Active locomotion, which is powered by skeletal muscles in vertebrates, is critical for animals to survive. Vertebrate skeletal muscles consist of axial muscles (head, trunk, and tail muscles) and appendicular muscles (limb muscles). Axial muscles first arose in the chordates to stabilize and enable side-to-side movement of the body axis. In jawed vertebrates, the subdivision of axial muscles into epaxial (dorsal) and hypaxial (ventral) muscles led to an increased range of movement: dorsoventral undulation in fish and lateral movements in terrestrial vertebrates (Goodrich, 2004; Romer and Parsons, 1986; Fetcho, 1987; Sefton and Kardon, 2019). Among these muscles, epaxial muscles are characterized by their unique anatomical structure, which extends dorsally and surrounds the vertebrae. This morphology also ensures mechanical support and protection of the vertebrae and the spinal cord inside. While we have a detailed understanding of how the myotome, precursors of epaxial and hypaxial muscles, differentiates from a somitic compartment called the dermomyotome (DM) (Kalcheim et al., 1999; Gros et al., 2009; Hollway and Currie, 2005), we only begin to understand the cellular and molecular mechanisms of the subsequent morphogenetic processes generating the epaxial muscles. Previous studies in rats and mice suggested that myocytes of the epaxial myotome do not actively migrate dorsally but are guided by external forces (Deries et al., 2010; Deries et al., 2012). However, what exerts such forces to drive extension of epaxial myotome is still unclear.

Fish have been extensively utilized to study myotome development thanks to the transparency of their bodies throughout embryonic development (Wolff et al., 2003; Stellabotte et al., 2007; Nguyen et al., 2017; Ganassi, 2018). Additionally, their epaxial trunk muscles have a simple structure consisting of only one anatomical unit (reviewed in Sefton and Kardon, 2019). Like other vertebrates, fish myotomes, on either side of the neural tube, extend dorsally after somite differentiation and eventually cover the neural tube by the end of embryonic development (Figure 1A). The spontaneous medaka (Oryzias latipes) mutant Double anal fin (Da) displays a particular epaxial myotome morphology, in which the dorsal ends of the left and right epaxial myotome fail to extend sufficiently and thus do not cover the neural tube at the end of embryonic development. Previous studies demonstrated that the dorsal trunk region of the Da mutant is transformed into the ventral one, including not only the myotome but also the body shape, skeletal elements, pigmentation, and fin morphology (Figure 1B and D; Ishikawa, 1990; Ohtsuka et al., 2004). Given the unique morphological features, the medaka Da mutant is an excellent model to study the morphogenesis of epaxial myotome. Genetic analysis of the Da mutant revealed that this phenotype is due to a dramatic reduction of the expression of the transcription factors zic1 and zic4 in the dorsal somites (Figure 1C and E), and identified zic1/zic4 as master regulators of trunk dorsalization (Ohtsuka et al., 2004; Kawanishi et al., 2013). The down-regulation of zic1/zic4 specifically in the dorsal somites is caused by the insertion of a large transposon, disrupting the dorsal somite enhancer of zic1/zic4 (Moriyama et al., 2012; Inoue et al., 2017). While the function and downstream targets of Zic1 and Zic4 have been studied in nervous system development and somitogenesis (Li, 2006; Pan et al., 2011; Himeda et al., 2013; Hong and Saint-Jeannet, 2017; Aruga and Millen, 2018), the molecular mechanism of how these Zic genes control dorsal trunk morphogenesis has not been investigated so far.

Figure 1 with 1 supplement see all

Download asset Open asset

Here, we describe the morphogenetic process of the formation of the epaxial myotome of the back, which we termed ‘dorsal somite extension’ (Figure 1A). By in vivo time-lapse imaging we uncovered its cellular dynamics; during dorsal somite extension, dorsal DM cells reduce their proliferative activity and subsequently form unique large protrusions extending dorsally, guiding the epaxial myotome towards the top of the neural tube. We also found that these DM cells form a subpopulation that gives rise to non-myotomal cell lineages during embryonic development. In the Da mutant, by contrast, DM cells keep their high proliferative activity and mainly form small protrusions. Mechanistically, we identified a Zic1 downstream-target gene, wnt11 (former wnt11r Postlethwait et al., 2019), as a crucial factor for dorsal somite extension, and demonstrated that Wnt11 regulates cellular behavior of dorsal DM cells by promoting protrusion formation and negatively regulating proliferation. We thus propose an unprecedented process of epaxial myotome morphogenesis driven by a non-myogenic population of DM cells during embryogenesis.

Our previous study showed that zic1 and zic4 expression starts at embryonic stages and persists throughout life (Kawanishi et al., 2013). Phenotypic analysis of homozygous adult Da mutants implies long-term participation of Zic-downstream genes in the formation of dorsal musculatures, which eventually affects the external appearance of the fish adult trunk. Here, we examined the initial phase of this long-term dorsalization process. In the following of the study, we will focus on zic1, since zic1 and zic4 are expressed in an identical fashion with overlapping functions in trunk dorsalization of medaka, and zic4 is expressed slightly weaker than zic1 (Moriyama et al., 2012; Kawanishi et al., 2013).

Proliferative activity of the dorsal DM cells is enhanced in the Da mutant

In fish, as in other vertebrates, the DM gives rise to muscle precursor cells, which ultimately differentiate into myofibers. In medaka the DM is a one cell-thick, Pax3/7-positive cell layer encompassing the myotome (Figure 2A–B’’; Hollway et al., 2007; Abe et al., 2019). A high proliferative activity of the dorsal DM could explain a larger cross-sectional area of the dorsal myotome of the Da mutant. To test this, we performed immunohistochemistry against the mitotic marker phospho-histone H3 (pH3) and the DM marker Pax3/7 on Wt and Da embryos (Figure 2C–E). In both Wt and Da embryos, pH3-positive cells were randomly distributed in the dorsal DM without obvious bias (Figure 2C–D). At the 12-somite stage (12 ss, 1.7 dpf stage 23), when zic1 expression in the somites becomes restricted to the dorsal region (Kawanishi et al., 2013), the number of pH3-positive DM cells per dorsal somite was not significantly different between Wt and Da. Remarkably, from 16 ss (1.8 dpf, stage 24) onwards, the number of pH3-positive cells became reduced in the Wt, whereas in Da, no such reduction was observed (Figure 2E). At 35 ss (3.4 dpf, stage 30), pH3-positive cells increased both in the Wt and Da, but the mutant DM cells were more proliferative (Figure 2E). Immunohistochemistry against another proliferation marker PCNA confirmed these findings (Figure 2—figure supplement 1A-C). The number of pH3-positive cells in the ventral DM was not significantly different in Wt and Da embryos at 22 ss (2.75 dpf, stage 26) (Figure 2—figure supplement 2A). These results suggest that zic1 reduces proliferative activity of the DM, which becomes evident following the confinement of its expression to the dorsal somite region.

Figure 2 with 2 supplements see all

Download asset Open asset

Wt dorsal DM cells form numerous large, motile protrusions at the onset of dorsal somite extension

The epaxial myotome, on either side of the neural tube, extends dorsally to cover the neural tube by the end of embryonic development. In the Da mutant, the myotome is unable to cover the neural tube despite increased dorsal myotome growth at the hatching stage (Figures 1L and 2). This suggest that additionally to physical extension an active process might support the dorsal movement of somites. To examine the behavior of zic1-positive cells underlying this dorsal somite extension, we performed in vivo time-lapse imaging of dorsal somites using the transgenic line Tg(zic1:GFP,zic4:DsRed), which expresses GFP under the control of the zic1 promoter and enhancers to visualize the dorsal somitic cells (Kawanishi et al., 2013) (hereafter called Tg(zic1:GFP) since the DsRed fluorescence was negligible in the following analyses). Intriguingly, around 22 ss and onwards, cells at the tip of the dorsal somites started to form numerous large protrusions extending dorsally towards the top of the neural tube (Figure 3A, Video 1). We defined the beginning of protrusion formation as the onset of dorsal somite extension. Close-up views of the time-lapse images (Figure 3A, Video 1) showed that these protrusions were motile and dynamically formed new branches at their dorsal tips (Figure 3J, Figure 3—figure supplement 1D). Immunohistochemistry revealed that the protrusion-forming cells belong to the DM (Figure 3—figure supplement 1A-A'').

Figure 3 with 2 supplements see all

Download asset Open asset

Video 1

Download asset

To characterize the protrusions, we classified them according to their length into small ( < 8 μm, Figure 3C arrowheads) and large ( ≥ 8 μm, Figure 3C arrow) protrusions (Figure 3—figure supplement 1B-C). Based on their shape, we reasoned that the small protrusions correspond to lamellipodia (Figure 3C, arrowheads), while the large protrusions appeared more complex. To investigate the nature of large protrusions, we injected Actin-Chromobody GFP mRNA to visualize the actin skeleton. The large protrusions were found to exhibit a complex architecture consisting of a lamellipodia-like core structure (Figure 3D, bracket) with additional multiple bundles of filopodia (protrusions with linear arranged actin filaments) branching out from their dorsal tips (Figure 3D, arrows, summarized in Figure 3E).

Interestingly, time-lapse in vivo imaging of Tg(zic1:GFP);Da showed that in the Da background, protrusions started to form later (Figure 3B, Video 2) and the number of large protrusions and protrusions in total was significantly lower than in Wt (Figure 3I–I’’). In addition, protrusions were transient and mostly failed to form new branches at their dorsal tips (Figure 3K, Figure 3—figure supplement 1E). While no difference in the actin skeleton of small protrusions could be observed, filament bundles branching out from large protrusions of Da DM cells contained fewer and shorter filopodia compared to Wt (Figure 3G, arrowheads, summarized in Figure 3H). These results indicate that the protrusive activity, especially the ability to form large protrusions, is significantly reduced in the Da mutant. The large protrusions of the dorsal DM cells continuously appeared at later stages of dorsal somite extension, too (Figure 3—figure supplement 2).

Video 2

Download asset

To investigate the role of large protrusions during the onset of dorsal somite extension, we inhibited filopodia formation using ML141 (Figure 3L and M). ML141 specifically inhibits Cdc42/Rac1 GTPases, which are critical for filopodia formation (Hong et al., 2013; Fantin, 2015). Intriguingly, dorsal somites of embryos treated with ML141 extended significantly less dorsally compared to control embryos treated with DMSO (Figure 3N). Taken together, our data suggest that the unique large protrusions of the dorsal DM are involved in guiding the epaxial myotome dorsally and zic1 might promote this function.

DM cells delaminate and accumulate between opposing somites during the late phase of dorsal somite extension

We continued to trace the behavior of the DM tip cells until the somites reach the top of the neural tube. Indeed, in vivo imaging of Tg(zic1:GFP) revealed that dorsal DM cells continued to form protrusions, and additionally, some of them delaminated to become Zic1-positive mesenchymal cells accumulating in the space between the dorsal ends of the left and the right somites (Figure 4A, star-shaped cells, arrowheads) from 4.5 dpf (stage 33) onwards. This is consistent with previous observations of strongly zic1 expressing mesenchymal cells in Wt at late embryonic stages (Ohtsuka et al., 2004). As dorsal somite extension proceeded, the number of these mesenchymal cells increased, filling the space between the two myotomes (Figure 4A–C, arrowheads, Videos 3–4, arrowheads indicate representative mesenchymal cells). These mesenchymal cells formed protrusions towards neighboring mesenchymal cells and DM cells at the tip of somites, creating a dense cellular network between the dorsal ends of the somites. Mosaic cell-labelling demonstrated that the mesenchymal cells originated from the DM (Figure 4—figure supplement 1A-F, arrowheads). Interestingly, while mesenchymal cells dynamically formed protrusions, they showed no extensive migratory behavior and were rather stationary (Videos 3–4, Figure 4—figure supplement 2). This could suggest that these protrusions fulfill a non-migratory function. When the opposing somites came in contact with each other at 8 dpf (stage 38, 1 day before hatching), the mesenchymal cells tended to attach to the nearest DM cells at the tip of the somite, bridging the gap between the left and right DM cells (Figure 4I, Figure 4—figure supplement 1M-M'''').

Figure 4 with 3 supplements see all

Download asset Open asset

Video 3

Download asset

Video 4

Download asset

In Da mutants, mesenchymal DM cells were also detected in the space between the two myotomes, but the timing of their appearance was delayed, that is between 5 dpf (stage 34) and 5.5 dpf (stage 35) (Figure 4D–F) (4.5 dpf in Wt). Additionally, Da mesenchymal cells exhibited a rounder morphology and formed significantly fewer protrusions compared to Wt while the number of mesenchymal cells was not largely affected (Figure 4G and H, Figure 4—figure supplement 1G-L).

To examine the function of mesenchymal DM cells, we ablated these cells between the 10^th somites of 5.5 dpf Tg(zic1:GFP) embryos using an UV laser (Figure 4J–K’, Figure 4—figure supplement 3A-B'). Intriguingly, the distance between the left and the right tips of the 10^th somites increased after ablating the mesenchymal DM cells while the neighboring 9^th somites continued to shorten the gap (Figure 4L). This suggests that mesenchymal DM cells hold the left and the right somite together to promote the dorsal somite extension at this late phase of myotome development. After 16 hr post ablation, dorsal extension of the 10^th somites eventually resumed as the mesenchymal DM cells regenerated at the ablation site (Figure 4L).

Collectively, dorsal DM cells and the mesenchymal cells derived from them seem to actively participate in the entire process of dorsal somite extension, from its onset to neural tube coverage at the end.

Finally, we examined the fate of the dorsal DM cells that derive the mesenchymal cells. We employed a photoconversion technique mediated by a photoconvertible protein KikGR to specifically label a single dorsal DM cell exhibiting protrusions at the tip of a 10th and 20th somites during dorsal somite extension (Figure 4M, arrowhead) and tracked them until the hatching stage. We found that the labeled dorsal DM cells eventually differentiated into cells surrounding blood vessels (likely mural cells, n = 8/9 and 4/8 for 10th and 20th somites, respectively; Figure 4N and N’, arrowheads) and mesenchymal cells in the dorsal fin fold (n = 4/8 for 20th somites; Figure 4O and O’, arrowheads); however, we did not observe any labeled axial muscles (n = 0/9 and 0/8 for 10th and 20th somites, respectively). This suggests that the dorsal DM cells at the tip of somites, guiding myotome extension, do not become myotomal cells themselves at least until the end of embryonic development (hatching stage, 9 dpf).

Zic1 regulates the expression of dorsal-specific genes during somite differentiation

We then addressed the molecular machinery controlling the dorsal somite extension investigated above. Since somite extension is impaired in the zic1-enhancer mutant Da (Moriyama et al., 2012; Kawanishi et al., 2013), we reasoned that downstream genes of Zic1 are regulators of this process.

First, we identified genes which are specifically expressed in the dorsal somites. For this, we collected somites of the transgenic line Tg(zic1:GFP) by removing other tissues after pancreatin treatment, FACS sorted them into the dorsal (GFP+) and ventral (GFP-) somitic cells including the DM cells, and performed RNA-seq and ATAC-seq on both cell populations (Figure 5A, Figure 5—figure supplement 1A). The RNA-seq identified 1,418 differentially expressed genes. Among them 694 genes showed higher expression in the dorsal somites (termed hereafter dorsal-high genes), and 724 genes showed higher expression in the ventral somites (termed hereafter dorsal-low genes) (Figure 5C). We confirmed that zic1 and zic4 were found among the dorsal-high genes (Figure 5C, Supplementary file 1).

Figure 5 with 2 supplements see all

Download asset Open asset

Next, we identified potential direct Zic1-target genes by investigating Zic1 binding sites. Since there were no suitable antibodies available to perform ChIP-seq against medaka Zic1, we created a transgenic line expressing a Myc tagged zic1 in the Da background under the control of zic1 promoter and enhancers (Tg(zic1:zic1-Myc,zic4:DsRed);Da, called Tg(zic1:zic1-Myc);Da hereafter). This transgene (zic1:zic1-Myc) efficiently rescued the ventralized phenotype (dorsal and anal fin shape, pigmentation pattern, and body shape) of the Da mutant (Figure 5—figure supplement 1B-C), verifying the full functionality of the tagged Zic1 protein. Somites of the transgenic line Tg(zic1:zic1-Myc);Da were dissected and subjected to ChIP-seq using antibodies against Myc (Figure 5B). Since somites contain a low number of cells, we applied ChIPmentation (Schmidl et al., 2015) to identify genome-wide Zic1 binding sites. From two biological replicates, 5247 reliable ChIP peaks were identified, and we confirmed that the enriched DNA motif among these peaks was showing high similarity with previously identified binding motifs of ZIC family proteins (Figure 5—figure supplement 2A-B). Then, we associated each Zic1 peak to the nearest gene within 50 kb and identified 3232 genes as Zic1 target genes. By comparing the ChIPmentation with RNA-seq data, we found that Zic1 target genes are overrepresented in dorsal-high and dorsal-low genes. While 47% of the dorsal-high genes and 27% of the dorsal-low genes were found to be Zic1 target genes, only 12% of genes which showed no differential expression in dorsal or ventral somites were potential Zic1 downstream target genes (Figure 5D, Supplementary file 1). This suggests that Zic1 can function as transcriptional activator and repressor of versatile genes, but the former role seems to be dominant.

wnt11 is a direct downstream target of Zic1 and down-regulated in the dorsal somites of the Da mutant

To identify potential regulators of dorsal somite extension, we further investigated the differentially expressed Zic1 target genes. Gene Ontology (GO) analysis indicated that both dorsal-high and dorsal-low gene groups, regardless of whether they are Zic1 targets or non-Zic1 targets, were significantly enriched in development related GO terms (Figure 6A, Figure 6—figure supplement 1A, Supplementary files 2 and 3). This indicates that Zic1 regulates a number of developmental genes both directly and indirectly. These results are consistent with the fact that Zic1 regulates various dorsal-specific morphologies of somite-derivatives (Kawanishi et al., 2013).

Figure 6 with 1 supplement see all

Download asset Open asset

In the dorsal-high Zic1 targets, GO terms related to cell migration showed higher enrichment (e.g. ‘chemotaxis’ (p = 5.62E-10), ‘locomotion’ (6.65E-15), ‘ameboidal-type cell migration’ (p = 9.32E-10)) than dorsal-low genes or non-Zic1 target genes (Supplementary file 3). Interestingly, Wnt signaling pathway genes (e.g.: axin2, wnt11, sp5, lrp5, fzd10, prickle1a) and semaphorin-plexin signaling pathway genes (e.g.: sema3a, sema3c, sema3g, plxna1, plxna2, plxnb2, plxnb3, nrp2) were included in these gene groups. Indeed, the GO terms ‘Wnt signaling pathway’ and ‘Semaphorin-plexin signaling pathway’ themselves were also significantly enriched (p = 4.45E-7 and 1.94E-8, respectively) in dorsal-high Zic1 target genes (Supplementary file 2). This suggests that Zic1 directly regulates Wnt pathway, semaphorin and plexin genes, possibly to regulate cell movement in the dorsal somites. We also noticed that the terms ‘extracellular matrix organization’ (p = 7.74E-7; e.g.: adamts20, fbln1), ‘cell communication’ (p = 1.95E-5; e.g.: efna5, epha3) are enriched, suggesting that these genes also affect dorsal somite cell behavior.

Wnt signaling pathway components also ranked high among the dorsal-high Zic1 target genes by pathway enrichment analysis (Figure 6B, Figure 6—figure supplement 1B). Among them, we focused on wnt11 for further analyses due to the following reasons: First, wnt11 was one of the most differentially expressed genes in dorsal somites (Figure 5C, Supplementary file 1). Second, previous studies implicated Wnt11 in protrusion formation and cell migration (Ulrich et al., 2003; De Calisto et al., 2005; Garriock and Krieg, 2007; Matthews et al., 2008). This is particularly interesting since DM cells also exhibit protrusions and migration activity during dorsal somite extension, which are defective in Da mutants.

At the wnt11 locus, peaks of the Zic1-ChIP overlapped with intergenic open chromatin regions downstream of wnt11. These sites were more accessible in dorsal somites than in ventral somites, suggesting that Zic1 regulates wnt11 directly (Figure 6C). Additionally, in situ hybridizations against wnt11 performed on Wt and Da embryos indicated that wnt11 expression is significantly reduced at the dorsal tip of Da dorsal somites including the DM (Figure 6—figure supplement 1C-D). Already when the zic1 expression becomes restricted in the dorsal somites (12 ss) and with proceeding development when zic1 expression gets further restricted to the most dorsal part of the somites and mesenchymal DM cells (Ohtsuka et al., 2004), the expression of wnt11 in the dorsal somites of the Da mutant was reduced compared to Wt (Figure 6D–F, Figure 6—figure supplement 1C-F').

Taken together, we identified wnt11 as promising downstream target gene of Zic1, which could be a novel somite dorsalization factor and play a role in dorsal somite extension.

Wnt11 regulates cell behavior of dorsal DM

Our previous RNA-seq dataset revealed that the expression of wnt11 starts before gastrulation and increases as development proceeds (Figure 7—figure supplement 1A; Nakamura et al., 2021). Injection of single guide RNAs (sgRNAs) against wnt11 to knock out the gene revealed that the resultant genetically mosaic F0 embryos displayed delayed epiboly movement and subsequent morphological defects, including shorter body axis and impaired trunk development (Figure 7—figure supplement 2), implying the difficulty of assessment of the wnt11 function at later stages using the wnt11 mutant. To circumvent the problem, we took two different approaches to knock-down wnt11 during dorsal somite extension. Firstly, we used a wnt11 anti-sense morpholino (wnt11 MO) and determined a concentration which resulted in maximal knock-down effects during dorsal somite extension with minimal gastrulation phenotypes (see Materials and methods). Secondly, we performed temporally controlled knock-down of wnt11 after gastrulation using photo-cleavable Photo-Morpholinos (PhotoMOs) (Tallafuss, 2012; Figure 7A). Strikingly, in wnt11 Photo-Morphants with severe phenotypes the dorsal myotome failed to cover the neural tube (n = 7, Figure 7B–C), a phenotype similar to the Da mutant myotome. Additionally, the myofibers of the Photo-Morphants at 9 dpf were shorter and less organized. This phenotype is consistent with a previously reported role of Wnt11 during early myogenesis, where it regulates the elongation and orientation of myoblasts (Gros et al., 2009).

Figure 7 with 2 supplements see all

Download asset Open asset

Next, we investigated the proliferative activity of dorsal DM cells in wnt11 morphants and found that knock-down of wnt11 (either by conventional MO or by PhotoMO) induced significantly more pH3-positive cells per somite, compared to control embryos at 22 ss (Figure 7D). These findings are similar to the observations previously made in the dorsal DM of Da mutants, and suggest that Wnt11 is negatively regulating proliferation of dorsal DM cells.

We then explored whether the onset of dorsal somite extension in wnt11 morphants is similarly impaired as in the Da by time-lapse in vivo imaging. Remarkably, we observed a protrusion formation behavior of wnt11 morphant DM cells similar to Da DM cells, namely delayed onset of protrusion formation and the formation of fewer, shorter protrusions (Figure 7E, Video 5). Quantification of protrusions indicated that wnt11 morphants have significantly fewer large protrusions and protrusions in total, compared to control embryos (Figure 7F–H’’).

Video 5

Download asset

Overall, knock-down of the Zic1 target gene wnt11 recapitulated the phenotype of Da DM cells (Figure 3G–G’’), suggesting the crucial role of Wnt11 in regulating protrusion formation of DM cells.

To further confirm the importance of Wnt11 during dorsal somite extension, we performed rescue experiments in Da embryos. At 18 ss (2.1 dpf, stage 25), we injected a mix of human recombinant Wnt11 (hrWnt11) protein (or BSA in the control group) and Dextran Rhodamine onto the top of the 10^th somite of Tg(zic1:GFP);Da embryos (Figure 8A–B’’). Strikingly, the dorsal DM of Da embryos injected with Wnt11 formed significantly more large protrusions and more protrusions in total compared to Da embryos injected with BSA only (Figure 8C–C’’). This indicates that the ventralized Da DM protrusion phenotype can be partially rescued by Wnt11 protein injections and further emphasizes the importance of Wnt11 in somite dorsalization and during dorsal somite extension.

Figure 8

Download asset Open asset

Wnt11 acts through the Wnt/Ca²⁺ signaling pathway at the onset of somite extension

Finally, we investigated through which signaling pathway the non-canonical Wnt11 acts. In Xenopus, Wnt11 acts through the Wnt/Ca²⁺ pathway regulating the migration of cells from the dorsal somite and the neural crest into the dorsal fin fold (Garriock and Krieg, 2007). To examine whether this signaling pathway also plays a role during dorsal somite extension, we inhibited the Wnt/Ca²⁺ signaling pathway using KN-93 in Tg(zic1:GFP) embryos from 4 ss (1.3 dpf, stage 20)–22 ss (Figure 9A). KN-93 specifically inhibits CaMKII, a component of the Wnt/Ca²⁺ pathway, by binding to it and preventing its phosphorylation, thus keeping it in an inactivated state (Sumi et al., 1991; Tombes et al., 1995; Wu and Cline, 1998; Garriock and Krieg, 2007; Rothschild et al., 2013). Embryos treated with KN-93 showed a higher number of pH3-positive dorsal DM cells per somite, compared to embryos in the control group (Figure 9B). Furthermore, DM cells at the tip of the dorsal somite of embryos treated with KN-93 formed significantly fewer large and fewer protrusions in total, compared to embryos in the control group (Figure 9C–C’’), although the effect was less significant compared to the wnt11 morphants (Figure 7).

Figure 9

Download asset Open asset

From these results, we suggested that Wnt11 potentially acts through the Wnt/Ca²⁺ signaling pathway at the onset of somite extension. Wnt/Ca²⁺ signaling pathway is known to regulate actin polymerization (Choi and Han, 2002; Kohn and Moon, 2005) which could explain the dynamic protrusive activity of the DM cells (Figure 3), emphasizing the importance of Wnt11 during epaxial myotome morphogenesis.

Here, we elucidated the key developmental process underlying the epaxial myotome morphogenesis to cover the neural tube in the teleost, medaka. DM cells have been known to serve as a progenitor pool for myotomal and dermal cells (Ben-Yair and Kalcheim, 2005; Hollway et al., 2007; Stellabotte and Devoto, 2007). In our study, we showed that DM cells at the tip of the dorsal somite form unique large protrusions, guiding the myotome towards the top of the neural tube. Furthermore, these DM cells do not give rise to myotomal cells during embryonic development. We found that zic1 and its direct downstream target wnt11 are crucial factors for this morphogenetic process. We thus revealed a novel role of non-myogenic DM cells during epaxial myotome morphogenesis. Furthermore, to our best knowledge, this work demonstrates for the first time that the neural tube coverage by myotomes is driven by active cell movement, rather than passively by muscle growth. Consistent with this, in the absence of Zic1 activity, the myotome is unable to cover the neural tube at the hatching stage, despite increased dorsal myotome growth (Figures 1L and 2), supporting the involvement of the active cellular mechanism in this process. The model we propose for dorsal somite extension is shown in Figure 10.

Figure 10

Download asset Open asset

In the present study, we described the characteristic behavior of dorsal DM cells and propose their guiding role for the epaxial myotome moving towards the top of the neural tube. During dorsal somite extension, DM cells at the tip of the dorsal somites form large, motile protrusions, which contain multiple bundles of filopodia dynamically branching out from the tip of the protrusions. These long protrusions could be beneficial for invading the restricted open space between the neural tube and the ectoderm. Additionally, previous studies showed that in migrating mesenchymal cells, the formation of lamellipodia is associated with higher migratory speed, whereas filopodia play an exploratory role and are associated with high directionality (Leithner et al., 2016; Innocenti, 2018). Large protrusions of wild-type DM cells consist of a lamellipodia-like core and multiple filopodia, which could account for fast dorsal somite extension with high accuracy. The detailed structure and function of the large protrusions needs to be further investigated in the future studies.

In zebrafish, hypaxial muscle precursors delaminate from the ventral tip of the DM and migrate collectively in a cell stream to the prospective pectoral fin bud region while the cells at the leading edge form long filopodia (Haines et al., 2004; Talbot et al., 2019). Thus, DM cells, regardless of whether dorsal or ventral, seem to have the potential to form protrusions and to exhibit migratory behavior. However, regulation of migratory behavior could be different as the migration of hypaxial muscle precursors is mediated by the receptor tyrosine kinase Met (Haines et al., 2004) which is not expressed in zebrafish dorsal DM cells.

At later stages of dorsal somite extension, we observed that DM cells delaminate from the tip of the dorsal somites and progressively occupy the space between the opposing dorsal somites. These mesenchymal DM cells actively form protrusions towards neighboring mesenchymal DM cells and DM cells of the somites, thus forming a dense cellular network on top of the neural tube. This could provide a communication platform for the opposing somites to meet at the right position, exactly on top of the neural tube. Indeed, ablation of mesenchymal DM cells resulted in transient opening of the gap between the somites, suggesting that these cells at least exert a tension to hold the positions of the dorsal tips of the left and right somite together during neural tube coverage. Interestingly, our lineage tracing experiment revealed that the dorsal DM cells at the tip of somites differentiated into cells of non-myotomal lineages including mural cells of blood vessels and mesenchymal cells in the dorsal fin fold. These observations are consistent with previous studies reporting their somitic origins in zebrafish and Xenopus (Garriock and Krieg, 2007; Lee et al., 2013; Ando et al., 2016). However, our data also showed that these DM cells do not appear to contribute to the myotome at the hatching stage. We thus propose that the neural tube coverage is driven by a specific population of dorsal DM cells, which do not contribute to myotomal cells during embryonic development.

The ability of the motile DM cells to guide the underlying myotome dorsally assures complete coverage of the neural tube by the epaxial myotomes. Considering the fact that DM cells are in close cell-cell contact with the underlying myotome, it is plausible that they are biomechanically coupled with myotomal cells, facilitating dorsal myotome extension. A similar mechanism has been investigated in mouse neural tube formation. During mouse neural tube closure, the surface ectoderm, overlying the neuroepithelium, forms cell protrusions towards the midline. Disruption of membrane ruffles, a form of lamellipodia, in the surface ectoderm results in incomplete fusion of the subjacent neuroepithelium (Rolo et al., 2016).

Our data suggest that in medaka dorsal somites Wnt11 exerts its effect through promotion of protrusion formation and down-regulation of cell proliferation in the dorsal DM. Regarding the protrusion formation, previous studies of migrating mesendodermal cells in the zebrafish mutant silberblick and neural crest cells in Xenopus embryos consistently reported that Wnt11 is involved in the oriented elongation and stabilization of protrusions (Ulrich et al., 2003; De Calisto et al., 2005; Matthews et al., 2008). Similarly, myocardial cells in the heart of Wnt11 mutant mice also form fewer protrusions (Zhou et al., 2007). Thus, regulation of cell protrusions is a conserved function of Wnt11, observed in various developmental processes. However, the relationship between Wnt11 and cell proliferation could be context-dependent, as it negatively regulates proliferation in mouse neonatal hearts (Touma, 2017), while it promotes cell proliferation in mouse intestinal epithelial cell culture (Ouko et al., 2004). Hence, the function of Wnt11 in medaka somites is unique in that it promotes tissue elongation by regulating a balance between proliferative and migrative activity.

By inhibiting the Wnt/Ca²⁺ signaling pathway we showed that Wnt11 probably acts through this non-canonical Wnt signaling pathway during dorsal somite extension. Likewise, previous studies in Xenopus have shown that Wnt11 acts through this pathway during cell migration into the dorsal fin fold. There it regulates epithelial–mesenchymal transition in a distinct dorsal somite cell population which, together with a population of neural crest cells, contribute to the mesenchyme of the dorsal fin fold (Garriock and Krieg, 2007). Furthermore, during convergent extension in vertebrate gastrulation, Wnt/Ca2⁺ signaling pathway can regulate cell adhesion by promoting actin polymerization (Choi and Han, 2002; Kohn and Moon, 2005). Whether Wnt11 functions cell-autonomously or not is context dependent (De Calisto et al., 2005; Garriock and Krieg, 2007; Gros et al., 2009). In our study, administration of exogenous Wnt11 protein rescued the protrusive behavior of the surrounding DM cells, suggesting that Wnt11 can function cell non-autonomously during dorsal somite extension.

Importantly, our RNA-seq and ChIPmentation analyses revealed that Zic1 has diverse downstream target genes including various developmental genes. This suggests that the dorsal myotome is established via pleiotropic actions of Zic1; Wnt11 may not be a sole factor for dorsal somite extension, although it was suggested to be crucial in the present study. The semaphorin-plexin pathway may play a role, since previous studies suggested, besides its implication in axon guidance and neural cell migration, a role in non-neural cell migration (Alto and Terman, 2017). Furthermore, cell migration and tissue deformation are often linked with the extracellular matrix (ECM), which provides guiding or restraining cues influencing cell movements. Previous studies showed that during mouse epaxial myotome development, ECM composition changes dynamically, which is tightly accompanied by epaxial muscle morphogenesis; while the laminin content in the ECM decreases, increasing tenascin and stable fibronectin contents potentially promotes the alignment of myofibers and their final organization (Deries et al., 2012). Furthermore, recent studies suggested that cells can remodel the surrounding ECM and thereby increase their own motility. For example, during zebrafish gastrulation, the metalloproteinase mmp14 is expressed by migrating endoderm cells and degrades laminin and fibronectin, components of the ECM (Hu et al., 2018). In this context, of particular importance is our identification of Adamts20, encoding a proteoglycanase, as a dorsal-high Zic1-target gene in the somites (Supplementary file 1). Since Adamts20 is known to play a pivotal role in embryonic melanoblast migration by remodeling the dermal ECM (Rao et al., 2003; Silver et al., 2008), Adamnts20 could thus facilitate migration of Wnt11-expressing DM cells through remodeling of the ECM. Further functional analysis of dorsal-high Zic1-target genes identified in somites will provide useful insight into the molecular network driving dorsal somite extension.

Finally, in vertebrates and especially in fish, body shape and muscle morphology are closely linked, since a majority of the body mass consist of muscular tissue. Previous studies in fish populations have shown that speciation and adaptation to a specific aquatic habitat are associated with a changes in body depth, a measurement of the trunk dorsoventral axis (Tobler, 2008; Elmer et al., 2010; Weese et al., 2012; Fruciano, 2016). In this context, the external appearance of the adult Da mutant is intriguing, in that it exhibits a teardrop body shape (typical for fish swimming in the middle layer like tuna), instead of a dorsally flattened one (typical for surface swimming fish like medaka). Since our study shows that the activity of wnt11 could influence the body shape by regulating cell proliferation and the behavior of muscle progenitor cells, Wnt11 could be one of the crucial factors in evolution and diversity of body shape in fish. Furthermore, the expression of zic1 (Ohtsuka et al., 2004; Sun Rhodes and Merzdorf, 2006; Houtmeyers et al., 2013) and wnt11 (Marcelle et al., 1997; Olivera-Martinez et al., 2004; Garriock et al., 2005; Matsui et al., 2005) are strongly conserved among vertebrates, and we hypothesize that Wnt11-mediated morphogenesis of the somites represents an evolutionarily conserved mechanism that acts across vertebrates.

Sequencing data generated in this study have been submitted to the DDBJ BioProject database under accession number PRJDB11712.

1. 1. Helig AK
   2. Nakamura R
   3. Hashimoto Y

   (2022) DDBJ BioProject

   ID PRJDB11712. The function of transcription factor Zic1 in dorsalization of the medaka somite.

   https://ddbj.nig.ac.jp/resource/bioproject/PRJDB11712

1. 1. Abe K
   2. Shimada A
   3. Tayama S
   4. Nishikawa H
   5. Kaneko T
   6. Tsuda S
   7. Karaiwa A
   8. Matsui T
   9. Ishitani T
   10. Takeda H

   (2019) Horizontal Boundary Cells, a Special Group of Somitic Cells, Play Crucial Roles in the Formation of Dorsoventral Compartments in Teleost Somite

   Cell Reports 27:928–939.

   https://doi.org/10.1016/j.celrep.2019.03.068

   • PubMed
   • Google Scholar
2. 1. Alto LT
   2. Terman JR

   (2017) Semaphorins and their Signaling Mechanisms

   Methods in Molecular Biology 22:11–14.

   https://doi.org/10.1016/s0896-6273(00)80672-8

   • Google Scholar
3. 1. Ando K
   2. Fukuhara S
   3. Izumi N
   4. Nakajima H
   5. Fukui H
   6. Kelsh RN
   7. Mochizuki N

   (2016) Clarification of mural cell coverage of vascular endothelial cells by live imaging of zebrafish

   Development (Cambridge, England) 143:1328–1339.

   https://doi.org/10.1242/dev.132654

   • PubMed
   • Google Scholar
4. 1. Aruga J
   2. Millen KJ

   (2018) ZIC1 Function in Normal Cerebellar Development and Human Developmental Pathology

   Advances in Experimental Medicine and Biology 1046:249–268.

   https://doi.org/10.1007/978-981-10-7311-3_13

   • PubMed
   • Google Scholar
5. 1. Ashburner M
   2. Ball CA
   3. Blake JA
   4. Botstein D
   5. Butler H
   6. Cherry JM
   7. Davis AP
   8. Dolinski K
   9. Dwight SS
   10. Eppig JT
   11. Harris MA
   12. Hill DP
   13. Issel-Tarver L
   14. Kasarskis A
   15. Lewis S
   16. Matese JC
   17. Richardson JE
   18. Ringwald M
   19. Rubin GM
   20. Sherlock G

   (2000) Gene Ontology: tool for the unification of biology

   Nature Genetics 25:25–29.

   https://doi.org/10.1038/75556

   • Google Scholar
6. 1. Ben-Yair R
   2. Kalcheim C

   (2005) Lineage analysis of the avian dermomyotome sheet reveals the existence of single cells with both dermal and muscle progenitor fates

   Development (Cambridge, England) 132:689–701.

   https://doi.org/10.1242/dev.01617

   • PubMed
   • Google Scholar
7. 1. Bolger AM
   2. Lohse M
   3. Usadel B

   (2014) Trimmomatic: A flexible trimmer for Illumina sequence data

   Bioinformatics (Oxford, England) 30:2114–2120.

   https://doi.org/10.1093/bioinformatics/btu170

   • PubMed
   • Google Scholar
8. 1. Buenrostro JD
   2. Giresi PG
   3. Zaba LC
   4. Chang HY
   5. Greenleaf WJ

   (2013) Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position

   Nature Methods 10:1213–1218.

   https://doi.org/10.1038/nmeth.2688

   • PubMed
   • Google Scholar
9. 1. Choi SC
   2. Han JK

   (2002) Xenopus Cdc42 regulates convergent extension movements during gastrulation through Wnt/Ca2+ signaling pathway

   Developmental Biology 244:342–357.

   https://doi.org/10.1006/dbio.2002.0602

   • PubMed
   • Google Scholar
10. Preprint

    1. Collins ZM
    2. Ishimatsu K
    3. Tsai TYC
    4. Megason SG

    (2018) A Scube2-Shh Feedback Loop Links Morphogen Release to Morphogen Signaling to Enable Scale Invariant Patterning of the Ventral Neural Tube

    bioRxiv.

    https://doi.org/10.1101/469239

    • Google Scholar
11. 1. Daetwyler S
    2. Modes CD
    3. Fiolka R

    (2020) Fiji plugin for annotating movies with custom arrows

    Biology Open 9:3–6.

    https://doi.org/10.1242/bio.056200

    • PubMed
    • Google Scholar
12. 1. Davis GK
    2. Jaramillo CA
    3. Patel NH

    (2001) Pax group III genes and the evolution of insect pair-rule patterning

    Development (Cambridge, England) 128:3445–3458.

    https://doi.org/10.1242/dev.128.18.3445

    • Google Scholar
13. 1. De Calisto J
    2. Araya C
    3. Marchant L
    4. Riaz CF
    5. Mayor R

    (2005) Essential role of non-canonical Wnt signalling in neural crest migration

    Development (Cambridge, England) 132:2587–2597.

    https://doi.org/10.1242/dev.01857

    • PubMed
    • Google Scholar
14. 1. Deries M.
    2. Schweitzer R
    3. Duxson MJ

    (2010) Developmental fate of the mammalian myotome

    Developmental Dynamics 239:2898–2910.

    https://doi.org/10.1002/dvdy.22425

    • PubMed
    • Google Scholar
15. 1. Deries M
    2. Gonçalves AB
    3. Vaz R
    4. Martins GG
    5. Rodrigues G
    6. Thorsteinsdóttir S

    (2012) Extracellular matrix remodeling accompanies axial muscle development and morphogenesis in the mouse

    Developmental Dynamics 241:350–364.

    https://doi.org/10.1002/dvdy.23703

    • PubMed
    • Google Scholar
16. 1. Dobin A
    2. Davis CA
    3. Schlesinger F
    4. Drenkow J
    5. Zaleski C
    6. Jha S
    7. Batut P
    8. Chaisson M
    9. Gingeras TR

    (2013) STAR: ultrafast universal RNA-seq aligner

    Bioinformatics (Oxford, England) 29:15–21.

    https://doi.org/10.1093/bioinformatics/bts635

    • PubMed
    • Google Scholar
17. 1. Elmer KR
    2. Kusche H
    3. Lehtonen TK
    4. Meyer A

    (2010) Local variation and parallel evolution: morphological and genetic diversity across a species complex of neotropical crater lake cichlid fishes

    Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences 365:1763–1782.

    https://doi.org/10.1098/rstb.2009.0271

    • PubMed
    • Google Scholar
18. 1. Fantin A

    (2015) NRP1 Regulates CDC42 Activation to Promote Filopodia Formation in Endothelial Tip Cells

    Cell Reports 11:1577–1590.

    https://doi.org/10.1016/j.celrep.2015.05.018

    • Google Scholar
19. 1. Fetcho JR

    (1987) A review of the organization and evolution of motoneurons innervating the axial musculature of vertebrates

    Brain Research Reviews 434:243–280.

    https://doi.org/10.1016/0165-0173(87)90001-4

    • Google Scholar
20. 1. Fruciano C

    (2016) Genetic linkage of distinct adaptive traits in sympatrically speciating crater lake cichlid fish

    Nature Communications 6:12736.

    https://doi.org/10.1038/ncomms12736

    • Google Scholar
21. 1. Ganassi M

    (2018) Myogenin promotes myocyte fusion to balance fibre number and size

    Nature Communications 9:4232.

    https://doi.org/10.1038/s41467-018-06583-6

    • Google Scholar
22. 1. Garriock RJ
    2. D’Agostino SL
    3. Pilcher KC
    4. Krieg PA

    (2005) Wnt11-R, a protein closely related to mammalian Wnt11, is required for heart morphogenesis in Xenopus

    Developmental Biology 279:179–192.

    https://doi.org/10.1016/j.ydbio.2004.12.013

    • PubMed
    • Google Scholar
23. 1. Garriock RJ
    2. Krieg PA

    (2007) Wnt11-R signaling regulates a calcium sensitive EMT event essential for dorsal fin development of Xenopus

    Developmental Biology 304:127–140.

    https://doi.org/10.1016/j.ydbio.2006.12.020

    • PubMed
    • Google Scholar
24. 1. Gene Ontology Consortium

    (2021) The Gene Ontology resource: enriching a GOld mine

    Nucleic Acids Research 49:D325–D334.

    https://doi.org/10.1093/nar/gkaa1113

    • PubMed
    • Google Scholar
25. 1. Goodrich ES

    (2004) A two-Step mechanism for myotome formation in chick

    Developmental Cell 6:875–882.

    https://doi.org/10.1016/j.devcel.2004.05.006

    • Google Scholar
26. 1. Gros J
    2. Serralbo O
    3. Marcelle C

    (2009) WNT11 acts as a directional cue to organize the elongation of early muscle fibres

    Nature 457:589–593.

    https://doi.org/10.1038/nature07564

    • PubMed
    • Google Scholar
27. 1. Haines L
    2. Neyt C
    3. Gautier P
    4. Keenan DG
    5. Bryson-Richardson RJ
    6. Hollway GE
    7. Cole NJ
    8. Currie PD

    (2004) Met and Hgf signaling controls hypaxial muscle and lateral line development in the zebrafish

    Development (Cambridge, England) 131:4857–4869.

    https://doi.org/10.1242/dev.01374

    • PubMed
    • Google Scholar
28. 1. Heinz S
    2. Benner C
    3. Spann N
    4. Bertolino E
    5. Lin YC
    6. Laslo P
    7. Cheng JX
    8. Murre C
    9. Singh H
    10. Glass CK

    (2010) Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities

    Molecular Cell 38:576–589.

    https://doi.org/10.1016/j.molcel.2010.05.004

    • PubMed
    • Google Scholar
29. 1. Himeda CL
    2. Barro MV
    3. Emerson CP

    (2013) Pax3 synergizes with Gli2 and Zic1 in transactivating the Myf5 epaxial somite enhancer

    Developmental Biology 383:7–14.

    https://doi.org/10.1016/j.ydbio.2013.09.006

    • PubMed
    • Google Scholar
30. 1. Hollway G
    2. Currie P

    (2005) Vertebrate myotome development

    Birth Defects Research. Part C, Embryo Today 75:172–179.

    https://doi.org/10.1002/bdrc.20046

    • PubMed
    • Google Scholar
31. 1. Hollway GE
    2. Bryson-Richardson RJ
    3. Berger S
    4. Cole NJ
    5. Hall TE
    6. Currie PD

    (2007) Whole-somite rotation generates muscle progenitor cell compartments in the developing zebrafish embryo

    Developmental Cell 12:207–219.

    https://doi.org/10.1016/j.devcel.2007.01.001

    • PubMed
    • Google Scholar
32. 1. Hong L
    2. Kenney SR
    3. Phillips GK
    4. Simpson D
    5. Schroeder CE
    6. Nöth J
    7. Romero E
    8. Swanson S
    9. Waller A
    10. Strouse JJ
    11. Carter M
    12. Chigaev A
    13. Ursu O
    14. Oprea T
    15. Hjelle B
    16. Golden JE
    17. Aubé J
    18. Hudson LG
    19. Buranda T
    20. Sklar LA
    21. Wandinger-Ness A

    (2013) Characterization of a Cdc42 protein inhibitor and its use as a molecular probe

    The Journal of Biological Chemistry 288:8531–8543.

    https://doi.org/10.1074/jbc.M112.435941

    • PubMed
    • Google Scholar
33. 1. Hong CS
    2. Saint-Jeannet JP

    (2017) Znf703, a novel target of Pax3 and Zic1, regulates hindbrain and neural crest development in Xenopus

    Genesis (New York, N.Y 55:12.

    https://doi.org/10.1002/dvg.23082

    • PubMed
    • Google Scholar
34. 1. Houtmeyers R
    2. Souopgui J
    3. Tejpar S
    4. Arkell R

    (2013) The ZIC gene family encodes multi-functional proteins essential for patterning and morphogenesis

    Cellular and Molecular Life Sciences 70:3791–3811.

    https://doi.org/10.1007/s00018-013-1285-5

    • PubMed
    • Google Scholar
35. 1. Hu B
    2. Gao Y
    3. Davies L
    4. Woo S
    5. Topczewski J
    6. Jessen JR
    7. Lin F

    (2018) Glypican 4 and Mmp14 interact in regulating the migration of anterior endodermal cells by limiting extracellular matrix deposition

    Development (Cambridge, England) 145:17.

    https://doi.org/10.1242/dev.163303

    • PubMed
    • Google Scholar
36. 1. Hwang WY
    2. Fu Y
    3. Reyon D
    4. Maeder ML
    5. Tsai SQ
    6. Sander JD
    7. Peterson RT
    8. Yeh J-RJ
    9. Joung JK

    (2013) Efficient genome editing in zebrafish using a CRISPR-Cas system

    Nature Biotechnology 31:227–229.

    https://doi.org/10.1038/nbt.2501

    • PubMed
    • Google Scholar
37. 1. Innocenti M

    (2018) New insights into the formation and the function of lamellipodia and ruffles in mesenchymal cell migration

    Cell Adhesion & Migration 12:401–416.

    https://doi.org/10.1080/19336918.2018.1448352

    • PubMed
    • Google Scholar
38. 1. Inoue Y
    2. Saga T
    3. Aikawa T
    4. Kumagai M
    5. Shimada A
    6. Kawaguchi Y
    7. Naruse K
    8. Morishita S
    9. Koga A
    10. Takeda H

    (2017) Complete fusion of a transposon and herpesvirus created the Teratorn mobile element in medaka fish

    Nature Communications 8:1–14.

    https://doi.org/10.1038/s41467-017-00527-2

    • PubMed
    • Google Scholar
39. 1. Ishikawa Y

    (1990) Development of caudal structures of a morphogenetic mutant (Da) in the teleost fish, medaka (Oryzias latipes

    Journal of Morphology 205:219–232.

    https://doi.org/10.1002/jmor.1052050209

    • PubMed
    • Google Scholar
40. 1. Kalcheim C
    2. Cinnamon Y
    3. Kahane N

    (1999) Myotome formation: A multistage process

    Cell and Tissue Research 296:161–173.

    https://doi.org/10.1007/s004410051277

    • PubMed
    • Google Scholar
41. 1. Kawanishi T
    2. Kaneko T
    3. Moriyama Y
    4. Kinoshita M
    5. Yokoi H
    6. Suzuki T
    7. Shimada A
    8. Takeda H

    (2013) Modular development of the teleost trunk along the dorsoventral axis and zic1/zic4 as selector genes in the dorsal module

    Development (Cambridge, England) 140:1486–1496.

    https://doi.org/10.1242/dev.088567

    • PubMed
    • Google Scholar
42. 1. Kohn AD
    2. Moon RT

    (2005) Wnt and calcium signaling: beta-catenin-independent pathways

    Cell Calcium 38:439–446.

    https://doi.org/10.1016/j.ceca.2005.06.022

    • PubMed
    • Google Scholar
43. 1. Lee RTH
    2. Knapik EW
    3. Thiery JP
    4. Carney TJ

    (2013) An exclusively mesodermal origin of fin mesenchyme demonstrates that zebrafish trunk neural crest does not generate ectomesenchyme

    Development (Cambridge, England) 140:2923–2932.

    https://doi.org/10.1242/dev.093534

    • PubMed
    • Google Scholar
44. 1. Lee M-S
    2. Wan J
    3. Goldman D

    (2020) Tgfb3 collaborates with PP2A and notch signaling pathways to inhibit retina regeneration

    eLife 9:e55137.

    https://doi.org/10.7554/eLife.55137

    • PubMed
    • Google Scholar
45. 1. Leithner A
    2. Eichner A
    3. Müller J
    4. Reversat A
    5. Brown M
    6. Schwarz J
    7. Merrin J
    8. de Gorter DJJ
    9. Schur F
    10. Bayerl J
    11. de Vries I
    12. Wieser S
    13. Hauschild R
    14. Lai FPL
    15. Moser M
    16. Kerjaschki D
    17. Rottner K
    18. Small JV
    19. Stradal TEB
    20. Sixt M

    (2016) Diversified actin protrusions promote environmental exploration but are dispensable for locomotion of leukocytes

    Nature Cell Biology 18:1253–1259.

    https://doi.org/10.1038/ncb3426

    • PubMed
    • Google Scholar
46. 1. Li S

    (2006) The Xfeb gene is directly upregulated by Zic1 during early neural development

    Developmental Dynamics 235:2817–2827.

    https://doi.org/10.1002/dvdy.20896

    • Google Scholar
47. Website

    1. Li K

    (2008) The image stabilizer plugin for ImageJ

    Accessed February 7, 2008.

    http://www.cs.cmu.edu/~kangli/code/Image_Stabilizer.html
48. 1. Li H
    2. Durbin R

    (2009) Fast and accurate short read alignment with Burrows-Wheeler transform

    Bioinformatics (Oxford, England) 25:1754–1760.

    https://doi.org/10.1093/bioinformatics/btp324

    • PubMed
    • Google Scholar
49. 1. Lischik CQ
    2. Adelmann L
    3. Wittbrodt J

    (2019) Enhanced in vivo-imaging in medaka by optimized anaesthesia, fluorescent protein selection and removal of pigmentation

    PLOS ONE 14:e0212956.

    https://doi.org/10.1371/journal.pone.0212956

    • PubMed
    • Google Scholar
50. 1. Marcelle C
    2. Stark MR
    3. Bronner-Fraser M

    (1997) Coordinate actions of BMPs, Wnts, Shh and noggin mediate patterning of the dorsal somite

    Development (Cambridge, England) 124:3955–3963.

    https://doi.org/10.1242/dev.124.20.3955

    • PubMed
    • Google Scholar
51. 1. Matsui T
    2. Raya A
    3. Kawakami Y
    4. Callol-Massot C
    5. Capdevila J
    6. Rodríguez-Esteban C
    7. Izpisúa Belmonte JC

    (2005) Noncanonical Wnt signaling regulates midline convergence of organ primordia during zebrafish development

    Genes & Development 19:164–175.

    https://doi.org/10.1101/gad.1253605

    • PubMed
    • Google Scholar
52. 1. Matthews HK
    2. Broders-Bondon F
    3. Thiery JP
    4. Mayor R

    (2008) Wnt11r is required for cranial neural crest migration

    Developmental Dynamics 237:3404–3409.

    https://doi.org/10.1002/dvdy.21758

    • PubMed
    • Google Scholar
53. 1. Moriyama Y
    2. Kawanishi T
    3. Nakamura R
    4. Tsukahara T
    5. Sumiyama K
    6. Suster ML
    7. Kawakami K
    8. Toyoda A
    9. Fujiyama A
    10. Yasuoka Y
    11. Nagao Y
    12. Sawatari E
    13. Shimizu A
    14. Wakamatsu Y
    15. Hibi M
    16. Taira M
    17. Okabe M
    18. Naruse K
    19. Hashimoto H
    20. Shimada A
    21. Takeda H

    (2012) The medaka zic1/zic4 mutant provides molecular insights into teleost caudal fin evolution

    Current Biology 22:601–607.

    https://doi.org/10.1016/j.cub.2012.01.063

    • PubMed
    • Google Scholar
54. 1. Morsch M
    2. Radford RAW
    3. Don EK
    4. Lee A
    5. Hortle E
    6. Cole NJ
    7. Chung RS

    (2017) Triggering Cell Stress and Death Using Conventional UV Laser Confocal Microscopy

    Journal of Visualized Experiments 120:54983.

    https://doi.org/10.3791/54983

    • PubMed
    • Google Scholar
55. 1. Nakamura R
    2. Tsukahara T
    3. Qu W
    4. Ichikawa K
    5. Otsuka T
    6. Ogoshi K
    7. Saito TL
    8. Matsushima K
    9. Sugano S
    10. Hashimoto S
    11. Suzuki Y
    12. Morishita S
    13. Takeda H

    (2014) Large hypomethylated domains serve as strong repressive machinery for key developmental genes in vertebrates

    Development (Cambridge, England) 141:2568–2580.

    https://doi.org/10.1242/dev.108548

    • PubMed
    • Google Scholar
56. 1. Nakamura R
    2. Motai Y
    3. Kumagai M
    4. Wike CL
    5. Nishiyama H
    6. Nakatani Y
    7. Durand NC
    8. Kondo K
    9. Kondo T
    10. Tsukahara T
    11. Shimada A
    12. Cairns BR
    13. Aiden EL
    14. Morishita S
    15. Takeda H

    (2021) CTCF looping is established during gastrulation in medaka embryos

    Genome Research 31:968–980.

    https://doi.org/10.1101/gr.269951.120

    • PubMed
    • Google Scholar
57. 1. Nguyen PD
    2. Gurevich DB
    3. Sonntag C
    4. Hersey L
    5. Alaei S
    6. Nim HT
    7. Siegel A
    8. Hall TE
    9. Rossello FJ
    10. Boyd SE
    11. Polo JM
    12. Currie PD

    (2017) Muscle Stem Cells Undergo Extensive Clonal Drift during Tissue Growth via Meox1-Mediated Induction of G2 Cell-Cycle Arrest

    Cell Stem Cell 21:107–119.

    https://doi.org/10.1016/j.stem.2017.06.003

    • PubMed
    • Google Scholar
58. 1. Ohtsuka M
    2. Kikuchi N
    3. Yokoi H
    4. Kinoshita M
    5. Wakamatsu Y
    6. Ozato K
    7. Takeda H
    8. Inoko H
    9. Kimura M

    (2004) Possible roles of zic1 and zic4, identified within the medaka Double anal fin (Da) locus, in dorsoventral patterning of the trunk-tail region (related to phenotypes of the Da mutant

    Mechanisms of Development 121:873–882.

    https://doi.org/10.1016/j.mod.2004.04.006

    • PubMed
    • Google Scholar
59. 1. Olivera-Martinez I
    2. Thélu J
    3. Dhouailly D

    (2004) Molecular mechanisms controlling dorsal dermis generation from the somitic dermomyotome

    The International Journal of Developmental Biology 48:93–101.

    https://doi.org/10.1387/ijdb.15272374

    • PubMed
    • Google Scholar
60. 1. Ouko L
    2. Ziegler TR
    3. Gu LH
    4. Eisenberg LM
    5. Yang VW

    (2004) Wnt11 signaling promotes proliferation, transformation, and migration of IEC6 intestinal epithelial cells

    The Journal of Biological Chemistry 279:26707–26715.

    https://doi.org/10.1074/jbc.M402877200

    • PubMed
    • Google Scholar
61. 1. Pan H
    2. Gustafsson MK
    3. Aruga J
    4. Tiedken JJ
    5. Chen JCJ
    6. Emerson CP Jr

    (2011) A role for Zic1 and Zic2 in Myf5 regulation and somite myogenesis

    Developmental Biology 351:120–127.

    https://doi.org/10.1016/j.ydbio.2010.12.037

    • PubMed
    • Google Scholar
62. 1. Postlethwait JH
    2. Navajas Acedo J
    3. Piotrowski T

    (2019) Evolutionary Origin and Nomenclature of Vertebrate Wnt11-Family Genes

    Zebrafish 16:469–476.

    https://doi.org/10.1089/zeb.2019.1760

    • PubMed
    • Google Scholar
63. 1. Rao C
    2. Foernzler D
    3. Loftus SK
    4. Liu S
    5. McPherson JD
    6. Jungers KA
    7. Apte SS
    8. Pavan WJ
    9. Beier DR

    (2003) A defect in A novel ADAMTS family member is the cause of the belted white-spotting mutation

    Development (Cambridge, England) 130:4665–4672.

    https://doi.org/10.1242/dev.00668

    • PubMed
    • Google Scholar
64. 1. Rolo A
    2. Savery D
    3. Escuin S
    4. de Castro SC
    5. Armer HEJ
    6. Munro PMG
    7. Molè MA
    8. Greene NDE
    9. Copp AJ

    (2016) Regulation of cell protrusions by small GTPases during fusion of the neural folds

    eLife 5:e13273.

    https://doi.org/10.7554/eLife.13273

    • PubMed
    • Google Scholar
65. Book

    1. Romer A
    2. Parsons TS

    (1986)

    The Vertebrate Body

    Saunders College Pub.

    • Google Scholar
66. 1. Rothschild SC
    2. Lahvic J
    3. Francescatto L
    4. McLeod JJA
    5. Burgess SM
    6. Tombes RM

    (2013) CaMK-II activation is essential for zebrafish inner ear development and acts through Delta-Notch signaling

    Developmental Biology 381:179–188.

    https://doi.org/10.1016/j.ydbio.2013.05.028

    • PubMed
    • Google Scholar
67. 1. Schindelin J
    2. Arganda-Carreras I
    3. Frise E
    4. Kaynig V
    5. Longair M
    6. Pietzsch T
    7. Preibisch S
    8. Rueden C
    9. Saalfeld S
    10. Schmid B
    11. Tinevez J-Y
    12. White DJ
    13. Hartenstein V
    14. Eliceiri K
    15. Tomancak P
    16. Cardona A

    (2012) Fiji: an open-source platform for biological-image analysis

    Nature Methods 9:676–682.

    https://doi.org/10.1038/nmeth.2019

    • PubMed
    • Google Scholar
68. 1. Schmidl C
    2. Rendeiro AF
    3. Sheffield NC
    4. Bock C

    (2015) ChIPmentation: fast, robust, low-input ChIP-seq for histones and transcription factors

    Nature Methods 12:963–965.

    https://doi.org/10.1038/nmeth.3542

    • PubMed
    • Google Scholar
69. 1. Sefton EM
    2. Kardon G

    (2019) Connecting muscle development, birth defects, and evolution: An essential role for muscle connective tissue

    Current Topics in Developmental Biology 132:137–176.

    https://doi.org/10.1016/bs.ctdb.2018.12.004

    • PubMed
    • Google Scholar
70. 1. Silver DL
    2. Hou L
    3. Somerville R
    4. Young ME
    5. Apte SS
    6. Pavan WJ

    (2008) The secreted metalloprotease ADAMTS20 is required for melanoblast survival

    PLOS Genetics 4:e1000003.

    https://doi.org/10.1371/journal.pgen.1000003

    • PubMed
    • Google Scholar
71. 1. Stellabotte F
    2. Devoto SH

    (2007) The teleost dermomyotome

    Developmental Dynamics 236:2432–2443.

    https://doi.org/10.1002/dvdy.21253

    • PubMed
    • Google Scholar
72. 1. Stellabotte F
    2. Dobbs-McAuliffe B
    3. Fernández DA
    4. Feng X
    5. Devoto SH

    (2007) Dynamic somite cell rearrangements lead to distinct waves of myotome growth

    Development (Cambridge, England) 134:1253–1257.

    https://doi.org/10.1242/dev.000067

    • PubMed
    • Google Scholar
73. 1. Stemmer M
    2. Thumberger T
    3. Del Sol Keyer M
    4. Wittbrodt J
    5. Mateo JL

    (2015) CCTop: An Intuitive, Flexible and Reliable CRISPR/Cas9 Target Prediction Tool

    PLOS ONE 10:e0124633.

    https://doi.org/10.1371/journal.pone.0124633

    • PubMed
    • Google Scholar
74. 1. Sumi M
    2. Kiuchi K
    3. Ishikawa T
    4. Ishii A
    5. Hagiwara M
    6. Nagatsu T
    7. Hidaka H

    (1991) The newly synthesized selective Ca2+/calmodulin dependent protein kinase II inhibitor KN-93 reduces dopamine contents in PC12h cells

    Biochemical and Biophysical Research Communications 181:968–975.

    https://doi.org/10.1016/0006-291x(91)92031-e

    • PubMed
    • Google Scholar
75. 1. Sun Rhodes LS
    2. Merzdorf CS

    (2006) The zic1 gene is expressed in chick somites but not in migratory neural crest

    Gene Expression Patterns 6:539–545.

    https://doi.org/10.1016/j.modgep.2005.10.006

    • PubMed
    • Google Scholar
76. 1. Swinburne IA
    2. Mosaliganti KR
    3. Green AA
    4. Megason SG

    (2015) Improved Long-Term Imaging of Embryos with Genetically Encoded α-Bungarotoxin

    PLOS ONE 10:e0134005.

    https://doi.org/10.1371/journal.pone.0134005

    • PubMed
    • Google Scholar
77. 1. Takashima S
    2. Shimada A
    3. Kobayashi D
    4. Yokoi H
    5. Narita T
    6. Jindo T
    7. Kage T
    8. Kitagawa T
    9. Kimura T
    10. Sekimizu K
    11. Miyake A
    12. Setiamarga DHE
    13. Murakami R
    14. Tsuda S
    15. Ooki S
    16. Kakihara K
    17. Hojo M
    18. Naruse K
    19. Mitani H
    20. Shima A
    21. Ishikawa Y
    22. Araki K
    23. Saga Y
    24. Takeda H

    (2007) Phenotypic analysis of a novel chordin mutant in medaka

    Developmental Dynamics 236:2298–2310.

    https://doi.org/10.1002/dvdy.21245

    • PubMed
    • Google Scholar
78. 1. Talbot JC
    2. Teets EM
    3. Ratnayake D
    4. Duy PQ
    5. Currie PD
    6. Amacher SL

    (2019) Muscle precursor cell movements in zebrafish are dynamic and require Six family genes

    Development (Cambridge, England) 146:10.

    https://doi.org/10.1242/dev.171421

    • PubMed
    • Google Scholar
79. 1. Tallafuss A

    (2012) Turning gene function ON and OFF using sense and antisense photo-morpholinos in zebrafish

    Development (Cambridge, England) 139:1691–1699.

    https://doi.org/10.1242/dev.072702

    • Google Scholar
80. 1. Thermes V

    (2002) I-SceI meganuclease mediates highly efficient transgenesis in fish

    Mechanisms of Development 118:91–98.

    https://doi.org/10.1016/S0925-4773(02)00218-6

    • Google Scholar
81. 1. Tobler M

    (2008) Toxic hydrogen sulfide and dark caves: Phenotypic and genetic divergence across two abiotic environmental gradients in Poecilia mexicana

    Evolution; International Journal of Organic Evolution 62:2643–2659.

    https://doi.org/10.1111/j.1558-5646.2008.00466.x

    • Google Scholar
82. 1. Tombes RM
    2. Grant S
    3. Westin EH
    4. Krystal G

    (1995)

    G1 cell cycle arrest and apoptosis are induced in NIH 3T3 cells by KN-93, an inhibitor of CaMK-II (the multifunctional Ca2+/CaM kinase

    Cell Growth & Differentiation 6:1063–1070.

    • PubMed
    • Google Scholar
83. 1. Touma M

    (2017) Wnt11 regulates cardiac chamber development and disease during perinatal maturation

    JCI Insight 2:1–18.

    https://doi.org/10.1172/jci.insight.94904

    • Google Scholar
84. 1. Ulrich F
    2. Concha ML
    3. Heid PJ
    4. Voss E
    5. Witzel S
    6. Roehl H
    7. Tada M
    8. Wilson SW
    9. Adams RJ
    10. Soll DR
    11. Heisenberg C-P

    (2003) Slb/Wnt11 controls hypoblast cell migration and morphogenesis at the onset of zebrafish gastrulation

    Development (Cambridge, England) 130:5375–5384.

    https://doi.org/10.1242/dev.00758

    • PubMed
    • Google Scholar
85. 1. Volpe BA
    2. Fotino TH
    3. Steiner AB

    (2020) Confocal Microscope-Based Laser Ablation and Regeneration Assay in Zebrafish Interneuromast Cells

    Journal of Visualized Experiments 159:60966.

    https://doi.org/10.3791/60966

    • PubMed
    • Google Scholar
86. 1. Wan Y
    2. Otsuna H
    3. Chien C-B
    4. Hansen C

    (2009) An interactive visualization tool for multi-channel confocal microscopy data in neurobiology research

    IEEE Transactions on Visualization and Computer Graphics 15:1489–1496.

    https://doi.org/10.1109/TVCG.2009.118

    • PubMed
    • Google Scholar
87. 1. Weese DJ
    2. Ferguson MM
    3. Robinson BW

    (2012) Contemporary and historical evolutionary processes interact to shape patterns of within-lake phenotypic divergences in polyphenic pumpkinseed sunfish, Lepomis gibbosus

    Ecology and Evolution 2:574–592.

    https://doi.org/10.1002/ece3.72

    • PubMed
    • Google Scholar
88. 1. Wolff C
    2. Roy S
    3. Ingham PW

    (2003) Multiple muscle cell identities induced by distinct levels and timing of hedgehog activity in the zebrafish embryo

    Current Biology 13:1169–1181.

    https://doi.org/10.1016/s0960-9822(03)00461-5

    • PubMed
    • Google Scholar
89. 1. Wu GY
    2. Cline HT

    (1998) Stabilization of dendritic arbor structure in vivo by CaMKII

    Science (New York, N.Y.) 279:222–226.

    https://doi.org/10.1126/science.279.5348.222

    • PubMed
    • Google Scholar
90. 1. Xiong F
    2. Ma W
    3. Hiscock TW
    4. Mosaliganti KR
    5. Tentner AR
    6. Brakke KA
    7. Rannou N
    8. Gelas A
    9. Souhait L
    10. Swinburne IA
    11. Obholzer ND
    12. Megason SG

    (2014) Interplay of cell shape and division orientation promotes robust morphogenesis of developing epithelia

    Cell 159:415–427.

    https://doi.org/10.1016/j.cell.2014.09.007

    • PubMed
    • Google Scholar
91. Book

    1. Yamamoto T

    (1975)

    Medaka (Killifish): Biology and Strains

    Tokyo: Keigaku Publishing Company.

    • Google Scholar
92. 1. Zhang Y
    2. Liu T
    3. Meyer CA
    4. Eeckhoute J
    5. Johnson DS
    6. Bernstein BE
    7. Nusbaum C
    8. Myers RM
    9. Brown M
    10. Li W
    11. Liu XS

    (2008) Model-based analysis of ChIP-Seq (MACS

    Genome Biology 9:R137.

    https://doi.org/10.1186/gb-2008-9-9-r137

    • PubMed
    • Google Scholar
93. 1. Zhou W
    2. Lin L
    3. Majumdar A
    4. Li X
    5. Zhang X
    6. Liu W
    7. Etheridge L
    8. Shi Y
    9. Martin J
    10. Van de Ven W
    11. Kaartinen V
    12. Wynshaw-Boris A
    13. McMahon AP
    14. Rosenfeld MG
    15. Evans SM

    (2007) Modulation of morphogenesis by noncanonical Wnt signaling requires ATF/CREB family-mediated transcriptional activation of TGFbeta2

    Nature Genetics 39:1225–1234.

    https://doi.org/10.1038/ng2112

    • PubMed
    • Google Scholar

Author details

1. Ann Kathrin Heilig

   1. Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo, Japan
   2. Centre for Organismal Studies, Heidelberg University, Heidelberg, Germany
   3. Heidelberg Biosciences International Graduate School, Heidelberg, Germany

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

2. Ryohei Nakamura

   Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo, Japan

   Contribution

   Data curation, Formal analysis, Funding acquisition, Investigation, Validation, Writing - review and editing

   Competing interests

   No competing interests declared

3. Atsuko Shimada

   Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo, Japan

   Contribution

   Data curation, Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

4. Yuka Hashimoto

   Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo, Japan

   Contribution

   Data curation, Formal analysis, Investigation

   Competing interests

   No competing interests declared

5. Yuta Nakamura

   Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo, Japan

   Contribution

   Resources

   Competing interests

   No competing interests declared

6. Joachim Wittbrodt

   Centre for Organismal Studies, Heidelberg University, Heidelberg, Germany

   Contribution

   Supervision, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8550-7377

7. Hiroyuki Takeda

   Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo, Japan

   Contribution

   Conceptualization, Funding acquisition, Supervision, Writing - review and editing

   For correspondence

   htakeda@bs.s.u-tokyo.ac.jp

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-7932-6358

8. Toru Kawanishi

   Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo, Japan

   Contribution

   Conceptualization, Funding acquisition, Investigation, Resources, Supervision, Writing - review and editing

   For correspondence

   toru.kawanishi@bs.s.u-tokyo.ac.jp

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7038-9769

• 1,200

  views

• 138

  downloads

• 7

  citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.