all?

    We thank the reviewer for this comment. We have clarified the similarities and differences between Rlig1 and RtcB in the Introduction and Discussion. Although both enzymes catalyse RNA ligation, they act on distinct end chemistries. RtcB mediates 3′–5′ ligation of RNA ends generated during canonical tRNA splicing, joining a 5′-hydroxyl end to a 2′,3′-cyclic phosphate or 3′-phosphate end. In contrast, Rlig1 catalyses 5′–3′ ligation of RNA fragments bearing a 5′-phosphate and a 3′-hydroxyl group.

    These distinct substrate requirements make direct functional compensation by RtcB unlikely. RNA ends generated for ligation by Rlig1 would first require end processing to generate termini compatible with RtcB-mediated ligation. Nevertheless, indirect compensation or partial functional overlap after such processing cannot be excluded.

    We sought to address this question experimentally by obtaining rtcb mutants from the European Zebrafish Resource Center. However, subsequent genotyping showed that the supplied sperm did not contain the intended rtcb mutant alleles, precluding analysis in the present study. We have therefore explicitly acknowledged that the extent to which RtcB may compensate for loss of Rlig1 remains unresolved and will require analysis of validated rtcb mutant lines in future work.

    I examined the DEG tables, and I did not notice an obvious substantial enrichment of genes on chromosome 25 (White et al., 2022, https://doi.org/10.7554/eLife.72825). Were the different samples from different clutches or the same clutch? I may have missed it. Regardless, I would carefully check the DEGs that are important for conclusions and check that they are not on the same chromosome as rlig1. It is likely worth rerunning all of the GO/GSEA with genes on chromosome 25 excluded.

    We thank the reviewer for raising this potential confound. The RNA-seq samples were derived from independent clutches. To determine whether the observed transcriptional changes could be influenced by local effects associated with the rlig1 locus on chromosome 25, we performed two complementary analyses.

    First, we examined the chromosomal distribution of differentially expressed genes (DEGs) at each developmental stage. The chromosomal distribution was assessed using the original DEG analysis presented in the manuscript (no pre-filtering before DESeq2; DEGs defined as padj 1). Chromosome 25 contains 806 of 25,254 annotated protein-coding genes in the zebrafish genome, corresponding to 3.2% of all coding genes. Across developmental stages, the proportion of DEGs located on chromosome 25 ranged from 1.4% to 4.1% (cleavage: 12/419; sphere: 17/; shield: 37/892; bud: 26/781; 1 dpf: 3/216; 5 dpf: 8/587). Relative to the genomic expectation, this corresponds to enrichment values between 0.43- and 1.30-fold. Only the shield stage showed a modest increase in the proportion of chromosome 25 DEGs (1.30-fold), whereas all other stages were at or below the genomic expectation. Thus, genes on chromosome 25 are not globally overrepresented among the DEGs in the rlig1 mutant dataset.

    Second, we repeated the complete differential-expression analysis for each developmental stage after excluding all chromosome 25 genes before DESeq2 normalisation, size-factor estimation, and dispersion modelling. This re-analysis was performed using an updated workflow, including removal of genes with zero total counts prior to DESeq2, which changes the number of genes entering Benjamini–Hochberg correction and consequently the total number of detected DEGs; all other analysis parameters were identical to the original analysis. This approach ensured that chromosome 25 genes could not influence either normalisation or statistical inference for genes on other chromosomes. Using the same DEG thresholds as in the original analysis (padj 1), exclusion of chromosome 25 had only minimal effects on the remaining DEG sets.

    Stage

    Full DEGs

    Non-Chr25 DEGs

    Lost (Chr25)

    Lost (non-Chr25)

    Gained

    1 (4-cell)

    419

    415

    5

    0

    1

    2 (Sphere)

    913

    879

    34

    5

    5

    3 (Shield)

    592

    553

    37

    5

    3

    4 (Bud)

    349

    329

    20

    0

    0

    5 (1 dpf)

    7

    6

    1

    0

    0

    6 (5 dpf)

    168

    164

    4

    0

    0

    Across all six developmental stages, only ten non-chromosome-25 genes lost significance and nine genes gained significance. These minor changes were confined largely to the sphere and shield stages, which also showed the highest relative representation of chromosome 25 DEGs. At the 4-cell, bud, 1 dpf, and 5 dpf stages, no non-chromosome-25 genes lost significance after chromosome 25 was excluded.

    We also repeated the GO and GSEA analyses after excluding chromosome 25 genes. As expected, a small number of individual terms changed; however, the principal enrichment patterns and overall biological interpretation remained unchanged. Together, these analyses indicate that the transcriptomic phenotype is not substantially driven by chromosome 25-linked DEGs or by local effects associated with the edited rlig1 locus. While this analysis cannot exclude effects on individual linked genes, it shows that such effects do not substantially affect the main transcriptional or pathway-level conclusions of the study.

    **Referees cross-commenting**

    I missed the point about the RNA-seq samples being cousin-matched. While I am optimistic that the results won't change, I agree with Reviewer #3 that some confirmation is necessary. It was unclear to me whether the samples were from the same or different clutches - if they are from different clutches and share overlapping genes, that would also add support to the results. I think that detail was missing from the methods, and I had pointed it out. Either additional RNA-seq or even qPCR of some top genes from a heterozygous incross is a reasonable request.

    We thank the reviewer for raising this point and apologise that the breeding design for the transcriptomic experiments was not described sufficiently clearly. The developmental RNA-seq samples were not cousin-matched. Rather, WT and MZrlig1 embryos were collected from separate group matings and therefore originated from different clutches. Independent pooled samples were analysed at each developmental stage, as now described explicitly in the revised Methods.

    We agree that independent validation in a sibling-controlled genetic setting is important. We therefore performed RT-qPCR for eight genes selected from the 5 dpf mRNA-seq dataset using sibling-matched zygotic rlig1 mutants and WT larvae generated by heterozygous incrosses. For each genotype, three independent biological replicates were analysed, with four larvae per sample. Six of the eight selected genes showed changes in the same direction as in the original MZrlig1 RNA-seq dataset: cyp2p9, itln3, sult3st4, fabp7b, hamp, and rlig1 itself. In particular, itln3 remained strongly upregulated, whereas rlig1 expression was markedly reduced in the sibling-matched zygotic mutants. In contrast, gdf3 and gstp1.1 did not show the same directional change in this validation experiment.

    These results provide independent support that several of the transcriptional changes identified in the MZrlig1 RNA-seq dataset are also observed in sibling-matched zygotic mutants. At the same time, the incomplete concordance of individual genes is consistent with the fact that maternal-zygotic and zygotic mutants represent biologically distinct conditions and may differ in both effect size and molecular consequences. We have added these validation data as Supplementary Figure 7 and revised the Results and Methods accordingly.

    Reviewer #1 (Significance (Required)):

    • Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

    This study provides a conceptual and biological advance by identifying a role for a vertebrate RNA ligase in brain development, behavior, and transcriptional regulation.

    • Place the work in the context of the existing literature (provide references, where appropriate).

    Although RNA ligases from single-cell organisms and phage are well-characterized, the roles of RNA ligases in vertebrates are relatively understudied. There are only two, including the one the one that is the focus of this manuscript. This study demonstrates an in vivo function for Rlig1, linking molecular changes to neural development and function. The Rlig1 enzyme was only very recently discovered (2023), making this work timely and an important addition to an area with relatively few studies.

    A major strength of the study is its multi-level approach, integrating diverse techniques to coherently link this gene to organism-level phenotypes. This work provides a strong conceptual and functional advance by demonstrating a role for Rlig1 in vertebrate neural circuit function and behavior. A remaining mechanistic gap is that the direct RNA substrates of Rlig1 are not identified, and the observed transcriptomic changes in mRNA are likely downstream consequences of its loss. However, these points are clearly acknowledged in the discussion, making the study a well-balanced contribution. Given the existence of a mouse knockout model, further discussion comparing the zebrafish transcriptomic results and phenotypes to those observed in mouse would help place this work in the context of prior studies. Overall, the main conclusions are well supported, and the limitations do not undermine them. This study represents an important contribution that establishes a foundation for future mechanistic work linking Rlig1 substrates to the observed phenotypes.

    We thank the reviewer for this thoughtful and encouraging assessment.

    • State what audience might be interested in and influenced by the reported findings.

    Zebrafish basic science researchers, particuarly those studying how genes lead to altered neural circuits and behavior, are the most direct target audience. However, the work is of more broad interest to those in the fields of neurodevelopment, gene regulation, and RNA biology / processing.

    • Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

    I am comfortable evaluating zebrafish mutants, transcriptomics, and behavioral assay design. I have more limited experiment in neural circuit anaysis and interpretation of calcium imaging data, though this part of the manuscript was also clearly presented and understandable.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    Summary Klusman et al have investigated the function of the RNA ligase rlig1 in zebrafish. They first document expression of the gene, by quantitative RT-PCR and HCR-fluorescent in situ hybridization. They then test ligase activity of the Rlig1 protein in vitro. They next generate a null mutant and test function of the visual system using behaviour as well as calcium imaging. The data indicate that rlig1 is broadly expressed and capable of ligating RNA; loss of rlig1 has mild effects on overall development and pronounced effects on behavioural and neuronal response to visual stimuli. Finally, the authors use bulk transcriptome analysis to identify changes in gene expression in the mutants.

    We thank the reviewer for this accurate summary of our study and for recognising that the behavioural and calcium-imaging results together support a role for rlig1 in visual processing and visually guided behaviour.

    **Referees cross-commenting**

    I agree that more details are required about the crosses would be useful.

    We also agree that further detail on the breeding schemes is important. We have therefore expanded the Methods and figure legends to describe the crosses used for each experiment, including the relationship between mutant and control animals and whether samples were sibling- or cousin-matched.

    Reviewer #2 (Significance (Required)):

    Overall, the conclusions that rlig1 is required for normal development of the embryo, especially of a fully functioning visual system, are well supported. The optomotor response experiments have high power and, together with functional imaging, show a clear difference between mutant and wildtype.

    One limitation of this manuscript is in the characterization of gene expression. The gene expression database in Zfin contains one image of rlig1 (https://zfin.org/ZDB-IMAGE-060710-1925#image), which shows broad expression in cells of the embryo and larvae and no expression in the yolk. The images here, with the exception of the mutant in Figure 3C, show expression in the yolk. This would suggest that the yolk signal is not autofluorescence, which is inconsistent with the Thisses' data. Additonally, Figure S1 indicates a variable level of non-specific signal, especially in panel g. Thus, the distribution of rlig1 mRNA is unclear.

    We agree that the yolk-associated signal should not be interpreted as specific rlig1 expression.

    rlig1 transcripts are completely absent from the RNA-seq datasets of MZrlig1 mutants at all developmental stages analysed. Thus, the variable fluorescence observed in the yolk and in the no-probe controls (Supplementary Figure 1) cannot represent residual rlig1 expression, but must reflect non-specific background signal and/or autofluorescence. We have clarified this point in the revised manuscript.

    The transcriptome analysis identified changes in gene expression in the mutant. This establishes a role for rlig1 in development, and identifies several processes that are disrupted by loss of rlig1. However, the molecular analysis sheds little light on direct targets of the ligase. Given the established effects on tRNA, for example, it is unclear why RNA was analysed only by short reads on poly(A) RNA. The reader is left wondering whether zebrafish tRNA contains introns that require Rlig1 for processing. In this context, it would be useful for the authors to provide more background on tRNA splicing in vertebrates, including a mention of tricRNA, and potentially the role of TSEN complex in brain development.

    We have expanded the Introduction as suggested to provide additional context on tRNA splicing in vertebrates. We now explain that canonical tRNA splicing is initiated by the TSEN complex and completed by RtcB, which ligates RNA ends with chemistries distinct from those used by Rlig1. We also discuss that excised tRNA introns can form stable tRNA intronic circular RNAs (tricRNAs), and that defects in TSEN complex components are associated with neurodevelopmental disorders, underscoring the importance of RNA processing for nervous-system development.

    We agree that our poly(A)-enriched RNA-seq data do not identify direct RNA substrates of Rlig1. We have clarified throughout the manuscript that these experiments were designed to characterise downstream transcriptional consequences of rlig1 loss.

    We have additionally analysed tRNA and rRNA abundance in total RNA from 5 dpf WT and MZrlig1 larvae. These analyses identified altered levels of specific tRNA and 5S rRNA species in MZrlig1 larvae (Figure 5h,i; Supplementary Tables 8–11), supporting an association between Rlig1 loss and altered RNA homeostasis.

    To summarize, this manuscript extends work in the mouse and in cell lines that demonstrate a requirement for rlig1. It does not shed light on direct targets of Rlig1, but provides a strong foundation for future work on the role of RNA ligation in vertebrate development and brain function.

    This paper is expected to be of interest to a specialised audience.

    Minor points: The images showing gene expression in Figure 2 are not easy to see, due to the LUT used and low intensity of the signal. To aid the reader, the HCR channel should be shown in grayscale, possibly with the contrast enhanced (to the same extent in all images).

    To improve the visibility and interpretation of the HCR signal, we have added a new Supplementary Figure 2 showing the *rlig1 *channel in greyscale. Within comparable developmental-stage panels, identical contrast settings were applied to all images.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    Summary This paper provides good evidence that a newly described enzyme that catalyzes 5'-3' RNA ligation - rlig1 - plays some role in early vertebrate neurodevelopment. Using embryonic and larval zebrafish as a model, they found that, while rlig1 mRNA is highly maternally deposited and ubiquitously expressed early on, expression later in development localizes to the brain and eyes. They generated a stable CRISPR/Cas9 large deletion mutant spanning from upstream the 5'UTR past the start codon. By comparing wild type and maternal-zygotic (MZ) rlig1 mutants, the authors found that animals developed overtly normally but did show reduced behavioral responsiveness to a visual stimulus experimental paradigm. By combining calcium imaging and poly(A)-enriched RNA-sequencing transcriptomic analyses, they found that there was decreased neuronal activity in regions needed for visual processing, and that there was dysregulation of neural-related gene networks and metabolic and translational pathways.

    We thank the reviewer for this detailed and accurate summary of our study and for recognising the convergent evidence linking Rlig1 loss to altered neural activity and visually guided behaviour in developing zebrafish.

    Major comments

    1. My main major comment is that, because there is so much inherent variability in behavior and even development across different clutches, this study relies on comparing (cousin-matched) WT and maternal-zygotic rlig1 mutant animals. In most reliable peer-reviewed papers, this is not a fair comparison. While I appreciate that authors stated that they used parents that were siblings (so, offspring would be cousin-matched), I do not consider this scientifically rigorous enough for the claims presented. a. I do not consider it a reasonable request to ignore the massive amount of work that went into this paper using WT and MZrlig1 comparisons. However, at minimum, authors should consider performing essential behavior and RNA-seq (see point b) experiments with heterozygous incrosses of single-pair matings, and genotyping the animals post-hoc. Including this critical data in a main figure, as the basis for using MZ animals for the rest of the paper, would induce some confidence that the phenotypes and claims presented are not a result of inherent variability. If the authors already have adult heterozygous animals of mating age, I estimate that these experiments may be completed very reasonably within 3-4 weeks; if new animals need to be generated, this request would take ~4 months. Typically, these kinds of experiments would not be considered a financial burden to perform.

    Our central genetic condition was maternal-zygotic loss of rlig1, motivated by the strong maternal deposition of rlig1 mRNA during cleavage stages. A heterozygous incross would produce zygotic mutants that still receive maternal rlig1 transcript and protein, and would therefore test a related but biologically distinct condition. For the maternal–zygotic experiments, we used cousin-matched WT controls derived from the same parental family to minimise genetic-background differences, and we performed the behavioural assays with substantial numbers of larvae across independent experiments.

    We nevertheless repeated the behavioural analysis as suggested using zygotic rlig1 mutants and WT sibling controls obtained from heterozygous incrosses. This analysis revealed a qualitatively similar, although less pronounced, reduction in visually guided behaviour in zygotic mutants (new Supplementary Figure 4). We speculate that the reduced effect size is consistent with partial compensation by maternally supplied rlig1 transcript or protein in zygotic mutants.

    b. For transcriptomic analyses, I have two main points: i) again, it is difficult to statistically rigorously compare transcriptomes of nonsibling-matched animals with such low numbers of single 5 dpf brains. In line with point a, it would be essential to pool at least a few WT and rlig1 mutant siblings for at least 3 biological replicates per samples and compare those analyses with the results from MZ animals. ii) Typically this would not be a major concern, however given the nature of the gene of interest and published in vitro findings, I do consider that the rlig1 enzyme catalyzes 5'-3' RNA ligation, has been shown to be implicated in rRNA integrity and tRNA targeting, and is broadly essential for repair, splicing, and editing of RNAs. Thus, while the poly(A)-enriched RNA sequencing can provide context about gene networks that are affected (either primarily or secondarily), sequencing that enriches for tRNAs, polysome profiling or ribosome profiling, or some more targeted sequencing approach would be more appropriate to more rigorously support the claims in the paper. Depending on readiness of mating-age animals, this experiment and analyses may reasonably take up to 3 months; this approach may be considered a financial burden. Alternatively, with the current mRNA sequencing, the authors could delve into whether they can identify altered splicing or RNA editing dynamics in different RNA modules. I estimate that this alternative analysis approach may take up to one month to develop and interpret.

    We would like to clarify that the poly(A)-enriched RNA-seq was not performed on single 5 dpf brains, but on independent pools of 8–10 age- and genotype-matched whole embryos or larvae collected across six developmental stages. We have also validated eight selected 5 dpf RNA-seq candidates by RT-qPCR using sibling-matched zygotic rlig1 mutants and WT larvae generated by heterozygous incrosses. For each genotype, we analysed three independent biological replicates, each comprising a pool of four larvae. Six of the eight tested genes showed changes in the same direction as in the original MZrlig1 RNA-seq dataset, including cyp2p9, itln3, fabp7b, hamp, sult3st4, and rlig1 (new Supplementary Figure 7). Although zygotic mutants are not equivalent to maternal–zygotic mutants because they retain maternally supplied rlig1 transcript and protein, these results provide independent support for a substantial subset of the transcriptional changes identified in the MZrlig1 dataset. We have revised the Methods, Results, and Discussion to describe the breeding schemes and this limitation more explicitly.

    We also agree that poly(A)-enriched RNA-seq alone cannot identify direct Rlig1 substrates or adequately assess non-polyadenylated RNA classes. We therefore added targeted analyses of tRNA and rRNA abundance from total RNA isolated from 5 dpf WT and MZrlig1 larvae. The tRNA analysis identified seven tRNAs with increased and ten with decreased abundance in MZrlig1 larvae, including tRNA-Lys-CTT, previously found among RNAs enriched in human Rlig1 immunoprecipitates, and tRNA-Thr-CGT, which was reported to be increased in female rlig1 knockout mouse brains (Figure 5i; Supplementary Tables 8–9). In parallel, the rRNA analysis identified altered abundance of 122 5S rRNA species, with 86 increased and 36 decreased in MZrlig1 larvae (Figure 5h; Supplementary Tables 10–11).

    These new data provide additional evidence that loss of Rlig1 is associated with altered tRNA and rRNA homeostasis. At the same time, we explicitly state that neither the mRNA-, tRNA-, nor rRNA-seq datasets establish direct enzymatic substrates of Rlig1 or demonstrate altered tRNA splicing, RNA editing, or translation. Direct substrate mapping and analyses such as ribosome profiling will be important directions for future work. The revised manuscript frames the transcriptomic analyses accordingly.

    o The experiments as documented are adequately replicated and statistical analyses adequate (minus the nonsibling-matched point 1). I note that labels should more clearly state or denote individual (n) or experimental (N) numbers, some of which I provide in Minor comments below.

    We agree and have revised the figure legends accordingly. We now distinguish N for independent experiments or biological replicates from n for individual embryos, larvae, imaging planes, segmented cells or trials. Where pooled samples were used, the legends and Methods now state the number of embryos or larvae per pool and the number of independent pools or experiments.

    Minor comments Comments on figures or figure legends:

    1. Figure 1e, align the "#" labels better, they look diagonal.

    Thank you. We corrected the alignment of the labels in Figure 1e.

    1. For 1f, consider labeling independent replicates directly on the graph instead of just the label, otherwise not very clear to the reader.

    We have revised Figure 1f to make the independent replicates more transparent. The figure now clearly indicates the number of independent replicates used for quantification. Every replicate has a different colour now, and N = 3 is indicated in the figure.

    1. Figure 2a, consider adding the reference gene (eef1a) in the legend.

    We have added eef1a to the Figure 2a legend and clarified that relative rlig1 mRNA levels were calculated using eef1a as the reference gene.

    1. Figure 2a - if I understand the experiment correctly, the current label n=3 (which would mean 3 individual embryos/larvae) should read N=3 (three independent experiments of x number of embryos/larvae per run)

    Thank you very much for this suggestion. We have corrected the sample-size notation in Figure 2a. The label now uses N for independent experiments and specifies the number of embryos or larvae used per experiment where appropriate.

    1. Supplementary Figure 1 was very unconvincing comparing WT to MZ mutants, I'm sorry to say I really could not tell much difference. When compared to Figure 3c, they look quite different. The DRAQ7 labeling also appeared uneven in Supplementary Figure 1. Consider optimizing the imaging strategy and providing more interpretably images. A separate, aesthetic comment - magenta was very difficult for me to see against a black background, consider switching the rlig1 channel to grayscale or flip the colors so that rlig1 mRNA is cyan, for example.

    We thank the reviewer for this comment and apologise that the purpose of Supplementary Figure 1 was not sufficiently clear. This figure shows no-probe control samples imaged in the rlig1 detection channel to document stage-dependent background and autofluorescence. Because no rlig1 probe was applied, no genotype-dependent difference between WT and MZrlig1 samples is expected in these images. The variable signal, including the yolk-associated fluorescence, therefore represents background rather than specific rlig1 mRNA detection.

    In contrast, Figure 3c shows samples processed with the rlig1 HCR probe set. The marked reduction of punctate signal in MZrlig1 larvae in this experiment is therefore attributable to the absence of rlig1 transcripts, consistent with the RNA-seq and RT-qPCR data. We have clarified this distinction in the revised text and figure legends.

    The apparently uneven DRAQ7 signal in some no-probe control images reflects differences in embryo orientation and imaging planes rather than genotype-specific staining differences. To improve the visibility and interpretability of the HCR data, we have additionally included a new Supplementary Figure 2 showing the rlig1 channel in greyscale, with matched contrast settings within comparable developmental-stage panels.

    1. Calcium imaging - related to Major comments above, consider performing this experiment in sibling-matched animals, especially with only one copy of the transgene. If WT vs. sibling mutant results look similar to the WT vs MZ mutant results, this would be more convincing.

    We agree that calcium imaging in sibling-matched zygotic mutants would provide a valuable complementary dataset. However, zygotic mutants retain maternally supplied rlig1 transcript and protein and therefore represent a biologically distinct condition from the maternal–zygotic mutants examined in our principal imaging experiments. Consistent with this distinction, the behavioural phenotype in sibling-matched zygotic mutants was qualitatively similar but less pronounced than in maternal–zygotic mutants.

    A sufficiently powered brain-wide calcium-imaging analysis in sibling-matched animals would require generation, imaging, and analysis of a substantial additional cohort, while the expected smaller effect size would limit its ability to directly test the maternal–zygotic phenotype reported here. We therefore believe that this experiment extends beyond the scope of the present study.

    **Referees cross-commenting**

    I agree with Reviewer #1 that at least the raw code is uploaded to GitHub or Zenodo, and raw data to be uploaded to Zenodo.

    We agree and thank the reviewer for this important suggestion. To ensure that the study can be reproduced without the need to contact the authors, we have made the underlying data and custom analysis code publicly accessible. The RNA-seq data have been deposited in the GEO repository under accession number GSE308510 and are available at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE308510.

    In addition, the raw imaging data, behavioural and calcium-imaging datasets, processed data, and custom scripts used for the behavioural, calcium-imaging, as well as the tRNA and rRNA sequencing data have been deposited on KonDATA (DOI: 10.48606/vpwgm69277srrgaj) – together more than 190 GB – and can be accessed using this link: https://kondata.uni-konstanz.de/radar/en/dataset/vpwgm69277srrgaj?token=gLEaYEENHjmHBhjhHUHK.

    We have revised the Data and code availability statement in the manuscript accordingly (also see the response to Reviewer #1).

    I agree with Reviewer #1 that brain size can and should also be assessed, presumably using the same images already collected. For example, in Figure 5b, number of neural cells (even when normalized) could be lower if brain size is small. Reasonable control analysis.

    As suggested, we have quantified tectum width, tectum length, and hindbrain width from the existing calcium-imaging datasets in a blinded manner. Although MZrlig1 larvae showed modest reductions in tectum width and length, hindbrain width did not differ between genotypes. Thus, the reduced number of motion-responsive hindbrain cells is unlikely to be explained by a gross difference in hindbrain size. These control analyses are presented in the new Supplementary Figure 6 (also see the response to Reviewer #1).

    I agree with Reviewer #2 that addressing, either by writing or experimentally, a bit more about direct targets of the ligase (including tRNAs and rRNAs) will strengthen the manuscript significantly.

    We thank the reviewer for this helpful suggestion. To address this point, we have added new analyses of rRNA and tRNA abundance in 5 dpf WT and MZrlig1 larvae, together with an expanded discussion of their interpretation. These data provide additional evidence that loss of Rlig1 is associated with altered RNA homeostasis, while we distinguish such effects from the direct RNA substrates of the ligase, which remain to be identified (also see the response to Reviewer #2).

    I agree with Reviewer #1 first comment (last sentence) that, if RNA-seq (or other appropriate sequencing) of sibling-matched samples is financially prohibitive, then at least qPCR of some top genes would be acceptable.

    We have performed RT-qPCR validation of selected top differentially expressed genes using sibling-matched WT and zygotic rlig1 mutant larvae generated by heterozygous incrosses. These data provide independent support for the altered expression of several genes identified in the maternal–zygotic rlig1 RNA-seq dataset and are presented in new Supplementary Figure 9 (also see the response to Reviewer #1).

    I agree with the additional comment from Reviewer #1 - the manuscript details cousin-matched samples in lines 666-667, but I'd like to add a suggestion that the authors include details about "single-pair" versus "group-mating". For behavior and all analyses in these kinds of zebrafish experiments, it is very important that multiple replicates of single-pair (one female crossed to one male), sibling-matched groups are used.

    We appreciate the reviewer’s helpful suggestion. We agree that further detail on the breeding schemes is important. We have therefore expanded the Methods to specify, for each experiment, whether embryos or larvae were obtained from single-pair or group matings, the number of independent crosses or clutches, and whether mutant and control animals were sibling- or cousin-matched.

    Reviewer #3 (Significance (Required)):

    This study provides a good increase in our knowledge about a newly described RNA ligase enzyme - rlig1 - in vivo. The authors integrate their results across organismal behavior, brain cell activity, and transcriptomes using a newly generated stable genetic mutant to uncover a new link between neuronal RNA processing, development, and sensory-motor computation. Given that the human orthologue of this gene has been associated with neurological and cognitive conditions, including neurodevelopmental and neuroinflammatory disorders and Alzheimer's disease, the generation and characterization of this stable mutant line proves valuable. There are important technical limitations, specifically related to the comparison of wild type and maternal-zygotic mutant animals, that may not faithfully represent statistical differences compared to sibling-matched animals. Basic biological audiences, including in neurodevelopment, genetics, and RNA biology, would be interested in this research.

    We thank the reviewer for recognising the value of the stable rlig1 mutant line and for highlighting the importance of the breeding design. We agree that comparisons between cousin-matched WT and maternal–zygotic (MZ) mutant larvae require careful interpretation. However, a fully sibling-matched WT versus MZrlig1 comparison is not genetically possible. Maternal–zygotic mutants must be produced by homozygous mutant mothers, whereas WT siblings can only be obtained from a different maternal genotype. Thus, the maternal genotype and, critically, the presence or absence of maternally deposited rlig1 RNA and protein – necessarily differs between these conditions. This is not merely a technical limitation of the experimental design, but an intrinsic feature of testing maternal–zygotic gene function. A heterozygous incross instead produces sibling-matched zygotic mutants, which retain maternal rlig1 products and therefore represent a biologically distinct genetic condition rather than a direct replacement for the MZ comparison.

    For the MZ experiments, we minimised genetic-background differences by using cousin-matched controls derived from the same parental family and by analysing independent experimental replicates. Importantly, the principal behavioural finding was independently supported in sibling-matched zygotic mutants generated by heterozygous incrosses. These larvae showed a qualitatively similar reduction in visually guided behaviour, although with a smaller effect size (new Supplementary Figure 4). We also validated selected transcriptional changes in sibling-matched zygotic mutants by RT-qPCR (new Supplementary Figure 9). The weaker phenotype in zygotic mutants is consistent with partial buffering by maternal rlig1 transcript or protein. Future studies will be valuable to further separate how maternal and zygotic Rlig1 affects gene expression and visually guided behaviour.

    Insufficient expertise to evaluate: While I understand the first part of Figure 1, I do not have expertise in these sorts of assays. The rest of the experiments I do have sufficient expertise to evaluate. And thank you to the authors for providing direct DOI links to references.

    We are grateful for the reviewers’ detailed comments, which substantially improved the manuscript. We hope that the revised text and additional analyses address the central concerns and make the study more transparent and useful to the field.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary

    This paper provides good evidence that a newly described enzyme that catalyzes 5'-3' RNA ligation - rlig1 - plays some role in early vertebrate neurodevelopment. Using embryonic and larval zebrafish as a model, they found that, while rlig1 mRNA is highly maternally deposited and ubiquitously expressed early on, expression later in development localizes to the brain and eyes. They generated a stable CRISPR/Cas9 large deletion mutant spanning from upstream the 5'UTR past the start codon. By comparing wild type and maternal-zygotic (MZ) rlig1 mutants, the authors found that animals developed overtly normally but did show reduced behavioral responsiveness to a visual stimulus experimental paradigm. By combining calcium imaging and poly(A)-enriched RNA-sequencing transcriptomic analyses, they found that there was decreased neuronal activity in regions needed for visual processing, and that there was dysregulation of neural-related gene networks and metabolic and translational pathways.

    Major comments

    1. My main major comment is that, because there is so much inherent variability in behavior and even development across different clutches, this study relies on comparing (cousin-matched) WT and maternal-zygotic rlig1 mutant animals. In most reliable peer-reviewed papers, this is not a fair comparison. While I appreciate that authors stated that they used parents that were siblings (so, offspring would be cousin-matched), I do not consider this scientifically rigorous enough for the claims presented.

    a. I do not consider it a reasonable request to ignore the massive amount of work that went into this paper using WT and MZrlig1 comparisons. However, at minimum, authors should consider performing essential behavior and RNA-seq (see point b) experiments with heterozygous incrosses of single-pair matings, and genotyping the animals post-hoc. Including this critical data in a main figure, as the basis for using MZ animals for the rest of the paper, would induce some confidence that the phenotypes and claims presented are not a result of inherent variability. If the authors already have adult heterozygous animals of mating age, I estimate that these experiments may be completed very reasonably within 3-4 weeks; if new animals need to be generated, this request would take ~4 months. Typically, these kinds of experiments would not be considered a financial burden to perform.

    b. For transcriptomic analyses, I have two main points: i) again, it is difficult to statistically rigorously compare transcriptomes of nonsibling-matched animals with such low numbers of single 5 dpf brains. In line with point a, it would be essential to pool at least a few WT and rlig1 mutant siblings for at least 3 biological replicates per samples and compare those analyses with the results from MZ animals. ii) Typically this would not be a major concern, however given the nature of the gene of interest and published in vitro findings, I do consider that the rlig1 enzyme catalyzes 5'-3' RNA ligation, has been shown to be implicated in rRNA integrity and tRNA targeting, and is broadly essential for repair, splicing, and editing of RNAs. Thus, while the poly(A)-enriched RNA sequencing can provide context about gene networks that are affected (either primarily or secondarily), sequencing that enriches for tRNAs, polysome profiling or ribosome profiling, or some more targeted sequencing approach would be more appropriate to more rigorously support the claims in the paper. Depending on readiness of mating-age animals, this experiment and analyses may reasonably take up to 3 months; this approach may be considered a financial burden. Alternatively, with the current mRNA sequencing, the authors could delve into whether they can identify altered splicing or RNA editing dynamics in different RNA modules. I estimate that this alternative analysis approach may take up to one month to develop and interpret.

    The experiments as documented are adequately replicated and statistical analyses adequate (minus the nonsibling-matched point 1). I note that labels should more clearly state or denote individual (n) or experimental (N) numbers, some of which I provide in Minor comments below.

    Minor comments

    Comments on figures or figure legends:

    1. Figure 1e, align the "#" labels better, they look diagonal.
    2. For 1f, consider labeling independent replicates directly on the graph instead of just the label, otherwise not very clear to the reader.
    3. Figure 2a, consider adding the reference gene (eef1a) in the legend.
    4. Figure 2a - if I understand the experiment correctly, the current label n=3 (which would mean 3 individual embryos/larvae) should read N=3 (three independent experiments of x number of embryos/larvae per run)
    5. Supplementary Figure 1 was very unconvincing comparing WT to MZ mutants, I'm sorry to say I really could not tell much difference. When compared to Figure 3c, they look quite different. The DRAQ7 labeling also appeared uneven in Supplementary Figure 1. Consider optimizing the imaging strategy and providing more interpretably images. A separate, aesthetic comment - magenta was very difficult for me to see against a black background, consider switching the rlig1 channel to grayscale or flip the colors so that rlig1 mRNA is cyan, for example.
    6. Calcium imaging - related to Major comments above, consider performing this experiment in sibling-matched animals, especially with only one copy of the transgene. If WT vs. sibling mutant results look similar to the WT vs MZ mutant results, this would be more convincing.

    Referees cross-commenting

    I agree with Reviewer #1 that at least the raw code is uploaded to GitHub or Zenodo, and raw data to be uploaded to Zenodo.

    I agree with Reviewer #1 that brain size can and should also be assessed, presumably using the same images already collected. For example, in Figure 5b, number of neural cells (even when normalized) could be lower if brain size is small. Reasonable control analysis.

    I agree with Reviewer #2 that addressing, either by writing or experimentally, a bit more about direct targets of the ligase (including tRNAs and rRNAs) will strengthen the manuscript significantly.

    I agree with Reviewer #1 first comment (last sentence) that, if RNA-seq (or other appropriate sequencing) of sibling-matched samples is financially prohibitive, then at least qPCR of some top genes would be acceptable.

    I agree with the additional comment from Reviewer #1 - the manuscript details cousin-matched samples in lines 666-667, but I'd like to add a suggestion that the authors include details about "single-pair" versus "group-mating". For behavior and all analyses in these kinds of zebrafish experiments, it is very important that multiple replicates of single-pair (one female crossed to one male), sibling-matched groups are used.

    Significance

    This study provides a good increase in our knowledge about a newly described RNA ligase enzyme - rlig1 - in vivo. The authors integrate their results across organismal behavior, brain cell activity, and transcriptomes using a newly generated stable genetic mutant to uncover a new link between neuronal RNA processing, development, and sensory-motor computation. Given that the human orthologue of this gene has been associated with neurological and cognitive conditions, including neurodevelopmental and neuroinflammatory disorders and Alzheimer's disease, the generation and characterization of this stable mutant line proves valuable. There are important technical limitations, specifically related to the comparison of wild type and maternal-zygotic mutant animals, that may not faithfully represent statistical differences compared to sibling-matched animals. Basic biological audiences, including in neurodevelopment, genetics, and RNA biology, would be interested in this research.

    Insufficient expertise to evaluate: While I understand the first part of Figure 1, I do not have expertise in these sorts of assays. The rest of the experiments I do have sufficient expertise to evaluate. And thank you to the authors for providing direct DOI links to references.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary

    Klusman et al have investigated the function of the RNA ligase rlig1 in zebrafish. They first document expression of the gene, by quantitative RT-PCR and HCR-fluorescent in situ hybridization. They then test ligase activity of the Rlig1 protein in vitro. They next generate a null mutant and test function of the visual system using behaviour as well as calcium imaging. The data indicate that rlig1 is broadly expressed and capable of ligating RNA; loss of rlig1 has mild effects on overall development and pronounced effects on behavioural and neuronal response to visual stimuli. Finally, the authors use bulk transcriptome analysis to identify changes in gene expression in the mutants.

    Referees cross-commenting

    I agree that more details are required about the crosses would be useful.

    Significance

    Overall, the conclusions that rlig1 is required for normal development of the embryo, especially of a fully functioning visual system, are well supported. The optomotor response experiments have high power and, together with functional imaging, show a clear difference between mutant and wildtype.

    One limitation of this manuscript is in the characterization of gene expression. The gene expression database in Zfin contains one image of rlig1 (https://zfin.org/ZDB-IMAGE-060710-1925#image), which shows broad expression in cells of the embryo and larvae and no expression in the yolk. The images here, with the exception of the mutant in Figure 3C, show expression in the yolk. This would suggest that the yolk signal is not autofluorescence, which is inconsistent with the Thisses' data. Additonally, Figure S1 indicates a variable level of non-specific signal, especially in panel g. Thus, the distribution of rlig1 mRNA is unclear.

    The transcriptome analysis identified changes in gene expression in the mutant. This establishes a role for rlig1 in development, and identifies several processes that are disrupted by loss of rlig1. However, the molecular analysis sheds little light on direct targets of the ligase. Given the established effects on tRNA, for example, it is unclear why RNA was analysed only by short reads on poly(A) RNA. The reader is left wondering whether zebrafish tRNA contains introns that require Rlig1 for processing. In this context, it would be useful for the authors to provide more background on tRNA splicing in vertebrates, including a mention of tricRNA, and potentially the role of TSEN complex in brain development.

    To summarize, this manuscript extends work in the mouse and in cell lines that demonstrate a requirement for rlig1. It does not shed light on direct targets of Rlig1, but provides a strong foundation for future work on the role of RNA ligation in vertebrate development and brain function.

    This paper is expected to be of interest to a specialised audience.

    Minor points:

    The images showing gene expression in Figure 2 are not easy to see, due to the LUT used and low intensity of the signal. To aid the reader, the HCR channel should be shown in grayscale, possibly with the contrast enhanced (to the same extent in all images).

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate).

    This study characterizes the function of RNA ligase 1 (Rlig1) in the vertebrate model zebrafish. Rlig1 is one of only two known RNA ligases in vertebrates, and its biological roles remain poorly understood. The authors combine gene expression analysis, loss-of-function approaches, transcriptomic profiling, calcium imaging, and behavioral assays to investigate its function during development. They show that loss of rlig1 (including maternal-zygotic loss) has no major effects on development or morphology, but that it leads to impairments in visually-guided behavior and altered neuronal activity in response to visual stimuli. Transcriptomic analyses reveal widespread dysregulation across multiple developmental stages, nominating genes that may underly the observed neural phenotypes. Together, the findings support a role for Rlig1 in neural development and function in vertebrates.

    Major comments:

    • Are the key conclusions convincing?

    The key conclusion of this study is that Rlig1 plays an important role in the development and function of vertebrate neural circuits. Overall, this overarching conclusion, as well as the individual conclusions from each set of experiments, are well supported by the data presented. The combination of tissue-specific expression of rlig1, robust behavioral phenotypes in mutants, transcriptomic changes across multiple developmental stages, and circuit differences observed through calcium imaging provides a coherent, multi-faceted argument for the importance of this enzyme in brain development and function. While the precise RNA substrates of Rlig1 and the mechanistic link between transcriptomic changes and neural phenotypes remain to be defined, the authors clearly acknowledge these next steps and limitations. This study is a critical foundation for those future experiments.

    • Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

    The claims in the manuscript are generally well-supported. The authors clearly acknowledge limitations and future experiments to further dissect mechanism in the Discussion section.

    • Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

    No major additional experiments appear essential for supporting the current claims.

    • Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

    No experiments are required for the current claims of the manuscript.

    • Are the data and the methods presented in such a way that they can be reproduced?

    The methods are generally well described. I would suggest that the "raw images, data, and source code for custom scripts used in this work" be made accessible without having to request from the authors. Zenodo provides up to 50 GB of storage, which is likely sufficient for the data presented in this manuscript. In particular, I think it is important to share the behavior analysis, calcium imaging pipeline, and transcriptomics analysis. Even if all the data is too large, a sample dataset and analysis scripts should be publicly available.

    • Are the experiments adequately replicated and statistical analysis adequate?

    The experiments appear adequately replicated, and statistical analyses are appropriate for the types of data presented.

    Minor comments:

    • Specific experimental issues that are easily addressable.
    • Are prior studies referenced appropriately?
    • Are the text and figures clear and accurate?
    • Do you have suggestions that would help the authors improve the presentation of their data and conclusions?
    • Throughout the manuscript: use the prime symbol for 5/3 DNA/RNA instead of an apostrophe. The prime symbol is present in a small number of sentences, but mostly the apostrophe is used.
    • Line 227: "Next, we compared the total number of neurons". The elavl3 driver labels brain cells in addition to neurons.
    • The authors compared to the total number of brain cells, but can they make any comments on the size of the brain across the various areas? I imagine this data is also accessible by analyzing the imaging already collected.
    • Given that there is already a mouse mutant for this gene and transcriptomics, can the authors do a more thorough job comparing the transcriptomics from that study with their own?
    • A clearer statement on the similarities and differences of Rlig1 and RtcB would be helpful. Is it possible RtcB is compensating at all?
    • I examined the DEG tables, and I did not notice an obvious substantial enrichment of genes on chromosome 25 (White et al., 2022, https://doi.org/10.7554/eLife.72825). Were the different samples from different clutches or the same clutch? I may have missed it. Regardless, I would carefully check the DEGs that are important for conclusions and check that they are not on the same chromosome as rlig1. It is likely worth rerunning all of the GO/GSEA with genes on chromosome 25 excluded.

    Referees cross-commenting

    I missed the point about the RNA-seq samples being cousin-matched. While I am optimistic that the results won't change, I agree with Reviewer #3 that some confirmation is necessary. It was unclear to me whether the samples were from the same or different clutches - if they are from different clutches and share overlapping genes, that would also add support to the results. I think that detail was missing from the methods, and I had pointed it out. Either additional RNA-seq or even qPCR of some top genes from a heterozygous incross is a reasonable request.

    Significance

    • Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

    This study provides a conceptual and biological advance by identifying a role for a vertebrate RNA ligase in brain development, behavior, and transcriptional regulation.

    • Place the work in the context of the existing literature (provide references, where appropriate).

    Although RNA ligases from single-cell organisms and phage are well-characterized, the roles of RNA ligases in vertebrates are relatively understudied. There are only two, including the one the one that is the focus of this manuscript. This study demonstrates an in vivo function for Rlig1, linking molecular changes to neural development and function. The Rlig1 enzyme was only very recently discovered (2023), making this work timely and an important addition to an area with relatively few studies.

    A major strength of the study is its multi-level approach, integrating diverse techniques to coherently link this gene to organism-level phenotypes. This work provides a strong conceptual and functional advance by demonstrating a role for Rlig1 in vertebrate neural circuit function and behavior. A remaining mechanistic gap is that the direct RNA substrates of Rlig1 are not identified, and the observed transcriptomic changes in mRNA are likely downstream consequences of its loss. However, these points are clearly acknowledged in the discussion, making the study a well-balanced contribution. Given the existence of a mouse knockout model, further discussion comparing the zebrafish transcriptomic results and phenotypes to those observed in mouse would help place this work in the context of prior studies. Overall, the main conclusions are well supported, and the limitations do not undermine them. This study represents an important contribution that establishes a foundation for future mechanistic work linking Rlig1 substrates to the observed phenotypes.

    • State what audience might be interested in and influenced by the reported findings.

    Zebrafish basic science researchers, particuarly those studying how genes lead to altered neural circuits and behavior, are the most direct target audience. However, the work is of more broad interest to those in the fields of neurodevelopment, gene regulation, and RNA biology / processing.

    • Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

    I am comfortable evaluating zebrafish mutants, transcriptomics, and behavioral assay design. I have more limited experiment in neural circuit anaysis and interpretation of calcium imaging data, though this part of the manuscript was also clearly presented and understandable.

  5. Version published to 10.64898/2025.12.01.691575 on bioRxiv