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W4285377708 abstract "Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Abstract The dorsal axial muscles, or epaxial muscles, are a fundamental structure covering the spinal cord and vertebrae, as well as mobilizing the vertebrate trunk. To date, mechanisms underlying the morphogenetic process shaping the epaxial myotome are largely unknown. To address this, we used the medaka zic1/zic4-enhancer mutant Double anal fin (Da), which exhibits ventralized dorsal trunk structures resulting in impaired epaxial myotome morphology and incomplete coverage over the neural tube. In wild type, dorsal dermomyotome (DM) cells reduce their proliferative activity after somitogenesis. Subsequently, a subset of DM cells, which does not differentiate into the myotome population, begins to form unique large protrusions extending dorsally to guide the epaxial myotome dorsally. In Da, by contrast, DM cells maintain the high proliferative activity and mainly form small protrusions. By combining RNA- and ChIP-sequencing analyses, we revealed direct targets of Zic1, which are specifically expressed in dorsal somites and involved in various aspects of development, such as cell migration, extracellular matrix organization, and cell-cell communication. Among these, we identified wnt11 as a crucial factor regulating both cell proliferation and protrusive activity of DM cells. We propose that dorsal extension of the epaxial myotome is guided by a non-myogenic subpopulation of DM cells and that wnt11 empowers the DM cells to drive the coverage of the neural tube by the epaxial myotome. Editor's evaluation This study addresses an interesting and underexplored question in developmental biology, specifically cell migration and muscle development. It builds upon prior analysis of the medaka Double anal fin (Da) mutants by using detailed bioinformatic and time-lapse analysis to explain dorsal somite extension. The authors show that dorsal muscle morphogenesis is actively guided by dorsal dermomyotome cells, rather than being passively shaped by physical constraints alone. Looking downstream of Da, they show that Wnt signaling is central to dorsal extension of the epaxial myotome and propose that similar functions may shape the dorsal musculature across vertebrates. https://doi.org/10.7554/eLife.71845.sa0 Decision letter Reviews on Sciety eLife's review process Introduction 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 The epaxial myotome of the Da mutant fails to cover the neural tube at the end of embryonic development. (A) Schematic representation of dorsal somite extension which results in the full coverage of the neural tube by the epaxial myotomes at the end of embryonic development. (B) Lateral view of adult Wt medaka. Dorsal, caudal, and anal fins are outlined. (C) Lateral view of whole-mount in situ hybridization against zic1 in a 12 ss (1.7 dpf, stage 23) Wt embryo. zic1 expression can be observed in the brain, neural tissues and the dorsal somites. (D) Lateral view of adult Da mutant. Dorsal, anal and caudal fins are outlined. The dorsal trunk region resembles the ventral trunk region. (E) Lateral view of whole-mount in situ hybridization against zic1 of a 12 ss Da embryo. zic1 expression can be observed in the brain and the neural tissues, but is drastically decreased in the dorsal somites (arrowhead). (F, H) Cross-sections of tail regions of hematoxylin stained 9 dpf embryos. Dorsal ends of myotomes are outlined. In Wt, the left and the right myotome come in close contact at the top of the neural tube and form a gapless muscle layer (F). In the Da mutant, the left and right myotome fail to come in contact at the top of the neural tube (H). (G, I) Dorsal view of whole-mount Phalloidin (magenta) immunostaining labeling the myotome of Wt (G) and Da (I) embryos. Epaxial myotome is outlined, and asterisks label melanophores. The contour of the myotomes was drawn based on the Z-stack images of the dorsal myotomes to avoid ambiguity caused by melanophores. Anterior to the left. (J) Schematic representation of measurements to analyze the distance between the left and the right dorsal tip of the myotome (yellow) and the cross-sectional area of the dorsal myotome. For each measurement, three consecutive optical cross sections of the 10th somite were analyzed and averaged. (K) Distance between the left and right tip of the dorsal myotome 5.5 dpf – 9 dpf (n = 6 and 5 for Wt and Da embryos, respectively at 5.5 dpf (stage 35) (p = 0.097); n = 5 and 5 at 6 dpf (stage 36) (p = 0.075); n = 8 and 8 at 7 dpf (stage 37) (p = 0.0047); n = 5 and 6 at 8 dpf (stage 38) (p = 0.019); n = 7 and 6 at 9 dpf (stage 39) (p = 0.034); median, first and third quartiles are shown). (L) Cross-sectional area of the dorsal somites at 22 ss (2.25 dpf, stage 26; n = 10 somites of 5 Wt embryos, n = 12 somites of 6 Da embryos) (p = 0.038), 7 dpf (n = 10 somites of 5 Wt embryos, n = 10 somites of 5 Da embryos) (p = 0.044) and 9 dpf (n = 8 somites of 4 Wt embryos, n = 6 somites of 3 Da embryos) (p = 0.0019). Median, first and third quartiles are shown. HM, horizontal myoseptum; NT, neural tube. Scale bar = 50 μm. ** p < 0.01, * p < 0.05, ns, not significant. 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. Results 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). The dorsal myotome of the Da mutant fails to cover the neural tube In wild-type (Wt) medaka, the dorsal ends of the myotomes first came in contact at 7 days post fertilization (dpf, stage 37) and formed the tight, thick myotome layer covering the neural tube at the end of embryonic development (9 dpf, stage 39) (Figure 1F–G, Figure 1—figure supplement 1A-F). In the ventralized Da mutant, however, the dorsal ends of the myotomes did not extend sufficiently and failed to cover the neural tube at the end of embryonic development (Figure 1H–I, Figure 1—figure supplement 1G-L). The ends of the ventralized dorsal myotome in the mutant displayed a round shape (not pointed as found in Wt) which resembled the morphology of the ventral myotome. We wondered if there are other morphological differences between Wt dorsal myotome and the ventralized dorsal myotome of the Da mutant. Indeed, the cross-sectional area in the Da mutant was significantly larger compared to Wt (Figure 1L). Possible explanations for a larger cross-sectional area in the Da mutant could be a larger myofiber diameter or a higher number of myofibers, which make up the myotome. We measured the diameter of dorsal myotome muscle fibers in Wt and Da mutant embryos but could not observe a difference, suggesting that dorsal myotome of the Da mutant has a higher number of myotomal cells (Figure 1—figure supplement 1M). 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 show lower proliferative activity after the confinement of zic1 expression to the dorsal somite. (A) Lateral view of 35 ss (3.4 dpf, stage 30) embryo, 10th somite is positioned in the center. Pax3/7 (green) labels DM cells and Phalloidin (magenta) labels myotome. The horizontal myoseptum (HM) separates the myotome into epaxial myotome (dorsal) and hypaxial myotome (ventral). (B–B’’) Optical cross sections, myotome is labeled by Phalloidin (magenta) and encompassed by a one-cell thick layer of DM labeled with Pax3/7 (green). Asterisks mark neural crest cells which are highly Pax3/7-positive (B’’). (C, D) Lateral view of Wt (C) and Da (D) 35 ss embryos labeled with Pax3/7 (green) and pH3 (magenta; representatives are indicated by arrowheads). Asterisks mark neural crest cells. (E) Quantification of pH3-positive cells in Wt and Da at 12 ss (n = 46.5 somites from 4 Wt embryos, n = 95 somites from 7 Da embryos; p = 0.48), 16 ss (n = 54.5 somites from 5 Wt embryos, n = 42.5 somites from 4 Da embryos; p = 0.0038), 22 ss (n = 66 somites from 6 Wt embryos, n = 40.5 somites from 5 Da embryos; p = 0035) and 35 ss (n = 49 somites from 6 Wt embryos, n = 47.5 somites from 5 Da embryos; p = 0.0008). Median, first and third quartiles are shown. ns, not significant, **p < 0.01, ***p < 0.001. Anterior to the left. Dorsal to the top. DM, dermomyotome; HM, horizontal myoseptum; M, myotome; NT, neural tube; NC, notochord. Scale bar = 50 μm. 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 Wt dorsal DM cells form numerous large, motile protrusions at the onset of somite extension. (A, B) Dorsal view of time-lapse in vivo imaging of onset of dorsal somite extension at 24 ss (2.4 dpf, stage 27) of Tg(zic1:GFP) (A) and Tg(zic1:GFP);Da (B) embryos. 15th somite is positioned in the center, z-stacks were imaged every 10 min, time is displayed in min. Asterisks indicate migrating melanophore. Scale bar = 50 μm. (C, F) Lateral view of in vivo imaging of Tg(zic1:GFP) (C) and Tg(zic1:GFP);Da (F). Signals in dorsal somites are highlighted in green. Arrowheads indicate small protrusions, arrow indicates large protrusion. Scale bar = 50 μm. (D, G) Dorsal view of in vivo imaging of large protrusion in Wt (D) and Da (G). Embryos were injected with Actin-Chromobody GFP (AC-GFP) mRNA. Signals in somitic cells are highlighted in green. Brackets indicate a lamellipodia-like structure. Arrows indicate filopodia branching out from dorsal tips of lamellipodia-like core. Asterisks indicate sclerotome cells. Scale bar = 25 μm. (E, H) Summary of dorsal DM cell protrusions in Wt (E) and Da (H) embryos. Arrowheads indicate small protrusions, brackets indicate lamellipodia-like core structure of large protrusions, and arrows indicate filopodia bundles branching off from dorsal tips of large protrusions. (I-I’’) Quantification of protrusions from Tg(zic1:GFP) (n = 14 embryos) and Tg(zic1:GFP);Da (n = 11 embryos). Protrusions of the 8th-12th somite were counted (mean ± SD, p = 0.01 for small protrusions, p = 3.3e-08 for large protrusions, p = 2.1e-05 for total protrusions). (J–K) Lateral view of protrusions extracted from time-lapse imaging of (A–B). Arrowheads indicate tip of protrusions, time is displayed in min. (J) Protrusion observed in Tg(zic1:GFP). (K) Protrusion observed in Tg(zic1:GFP);Da. Scale bar = 25 μm. (L, M) Z-planes of large protrusions of 24 ss embryos treated with DMSO (L) or ML141 (M). Arrowheads indicate filopodia. Scale bar = 25 μm. (N) Distance between dorsal somite tip and top of neural tube in 24 ss embryos (n = 12 somites from 6 Wt embryos, n = 8 somites from 4 Da embryos, p = 6.8e-05; n = 10 somites from 5 embryos treated with DMSO, n = 14 somites from 7 embryos treated with ML141, p = 0.021). Median, first and third quartiles are shown. ****p < 0.0001, *p < 0.5. Anterior to the left. DM, dermomyotome; M, myotome; NT, neural tube. Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Onset of dorsal somite extension in Tg(zic1:GFP). Dorsal view of time-lapse in vivo imaging of 24 ss Tg(zic1:GFP) embryo. 15th somite is positioned in the center, z-stacks were imaged every 10 min, time is displayed in min. Anterior to the left. Scale bar = 50 μm. 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 This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Onset of dorsal somite extension in Tg(zic1:GFP);Da. Dorsal view of time-lapse in vivo imaging of 24 ss Tg(zic1:GFP);Da embryo. 15th somite is positioned in center, z-stacks were imaged every 10 min, time is displayed in min. Bright cell at the bottom migrating to the right is a melanophore. Anterior to the left. Scale bar = 50 μm. 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 DM cells delaminate from the dorsal somite at the end of dorsal somite extension and accumulate between somites. (A–F) Dorsal (top) and cross-sectional (bottom) views of Tg(zic1:GFP) at 4.5 dpf (A), 5 dpf (B), and 6 dpf (C) and Tg(zic1:GFP);Da at 4.5 dpf (D), 5 dpf (E) and 6 dpf (F). 10th somite is positioned in center. Arrowheads point to exemplary mesenchymal DM cells, and asterisks mark melanophores. (G, H) Dorsal view of mesenchymal DM cells of Tg(zic1:GFP) (G) and Tg(zic1:GFP);Da (H). (I) Z-plane of dorsal view of 10th somite of Tg(zic1:GFP) embryo. Mesenchymal DM (mes. DM) cells are colored green. Arrowheads indicate exemplary protrusions formed between somitic DM cell and mesenchymal DM cell. (J-K’) Cross-sections of 9th somite before ablation (J), 8 hr post ablation (hpa) (J’), and 10th somite before ablation (K) and 8 hpa (K’) of Tg(zic1:GFP) 5.5 dpf embryo. Green arrowhead indicates the ablation site, and asterisk indicates a pigment cell. (L) Quantification of relative distance between the left and the right tips of the 9th and 10th somites after laser ablation (n = 6 Tg(zic1:GFP) embryos). (M-O’) Fate mapping, using KikGR-mediated photoconversion, of dorsal DM cells with protrusions. (M) A single dorsal DM cell at the dorsal tip of a somite (arrowhead) was labeled at 3.5 dpf. Cross-section of the trunk is shown. Asterisk indicates an ectopically labeled epidermis cell. (N-O’) At hatching stage (9 dpf), descendants of the labeled DM cell contribute to blood vessels (arrowheads in N, N’) and fin mesenchyme in the dorsal fin fold (arrowheads in O, O’). (N) and (O), lateral views; (N’) and (O’), cross sections. Asterisks indicate autofluorescent pigment cells. Anterior to the left. M, myotome; NT, neural tube; S, somite. Scale bar = 25 μm. Video 3 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Mesenchymal DM cells during dorsal somite extension at 4.5 dpf. Dorsal view of time-lapse in vivo imaging of 4.5 dpf Tg(zic1:GFP) embryo. 10th somite positioned in center, z-stacks were imaged every 10 min, time is displayed in min. Arrowhead indicates representative mesenchymal DM cell. Anterior to the left. Scale bar = 50 μm. Video 4 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Mesenchymal DM cells during dorsal somite extension at 5.5 dpf. Dorsal view of time-lapse in vivo imaging of 5.5 dpf Tg(zic1:GFP) embryo. 10th somite is positioned in center, z-stacks were imaged every 10 min, time is displayed in min. Arrowhead indicates representative mesenchymal DM cell. Anterior to the left. Scale bar = 50 μm. 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 10th 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 10th somites increased after ablating the mesenchymal DM cells while the neighboring 9th 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 10th 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)" @default.
W4285377708 created "2022-07-14" @default.
W4285377708 date "2021-08-18" @default.
W4285377708 modified "2023-09-27" @default.
W4285377708 title "Decision letter: Wnt11 acts on dermomyotome cells to guide epaxial myotome morphogenesis" @default.
W4285377708 doi "https://doi.org/10.7554/elife.71845.sa1" @default.
W4285377708 hasPublicationYear "2021" @default.
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