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  1. Evaluation Summary:

    This study is of interest to researchers who study cell migration and or muscle development. It builds upon prior analysis of Double Anal fin (Da) mutants by using detailed bioinformatic and time-lapse analysis to explain dorsal somite extension and find evidence that dorsal muscle morphogenesis is actively guided, rather than being passively shaped by physical constraints alone. This work illustrates the dynamic behaviors of dorsal somitic cells, which form elaborate protrusions, delaminate from their parent somite, and bridge the gap between opposing epaxial myotomes. Looking downstream of Da, they show that Wnt signaling is central to dorsal extension of the epaxial myotome in medaka and propose that similar functions may shape the dorsal musculature across vertebrates.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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  2. Reviewer #1 (Public Review):

    We possess a detailed understanding of how the myotome, precursors of the epaxial and hypaxial muscles, differentiates from the somites, the molecular mechanisms underlying subsequent morphogenetic movements are not well understood. It was previously shown that in the Medaka double anal fin mutant the dorsal trunk region is transformed into a ventral one and that this phenotype is caused by reduced zic1/zic2 expression in the dorsal somites. The loss of zic1 also increases proliferation in the dorsal dermamyotome. However, the downstream effectors of zic1/zic4 were not known. Here the authors show take advantage of a zic1 reporter line which they use to perform in vivo imaging of dorsal myotome cells. They show that in contrast to prior belief, the somites of both sides of the embryo fuse dorsally of the neural tube by active cell migration. After crossing the reporter line with zic1/zic4 mutants they observed a reduction in protrusive activity and a decrease in proliferation, that explain the failure of myotome fusion at the dorsal midline. Photoconversion experiments show that specifically the dorsal DM cells and the mesenchymal cells derived from them seem to actively participate in the entire process of dorsal somite extension. To determine the molecular machinery controlling dorsal somite extension the authors performed RNASeq, ATACSeq and ChIP Seq using a transgenic zic1:myc line that they generated that allowed them to identify 3200 direct downstream targets. In this manuscript the authors specifically study the downstream target wnt11r. The findings that zic1 binds to wnt11r is interesting, as wnt11 has been previously shown to be involved in cell migration in other systems. However, the reliance on photomorpholinos to downregulate wnt11r dampens my enthusiasm a bit, as the judgement of its efficacy relies on circumstantial evidence that the phenotype resembles the zic1 phenotype and that exogenously supplied human wnt11 protein partially rescues the phenotype. The strength of the manuscript are the in vivo time lapse analyses and the discovery that dorsal epaxial myotome cells actively migrate, as well as the identification of downstream targets. However, how wnt11r via non-canonical Wnt signaling leads to an increase in polarized protrusion and migration needs to be further investigated in the future.

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  3. Reviewer #2 (Public Review):

    The Medaka double anal fin mutant provides a unique tool for studying epaxial myogenesis and the processes that expand body wall musculature to enclose the neural tube. In these mutants, the myotome fails to expand over the neural tube in the stages examined. The time lapse imaging provided by the authors reveal the intriguing migratory dynamics of dorsal somitic cells, which bridge the inter-somitic space between opposing myotomes. These data provide evidence of differences in the behavior of Zic1:GFP+ cells between the Da and Wt fish. The authors show that Da fish have a reduced ability to form large protrusions. However, the fate of dorsal somitic migratory cells and their putative role in epaxial myogenesis is not clear.

    In addition to documenting cell dynamics, the authors also performed dissections and FACS sorting with Tg(Zic1:GFP) transgenic fish to compare RNA-seq and ATAC-seq data sets between GFP+ (dorsal) and GFP- (ventral) regions of the somites. ChIP-seq experiments were also performed using a Myc-tagged Zic1 in the Da background to identify potential Zic1 target genes. Comparisons of Chip-seq data sets with genes highly expressed in the dorsal somite suggested the involvement of Wnt signaling in epaxial myogenesis. The authors specifically focused on Wnt11r, which they hypothesized to be a direct target of Zic1 and a key regulator of epaxial muscle expansion. In support of this hypothesis, the authors show Wnt11r expression is reduced in Da mutants. While their Chip-seq and ATACseq data sets suggest the position of a putative Wnt11r enhancers, enhancer function was not directly tested. Interestingly, morpholino knockdown of Wnt11r, or pharmacological inhibition of the Wnt/Ca2+ pathway, resulted in partial phenocopy of the Da phenotype, including reduced protrusions from dorsal somitic cells. Exogenous application of human recombinant Wnt11 in Da mutants rescued cell protrusion formation, further supporting a role for Wnt11 in epaxial myogenesis.

    Overall, this paper is well written and addresses an interesting and underexplored question in developmental biology. The identification of a migratory population of dorsal somitic cells is an intriguing observation. The fate and function of these cells in epaxial myogenesis, however, is not clear from the current data set and warrants further investigation.

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  4. Reviewer #3 (Public Review):

    This study is of interest to researchers who study cell migration and or muscle development. It builds upon prior analysis of Double Anal fin (Da) mutants by using detailed bioinformatic and time-lapse analysis to explain dorsal somite extension, and find evidence that dorsal muscle morphogenesis is actively guided, rather than being passively shaped by physical constraints alone. Looking downstream of Da, they show that Wnt signaling is central to to dorsal extension of the epaxial myotome in medaka and propose that similar functions may shape the dorsal musculature across vertebrates.

    Strengths:

    Previously, the authors found that the medaka Da mutant causes strong loss of zic1 and zic4 expression, loss of epaxial myotome growth, and a 'ventralized' fin phenotype. However, the cellular and molecular mechanisms underpinning these defects remained unclear. It also remained unclear whether cells of the epaxial myotome are actively guided to their destinations or simply forced dorsally by passive constraints.

    In this study, using high resolution time-lapse data provides important insights into epaxial myogenesis, particularly by providing evidence of active migration rather than simply constrained growth. The authors demonstrate that the Da mutant has a specific lack of large cell protrusions, which may explain the dorsal growth defect.

    The authors establish a dataset of genes with expression enriched in Zic1+ cells, and promoters bound by Zic1 in these cells, which will be a rich resource for further investigation of epaxial myogenesis.

    Using this dataset, the authors identify Wnt11r ad a candidate Da-dependent signaling factor that may explain dorsal defects in the mutant. Consistent with this model, they show that wnt11r-MO causes loss of long-protrusions, similar to the Da mutant itself. They chase the signal further downstream by blocking CamKII function and find that this elicits an effect similar to the Da mutant, namely, specific loss of long-protrusions. Critically, they are able to restore formation of long-protrusions by injecting human Wnt11 protein into a dorsal somite, bolstering their argument that activation of Wnt signaling is a central target of Zic1/4. Although one could argue against any one of these findings in isolation, they work together to make a compelling case for wnt11 in actively guiding dorsal myotome extension.

    The paper's discussion concludes with a strong argument that these findings are not limited to fish, but are likely in play across vertebrates.

    Concerns:

    It would be important to clarify in results and methods how somites were isolated prior to FACs sorting. One line in the manuscript implies that somites were dissected cleanly while another suggests that whole tails were included. If it's just the somite, please say a little more in the methods about how this tissue was separated from its surroundings. If the whole tail was used, then the language about dorsal vs. ventral somite sorts should be adjusted.

    This manuscript makes use of Wnt11r morpholino data without confirmation using a mutant, which is considered the gold standard. So, a few comments could be softened, such as the claim that Wnt11r is essential. Including a photo-MO helps, because at least the earliest non-specific functions can be ruled out. This concern is also offset by the authors protein injection and CamKII inhibitor experiments, which establish confidence that this pathway is involved.

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