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. 2013 Apr;140(8):1740-50.
doi: 10.1242/dev.092726.

The Transition From Differentiation to Growth During Dermomyotome-Derived Myogenesis Depends on Temporally Restricted Hedgehog Signaling

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Free PMC article

The Transition From Differentiation to Growth During Dermomyotome-Derived Myogenesis Depends on Temporally Restricted Hedgehog Signaling

Nitza Kahane et al. Development. .
Free PMC article

Abstract

The development of a functional tissue requires coordination of the amplification of progenitors and their differentiation into specific cell types. The molecular basis for this coordination during myotome ontogeny is not well understood. Dermomytome progenitors that colonize the myotome first acquire myocyte identity and subsequently proliferate as Pax7-expressing progenitors before undergoing terminal differentiation. We show that the dynamics of sonic hedgehog (Shh) signaling is crucial for this transition in both avian and mouse embryos. Initially, Shh ligand emanating from notochord/floor plate reaches the dermomyotome, where it both maintains the proliferation of dermomyotome cells and promotes myogenic differentiation of progenitors that colonized the myotome. Interfering with Shh signaling at this stage produces small myotomes and accumulation of Pax7-expressing progenitors. An in vivo reporter of Shh activity combined with mouse genetics revealed the existence of both activator and repressor Shh activities operating on distinct subsets of cells during the epaxial myotomal maturation. In contrast to observations in mice, in avians Shh promotes the differentiation of both epaxial and hypaxial myotome domains. Subsequently, myogenic progenitors become refractory to Shh; this is likely to occur at the level of, or upstream of, smoothened signaling. The end of responsiveness to Shh coincides with, and is thus likely to enable, the transition into the growth phase of the myotome.

Figures

Fig. 1.
Fig. 1.
Shh and Gli activity in the DM and myotome of chick and mouse embryos. (A-I) ISH on the flank of E2.5 chick embryos. Gli1-3, Boc, Gas1 and Cdo are expressed in DM; Ptc1, Hhip, Sulf1 and Cdo and low levels of Gli1 and Gli2 are in myotome. Dashed lines mark the myotome. (J-P′) GFP, Pax7 and DAPI (gray) staining on Tg(GBS-GFP) control or Shh-/- mouse embryos at the indicated stages and axial levels. O′ and P′ are higher magnifications of boxes in O and P, respectively. Arrows in L-N point at myotomal cells, those in P′ to pyknotic nuclei. All pictures are transverse sections. D, dermis; DM, dermomyotome; M, myotome; NT, neural tube; S, somite; Scl, sclerotome. Scale bars: 50 μm.
Fig. 2.
Fig. 2.
The dynamics of Gli activity defines the sequential development of the mouse myotome. (A-E) Immunostaining for Pax7, Arx, GFP or myosin on Tg(GBS-GFP) mouse embryos at the indicated stages and axial levels. B-B″′ are the same image with different marker combinations. Arrows point at Pax7+/GFP+ or ARX+/GFP+ cells, arrowheads Pax7+/GFP- cells. Dashed lines separate the differentiating, Gli-responsive medial part of the myotome from its more differentiated Arx+ lateral part. D-D″″ are higher magnifications of D showing that Arx and Pax7 are mutually exclusive (D″″) and that subsets of Arx+ and Pax7+ populations express low levels of GFP (D′-D″′). By E12.5, GBS-GFP signal is downregulated at brachial levels (E). Unbroken lines in B″, D and E mark the border between myotome and dermis. (F) Immunostaining for GFP and desmin on E4 chick embryo, in which the medial part of a somite has been electroporated with GFP-DNA at E2. GFP+/Desmin+ pioneer fibers occupy the lateral part of the myotome; younger desmin+/GFP- myocytes its medial domain. (G) Progression of molecular identities during maturation of two distinct myoblast populations. D, dermis, DM, dermomyotome, M, myotome, Scl, sclerotome. Scale bars: in A-C, 100 μm; in D,E, 200 μm; in F, 50 μm.
Fig. 3.
Fig. 3.
Shh is necessary to trigger the differentiation of Pax7+ DM-derived progenitors. (A-H) E2.5 embryos subjected for 16 hours to pluronic gel alone (control) or cyclopamine in pluronic gel. Desmin+ myotomes were smaller in the presence of cyclopamine (dorsal views in A,B), contained more Pax7+ progenitors (C,D and higher magnifications in C′,D′), and exhibited reduced expression of Myf5 and MyoD mRNAs (E-H). (I-T) Expression of PTCΔloop2 cell-autonomously represses myogenic differentiation in both the medial (I-N) and lateral (O-T) myotome. (U) Quantification of the percentage of Pax7+ cells in myotome out of total GFP+ cells. *P<0.003. Error bars represent s.e.m. Dashed line in R-T represents the lateral limit of the desmin+ myotome. DM, dermomyotome; M, myotome; VLL, ventrolateral lip. Scale bar: in C,D,O-T, 50 μm; in E-H, 60 μm; in I-N, 40 μm.
Fig. 4.
Fig. 4.
Misexpression of Hhip in avian sclerotome represses the myogenic activity of No/floor plate-derived Shh. Transverse sections of embryos for which ventral somites were electroporated at E2 and fixed 24 hours later. (A-F) Control GFP (A), Hhip/GFP (B) or PTCΔloop2 (C) embryos. Note in B absence of a lateral myotome with concomitant looping of the VLL (arrow). (D-F) Co-electroporation to the ventral somite (asterisk) of TRE-Hhip+rtTA2s-M2 followed either by immediate activation of expression with Dox (E) or delayed activation after 8 hours (F). Note the small and intermediate sizes of the myotomes in E and F, respectively, compared with controls (A,D). The lateral myotome is virtually absent in E, as in B (arrows). (G-I) The effect of Hhip is rescued by excess Shh. (G) Embryos co-transfected with Shh and Hhip to sclerotome. (H) Sequential electroporation of Shh to ectoderm (arrows) followed by Hhip to sclerotome. (I) Electroporation of Shh alone to ectoderm (arrows). In addition to myotomal expression, desmin is sometimes apparent in the basal side of the medial DM (D), where it is particularly upregulated following local Shh application (H,I). (J) Quantification of the area occupied by desmin+ myotomes in treated compared with intact contralateral sides. Letters under bars refer to treatments/groups described in A-I. Error bars represent s.e.m. *P<0.02, **P<0.0001. Dorsal root ganglia (DRG) are marked by a dashed line. Scale bar: 100 μm. DM, dermomyotome; M, myotome; NT, neural tube; Scl, sclerotome.
Fig. 5.
Fig. 5.
Relative contribution of Gli activator and Gli3 repressor activities to the mouse epaxial myotome developmental. (A-C) Expression of Myf5, myosin and GFP in E9.5 and E10.5 Tg(GBS-GFP) embryos at indicated axial levels. Myf5 in myotome appears before GBS-GFP (A, arrows). GBS-GFP activity is restricted to the epaxial myotome (B; arrowhead delimits epaxial/hypaxial boundary). (C-C″′) Magnifications of the box in C. In the epaxial myotome, Myf5+/myosin+/GBS-GFP- myocytes are found (arrows). (D,E) ISH for GFP in E9.5 Tg(GBS-GFP) control and Gli3-/- embryos. The levels of GFP transcripts in Gli3 mutant myotome are higher than in controls. (F,G) GBS-GFP, Pax7 and Arx expression in E10.5 Tg(GBS-GFP) and Tg(GBS-GFP);Gli3-/- embryos. Dashed lines separate medial and lateral parts of the myotome and unbroken line marks the border between myotome and dermis. In Gli3 mutants, GBS-GFP activity persists longer and is found within the lateral myotome. (H-P) Expression of Myf5, Pax7 and GFP in E11.0 control, Shh-/- and Shh-/-; Gli3-/- embryos all carrying Tg(GBS-GFP). Insets in O and I are magnifications of boxes in main panels. Both Shh-/- and Shh-/-; Gli3-/- embryos lack myotomal GBS-GFP activity; remaining GFP+ cells are blood cells. Yet, lack of Gli3 rescues the reduction of Myf5+ epaxial myotome in absence of Shh function. Lines in H and N demarcate the epaxial myotome. All pictures are transverse sections. DM, dermomyotome, M, myotome, Scl, sclerotome. Scale bar: in A-C,F,G, 50 μm; in D,E, 40 μm; in H-P, 125 μm.
Fig. 6.
Fig. 6.
Avian myotome development transits from a Shh-dependent to a Shh-refractory phase. (A-D) Grafting of a No fragment dorsal to the young somite (A,B), or the late DM (C,D). (E-H) Co-electroporation of Shh-GFP to the ectoderm (blue, arrowheads) of E2 (E,F) or E3 (G,H) embryos. Insets in E,F show desmin staining only. (C′,D′,E′,F′,G′,H′) Higher magnifications of respective boxed areas. Myotome differentiation in response to No or Shh is time restricted. D, dermis; DM, dermomyotome; M, myotome. Scale bar: in A,B, 85 μm; in C-H, 100 μm.
Fig. 7.
Fig. 7.
DM progenitors, although refractory to Shh ligand, respond to Shh signaling components acting at the level of or downstream of Smo. (A-P) Dorsal views of whole-mount preparations at E3.5 (A-H) and E4.5 (I-J) labeled for GFP and desmin. Control-GFP (A,B,I,J), MyoD (C,D,K,L), GliA (E,F,M,N) or SmoM2 (G,H,O,P) were electroporated within dorsal somites at E2 (A-H) or DM at E3 (I-P). All the components of Shh signaling enhanced DM-derived myogenic differentiation compared with control-GFP. Note that, at E3, control-GFP does not generate myofibers. (Q) Quantification of the proportion of Pax7+ progenitors in myotome. Error bars represent s.e.m. *P<0.001, **P<0.0001.

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