ECB-ART-43145Evodevo 2013 Dec 02;41:33. doi: 10.1186/2041-9139-4-33.
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Myogenesis in the sea urchin embryo: the molecular fingerprint of the myoblast precursors.
BACKGROUND: In sea urchin larvae the circumesophageal fibers form a prominent muscle system of mesodermal origin. Although the morphology and later development of this muscle system has been well-described, little is known about the molecular signature of these cells or their precise origin in the early embryo. As an invertebrate deuterostome that is more closely related to the vertebrates than other commonly used model systems in myogenesis, the sea urchin fills an important phylogenetic gap and provides a unique perspective on the evolution of muscle cell development. RESULTS: Here, we present a comprehensive description of the development of the sea urchin larval circumesophageal muscle lineage beginning with its mesodermal origin using high-resolution localization of the expression of several myogenic transcriptional regulators and differentiation genes. A few myoblasts are bilaterally distributed at the oral vegetal side of the tip of the archenteron and first appear at the late gastrula stage. The expression of the differentiation genes Myosin Heavy Chain, Tropomyosin I and II, as well as the regulatory genes MyoD2, FoxF, FoxC, FoxL1, Myocardin, Twist, and Tbx6 uniquely identify these cells. Interestingly, evolutionarily conserved myogenic factors such as Mef2, MyoR and Six1/2 are not expressed in sea urchin myoblasts but are found in other mesodermal domains of the tip of the archenteron. The regulatory states of these domains were characterized in detail. Moreover, using a combinatorial analysis of gene expression we followed the development of the FoxF/FoxC positive cells from the onset of expression to the end of gastrulation. Our data allowed us to build a complete map of the Non-Skeletogenic Mesoderm at the very early gastrula stage, in which specific molecular signatures identify the precursors of different cell types. Among them, a small group of cells within the FoxY domain, which also express FoxC and SoxE, have been identified as plausible myoblast precursors. Together, these data support a very early gastrula stage segregation of the myogenic lineage. CONCLUSIONS: From this analysis, we are able to precisely define the regulatory and differentiation signatures of the circumesophageal muscle in the sea urchin embryo. Our findings have important implications in understanding the evolution of development of the muscle cell lineage at the molecular level. The data presented here suggest a high level of conservation of the myogenic specification mechanisms across wide phylogenetic distances, but also reveal clear cases of gene cooption.
PubMed ID: 24295205
PMC ID: PMC4175510
Article link: Evodevo
Species referenced: Echinodermata
Genes referenced: ehfl foxc1 foxf1 foxl1 foxy gcml LOC100887844 LOC100893907 LOC105442066 LOC115919910 LOC753216 LOC753325 mafl mef2cl six1 sox4 sox9 tbx3l tcf21l tubgcp2
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|Figure 1. Progression of MHC-positive cell lineages during sea urchin development.MHC and Trop1 expression in the gastrula (40 to 48 h), prism, (48 to 60 h), early (60 to 72 h) and late pluteus stage (72 to 96 h) . Expression of MHC(A, F, K, P) and Trop1(B, G, L, Q) was localized by colorimetric in situ hybridization. The embryos in panels A and B are shown in a vegetal view; the ones in panels C-T are viewed along the animal top/vegetal down (A/V) axis. The inset in panel I is double fluorescent in situ hybridization (FISH) showing the co-expression of CapZ (green) and MHC (red) in myoblasts. Double FISH indicates that Trop1 (green) and MHC (red) are co-expressed in the muscles of a late pluteus (inset in Q). FISH was used to localize the expression of MHC (red; C, D, H, I, M, N, R, S). Bright field images (C, H, M, R) and confocal stacks (D, I, N, S) that include 4',6-diamidino-2-phenylindole (DAPI) (blue) staining are shown. Embryos in C, D, E and T are shown as lateral views with the oral side to the right. The ciliary band and gut internal cilia were stained anti-acetylated tubulin (shown as green in S). The inset is a magnified view of the muscle fibers from a single confocal plane. A schematic representation of the progression of MHC-positive cells in the formation of the muscle fibers is shown (E, J, O, T). Ventrolateral processes (v), the cardiac sphincter (cs; red arrow), the pyloric sphincter (ps; black arrow) and the anal sphincter (as; yellow arrow) are indicated. Muscle fibers are indicated as white (N, S) or black (O, T) arrows.|
|Figure 2. Whole mount in situ hybridization (WMISH) of sea urchin regulatory genes whose orthologs are known myogenic factors.Sum1/MyoD1, MyoD2, MyoR, Maf, Myocardin, Mef2, Twist and Tbx6 expression was localized using WMISH at the gastrula (44 to 48 h; A-H), prism (60 to 65 h; A’-H’), and pluteus larva stage (72 to 80 h; A”-H”). All embryos are viewed along the animal top/vegetal down axis with the exceptions of panel A, which is shown in a vegetal view with the oral side on the bottom, B’, C’, D’ and H are viewed in a lateral view with the oral side on the left (B’) or right (C’, D’ and H’). Domains of expression other than the tip of the archenteron and the coelomic pouches are indicated as follows: black arrow, primary mesenchyme cells (PMC); black arrowhead, the cardiac sphincter; white arrowhead, the apical organ; black asterisk, the blastopore.|
|Figure 3. Co-expression analysis of putative sea urchin myogenic factors and MHC by double confocal fluoresecent in situ hybridization (FISH). Relative spatial domain of expression of FoxF(A), FoxC(B), FoxY(C), FoxL1(D), Six1/2(E), SoxE(F), MaF(G), Myocardin(H), MyoR2(I), Tbx6(J), Mef2(K), and Twist(L) (green) with respect to MHC (red) by double FISH in the late gastrula stage, 48 to 50 h. Every picture is a full projection of merged confocal stacks. Aside each picture are separately placed single focal planes of each channel plus a merged picture of an enlarged detail at the tip of the archenteron, to clarify any misleading issue of co-expression domains. In E, a single brightfield slice is also superimposed to the red channel. Yellow circles indicated by yellow arrowheads show co-expressing cells, and the white ones point to absence of co-expression. The white arrows indicate other domains of expression. All the embryos are viewed along the animal top/vegetal down (A/V) axis from the oral or aboral surface, excluding the one reported in L which is shown in a lateral view along the A/V axis with the oral side on the right. Nuclei are stained blue with 4',6-diamidino-2-phenylindole (DAPI).|
|Figure 4. Co-expression analysis of sea urchin putative regulatory factors at the tip of the archenteron by double confocal fluorescent in situ hybridization (FISH). Relative disposition of FoxF(A-C, E, H and K), FoxC(A, D and G), FoxY(D-F and J), FoxL1(C), Six1/2(K and L), SoxE(B, F, I and L), Tbx6(J) and SoxC(G-I) transcripts by double FISH in the late gastrula stage, 48 to 50 h. Every picture is a full projection of merged confocal stacks. FoxF is stained in red, FoxY and FoxL1 in green, FoxC in purple, Six1/2 and Tbx6 in magenta, SoxE in cyan and SoxC in yellow. Panel K is the only exception where Six1/2 is depicted in green. Full projection of split channels showing enlarged details of the tip of the archenteron are placed aside each picture. Yellow circles indicated by yellow arrowheads show cells co-expressing the analyzed genes. White arrows in G-I indicate the position of the presumptive anal and cardiac sphincters. The ectodermal staining in C corresponds to ciliary band expression of the FoxL1 gene. All the embryos are viewed in a lateral view along the animal top/vegetal down axis with the oral side on the left. Nuclei are labeled blue with 4',6-diamidino-2-phenylindole (DAPI).|
|Figure 5. Map of the regulatory state of the main mesodermal domains of the tip of the sea urchin archenteron at the late gastrula stage. (A) Schematic representation of a 48-h sea urchin embryo archenteron in lateral view along the animal top/vegetal down axis. The cell fate of each region is indicated as follows: Ab, aboral; An, animal; BC, blastocoelar cells; GC, germ cells; HC, hydropore canal; M, muscles; O, oral; V, vegetal. (B) Different mesodermal regions identified by specific regulatory signatures at the tip of the archenteron are shown in different colors distributed over the three major domains defined in this study. Regions of partial overlaps and names of the genes expressed in each region are shown in colors associated with each domain.|
|Figure 6. Dynamics of gene expression in the putative myoblast precursors of the sea urchin embryo. Fluorescent whole mount in situ hybridization and relative position of FoxY (green), FoxC (red) and FoxF (magenta) transcripts in the interval from 24 to 45 h. Each picture is a full projection of merged confocal stacks. Yellow circles indicated by yellow arrowheads show co-expression cells; white circles show absence of co-expression. White arrows in panel A indicate the emergence of FoxY transcription in the NSMs. For single-channel full projections of the images reported in D-I see Additional file 11: Figure S9. All the embryos are viewed in lateral view along the animal top/vegetal down axis with the exception of A, B and C that are seen in a vegetal view. Nuclei are labeled blue with 4',6-diamidino-2-phenylindole (DAPI).|
|Figure 7. Co-expression analysis of sea urchin putative myogenic factors and non-skeletogenic mesoderm (NSM) and small micromere (SM) molecular markers. (A-I) Expression of FoxC, FoxY, Ese, Gcm, Six1/2, SoxE, Tbx6 and Nanos was localized by double confocal fluorescent in situ hybridization at the very early gastrula stage, 30 to 32 h. Every picture is a full projection of merged confocal stacks. Yellow circles indicated by yellow arrowheads show co-expressing cells, and the white ones show absence of co-expression. For single-channel projections of the images reported in C, F and I see Additional file 11: Figure S9. All the embryos are viewed in a vegetal view with the exception of A, D, E, G and H that are seen in lateral view along the animal top/vegetal down axis. Nuclei are labeled blue with 4',6-diamidino-2-phenylindole (DAPI). In panels J and K a schematic representation is shown of a regulatory stage map of NSM of a 30-h sea urchin embryo orientated along the oral right/aboral left (O/Ab) axis in both lateral (J) and vegetal views (K). The different mesodermal cell types identified by specific regulatory signatures at the vegetal plate are shown in different colors: Mp, putative myoblast precursors, green with orange and red horizontal lines; BCp, blastocoelar cell precursors, yellow; PCp, pigment cell precursors, blue; SMd + Y, small micromere derivatives (SMd) plus FoxY expressing NSM cells (Y), green with an orange horizontal line. A legend indicates the names of the genes expressed in each region. For the sake of simplicity, primary mesenchyme cells are not shown.|
References [+] :
Amin, A Zn-finger/FH2-domain containing protein, FOZI-1, acts redundantly with CeMyoD to specify striated body wall muscle fates in the Caenorhabditis elegans postembryonic mesoderm. 2007, Pubmed