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The two somite compartments, dorso-lateraldermomyotome and medio-ventralsclerotome are major vertebrate novelties, but little is known about their evolutionary origin. We determined that sclerotome cells in Xenopus come from lateral somitic frontier (LSF) by lineage tracing, ablation experiments and histological analysis. We identified Twist1 as marker of migrating sclerotome progenitors in two amphibians, Xenopus and axolotl. From these results, three conclusions can be drawn. First, LSF is made up of multipotent somitic cells (MSCs) since LSF gives rise to sclerotome but also to dermomytome as already shown in Xenopus. Second, the basic scheme of somite compartmentalization is conserved from cephalochordates to anamniotes since in both cases, lateral cells envelop dorsally and ventrally the ancestral myotome, suggesting that lateral MSCs should already exist in cephalochordates. Third, the transition from anamniote to amniote vertebrates is characterized by extension of the MSCs domain to the entire somite at the expense of ancestral myotome since amniote somite is a naive tissue that subdivides into sclerotome and dermomyotome. Like neural crest pluripotent cells, MSCs are at the origin of major vertebrate novelties, namely hypaxial region of the somite, dermomyotome and sclerotome compartments. Hence, change in MSCs properties and location is involved in somite evolution.
Fig. 1. Sclerotome cells originate from lateral somitic frontier. (A) Scheme of the experimental procedure: lineage tracing experiments with WGA-rhodamine injected in the medial segmental plate and with WGA-fluorescein injected in the Meox2 expression domain at LSF. a to c: results for three embryos double injected at stage 13. d to i: Transverse section at stage 28. WGA-rhodamine (d, e, f), WGA-fluorescein (g, h, i), merge (j, k, l). Dash lines indicate the position of bilateral symmetry plane. Horizontal lines define the limit between anterior and trunk regions. Anterior side on the top. Nc, notochord. Nt, neural tube. (B) Scheme of the experimental procedure: lineage tracing experiments with WGA-fluorescein injected in the Meox2 expression domain at LSF. Fixed embryos were stained with Hoechst 33258 nucleus marker. Transverse section was performed at stage 28. Blue Hoechst 33258 nucleus marker (a), green WGA-fluorescein (b), merge (c). Arrows designs cells lining the notochord and muscle cells. (C) Scheme of the experimental procedure: lineage tracing experiments with WGA-rhodamine injected in the medial segmental plate. Fixed embryos were stained with Hoechst 33258 nucleus marker and 12/101 specific antibody for muscle cells. Transverse section were performed at stage 28. Blue nuclear staining alone (a) with red WGA-rhodamine (b), with green muscle cells (c), merge (d). (D) Scheme of the experimental procedure: embryos were injected in LSF with WGA-rhodamine at stage 13 and then transverse trunk sections at stage 28 were submitted to indirect immunofluorescence with 12/101 (a) or MF20 antibodies (d). b and e, WGA-rhodamine staining; c and f, merge. Rectangle dotted line designs the area magnification at the right side (a’, b’, c’ and d’, e’, f’). (E) Scheme of the experimental procedure: embryos were injected in LSF with WGA-fluorescein (green) and then submitted to ISH with Twist1 probe or directly observed. a, Twist1 expression analysis in an uninjected embryo; b-c, serial sections submitted to ISH with Twist1 probe (b) or directly observed (c). (F) Scheme of the experimental procedure: Pax1 plus Pax9 expression analysis in an uninjected embryo (a). Serial sections of an embryo injected in LSF with WGA-rhodamine (red) submitted to ISH with Pax1 and Pax9 probes (c) compared to ISH negative control (b) or directly observed (d). Arrows design co-staining areas. Scale bars: 100 μm.
Fig. 2. Twist1 is expressed in seemingly migrating sclerotome cells. (A) Scheme of the experimental procedure: expression of the somite patterning genes Meox2, Tcf15, Foxc1, Foxc2 and Twist1 in comparison to Myod1 on trunk transverse sections between stages 20 and 34. The analysed gene is indicated at the bottom right of each picture. Arrows design the stained sclerotome. (B) Postero-anterior expression of Twist1 at stage 28 on transverse section (a to f). (C) Somitic ventral migrating cells (arrows) on postero-anterior transverse sections (a to e). Orderly serial transverse sections stained by myotome marker (12/101 antibody, green), membrane lipophilic diI (red), and Hoechst 33258 nucleus marker (blue). DiI is used here to stain all cells. Rectangle dotted line designs the area magnification at the right side of each section (a’ to e’). Scale bars: 100 μm.
Fig. 3. The WGA tracer co-localizes with Twist1-expressing cells in the ventral route. (A) Scheme of the experimental procedure: postero-anterior sections of embryos (a to d) at stages 18, 21, 23 and 25 were stained by a myotome marker (12/101 antibody, green), WGA-rodhamine (red), and Hoechst 33258 nucleus marker (blue). WGA is used here to mark all cells and to better visualize the notochord contour. Brackets design somitic ventral migrating cells. Rectangle dotted line designs the area magnification at the bottom (stage 18) or at the right side (stages 21, 23 and 25) of each section (a’ to d’). Scale bars: 100 μm. (B) Postero-anterior expression of Twist1 at stage 18, 21, 23 and 25 on transverse sections (a to d). (C) Scheme of the experimental procedure: lineage tracing experiments with WGA-rhodamine (red) injected in the Meox2 expression domain at stage 13, fixed and sectioned at stage 22/23, then sections were stained with a myotome marker (12/101 antibody, green) and Hoechst 33258 nucleus marker. (D) Scheme of the experimental procedure: lineage tracing experiments with WGA-rhodamine (red) injected in the Meox2 expression domain at stage 13, fixed and sectioned at stage 22/23, then slides were stained with Hoechst 33258 nucleus marker. The same slides were next submitted to in situ hybridization with Twist1 probe to evaluate co-staining of WGA tracer with Twist1 expression. Arrows design cells labelled by WGA-rhodamine. (E), (F) and (G). Ablation of lateral somitic domain prevents sclerotome and hypaxial muscles formation. Scheme of the experimental procedure: ablation experiments of the lateral somitic domain at stage 17. Embryos were fixed and analysed for Twist1 (E), MyhE3 (F) and Mef2c (G) expressions (arrows) and in comparison with 12/101 staining in E. Abl. C, sham-operated embryos. Abl. M, medial domain was removed. Abl. L, lateral domain was removed. For statistical analysis, number of embryos is as follows: same expression, faint decreased, strong decreased. Kruskal-Wallis test and Dunn's multiple comparisons test revealed significant difference between Abl. L with Abl. C or Abl. M, for both Twist1 p < 0.0001, (Abl. C 13, 2, 0; Abl. M 13, 3, 0; Abl. L 0, 2, 13), and Mef2c p < 0.0001, (Abl. C 24, 0, 0; Abl. M 15, 6, 0; Abl. L 3, 0, 16) expressions. Mann-Whitney test revealed significant difference between Abl. L with Abl. C for MyhE3 expression p < 0.0001 (Abl. C 15, 0, 0; Abl. L 0, 5, 10).
Fig. 4. Twist1 marks lateral migrating sclerotome cells in axolotl. Scheme of experimental procedure: postero-anterior transverse sections (a to g) of axolotl embryos at stage 31 after whole mount in situ hybridization with Twist1 (A) or immunohistochemistry with 12/101 antibody (B). Arrows design migrating sclerotome progenitors. Rectangle dotted line designs the area magnification at the bottom of each panel (A and B, a’ to g’). (C) Schematic representation of somite compartmentalization in Xenopus. MSC domain is located at LSF at early neurulation (stage 13). LateralMyod1 expression domain could overlap the MSC domain. At mid-neurulation (stage 17/18), differentiation of lateralmyotome is initiated and early markers of dermomyotome and sclerotome like Pax3 (della Gaspera et al., 2012b) and Twist1 begin to be expressed. At stage 22 lateral cells migrate dorsally to give rise the dermomyotome. Next, sclerotome progenitors expressing Twist1 migrate ventrally to give rise to the sclerotome located medially at stage 28. (D) Evolution of somite compartmentalization based on axochord hypothesis. The axochord hypothesis (Brunet et al., 2015) proposes that the notochord evolves from a ventromedian muscle (the axochord in annelids) present in Urbilateria, the last common ancestor of bilaterian animals, and suggests that transverse muscles attached to it, could give rise to the ancestral myotome in cephalochordates. The origin of MSCs in Urbilateria is unknown. MSCs probably already exist in cephalochordates. The transition from anamniotes to amniotes is characterized by extension of MSCs domain at the expense of ancestral myotome. The chordate dorso-ventral axis is inverted compared with Urbilateria. VM, ventromedian muscle; TM, transverse muscle; M, medialsomite; L, lateralsomite.
