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First published online 6 June 2007
doi: 10.1242/dev.02867

Development 134, 2579-2591 (2007)
Published by The Company of Biologists 2007
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Involvement of vessels and PDGFB in muscle splitting during chick limb development

Samuel Tozer1, Marie-Ange Bonnin1, Frédéric Relaix2, Sandrine Di Savino1, Pilar García-Villalba1, Pascal Coumailleau1 and Delphine Duprez1,*

1 Biologie du Développement, CNRS, UMR 7622, Université P. et M. Curie, 9 Quai Saint-Bernard, Bât. C, 6e E, Case 24, 75252 Paris Cedex 05, France.
2 INSERM U787, Université P. et M. Curie, UMR S787, Paris 75013, France.


Figure 1
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  		Fig. 1. Angiogenic cells are detected in wing somatopleure before muscle progenitor cells in avian embryos. (A-F) Adjacent transverse sections of chick embryos, at the wing level, at 19 (A,D), 25 (B,E) and 30 (C,F) -somite stages were hybridized with Vegfr2 (A-C) and Pax3 (D-F) probes. (G-I) Transverse sections of quail embryos, at the wing level, at 22 (G), 24 (H) and 32 (I) -somite stages were hybridized with Pax3 probe (blue) and then incubated with QH1 antibody (brown). Arrowheads point to endothelial progenitor cells visualized with Vegfr2 probe (A,B) or QH1 antibody (G,H). Arrows indicate the overlapping domain of expression of Vegfr2 (A) and Pax3 (D) in somites. NT, neural tube; Nc, notochord; So, somites.

 

Figure 2
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  		Fig. 2. The vascular network organizes itself independently of muscle. (A) Transverse sections of E9.5 forelimbs of Pax3GFP/+ mouse embryos were incubated with the PECAM antibody (red) to visualize vascular cells. GFP (green) reveals the Pax3-expressing cells. DAPI staining (blue) visualizes the nuclei. (B,C) E13.5 forelimbs from Pax3+/+ and Pax3-/- mutant mice were cut transversally and then incubated with the PECAM antibody. (D,E) E13.5 hindlimbs of Pax3+/+ and Pax3-/- mutant mice were cut transversally and hybridized with the Hif2{alpha} probe and then incubated with the MF20 antibody. The asterisks show ventral muscles in control limbs (B,D) and the equivalent sites corresponding to the absent muscles in Pax3-/- limbs (C,E). Both muscles and muscle-less sites are surrounded by the vasculature.

 

Figure 3
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  		Fig. 3. The location of endothelial cells delineates the future cleavage zones of muscle masses. (A-F) Transverse sections of wings from E5/HH26 (A,D), E5.5/HH27 (B,E) and E6/HH28 (C,F) chick embryos were hybridized with the Vegfr2 probe (blue) and then incubated with the MF20 antibody (brown). (D,E) High magnification of the future cleavage zone of the ventral masses from A,B. (F) High magnification of the cleavage zone of the ventral masses from C. (G-K) Wings from E5.5/HH27 chick embryos were cut transversely from proximal to distal areas along the forearm elements and then hybridized with the Hif2{alpha} probe (blue) followed by immunohistochemistry using the MF20 antibody (brown). The presence of Hif2{alpha}-positive cells at the future splitting site of the ventral muscle mass was estimated to extend over 300 µm in length (shown are example images running from proximal to distal). (L) At E6/HH28, the ventral muscle mass was well separated into the posterior and anterior masses. The arrow indicates the Hif2{alpha}-positive cells delineating a future cleavage zone in the anterior part of the central muscle mass - cleavage that will produce the central and proximal anterior masses.

 

Figure 4
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  		Fig. 4. Ectopic blood vessels inhibit muscle formation. (A) VEGF/RCAS-expressing cells were grafted to the dorsal aspect of E4/HH22 chick wings and the embryos were fixed at E8/HH32. Consecutive sections of the manipulated wing (B,D,F,H,J) and of the control left wing (C,E,G,I) were cut at the same proximodistal level in order to allow comparison. Sections were hybridized with Vegfa (B), Hif2{alpha} (C,D,G,H) and Fgfr4 (E,F,I,J) probes (blue), and then incubated with the MF20 antibody (brown) that recognizes myosins. (B) Vegfa transcripts show the extent of the infection in dorsal regions of the wings. Ectopic VEGFA leads to a dramatic increase in the density of blood vessels, visualized by the expression of Hif2{alpha} (D), compared with the normal vascular network of the control wing (C). In the dorsal regions displaying an excess of blood vessels, we observed a reduction of muscle size and even a loss of certain muscles (F), as compared with the normal muscle pattern of the left control wing (E). Asterisks label the control muscles (E) and their putative locations in the VEGF-treated limbs (F). (G-J) High magnifications of the ANC (Anconeus) muscle (arrowed in C-F) located in dorsal and posterior regions of the control and manipulated limbs, showing that the hypervascularized muscles contain fewer myogenic cells (J) than the corresponding control muscles (I). For all sections, the top is dorsal and left is posterior. u, ulna; r, radius.

 

Figure 5
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  		Fig. 5. Collagen I is associated with normal and ectopic vasculature in limb muscles. Transverse and consecutive sections of wings from E6.5/HH29 chick embryos were hybridized with the VE-cadherin (A) and collagen I (B) probes followed by immunohistochemistry with the MF20 antibody. collagen I transcripts are detected with endothelial cells in the recently cleaved sites of the ventral muscle masses. (C-H) VEGF/RCAS-expressing cells were grafted to the dorsal aspect of E4/HH22 wings and the embryos were fixed at E8/HH32. Consecutive sections of VEGF-infected right limbs (C-E) and control left wings (F-H) were hybridized with the Vegf (C,F), Hif2{alpha} (D,G) and collagen I (E,H) probes (blue) then incubated with the MF20 antibody (brown). High magnifications are shown of the same dorsal muscle in experimental wings (C-E) and in control wings (F-H) at the same level. Ectopic hypervascularization (C,D) leads to an obvious increase in collagen I expression in muscles (E) compared with control muscles (H).

 

Figure 6
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  		Fig. 6. Blocking vessel formation leads to muscle fusion. (A-D) Dorsal views of wings from E8 chick embryos, which have undergone ink injection before fixation in order to visualize vessel organization. PBS bead implantation at E6 does not modify vessel assembly (B) as compared with the non-grafted left wing (A). sFLT1 bead implantation locally inhibits vessel formation (D) compared with the control left wing (C). (E-H) Transverse sections of sFLT1-treated right wings (F,H) and control left wings (E,G) were hybridized with the MyoD probe in order to visualize muscle organization. (G,H) High magnification of E,F, respectively. The EMU and EDC, two dorsal muscles, are cleaved in control wings, whereas they are fused in sFLT1-treated wings. EMU, extensor metacarpi ulnans; EDC, extensor, digitorum communis; u, ulna; r, radius.

 

Figure 7
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  		Fig. 7. PDGFB inhibits the expression of muscle markers. (A) Transverse sections of wings from E5.5/HH27 chick embryos were hybridized with the Pdgfb probe followed by immunohistochemistry with the MF20 antibody. (B,C) Transverse and consecutive sections of wings from E5.5/HH27 chick embryos were hybridized with the Pdgfrß (B) and MyoD probe (C). (D-I) PDGFB or PBS beads were implanted into the dorsal regions of E5/HH26 wings and the embryos fixed 2 days or 2.5 days later, at E7/HH30 or E7.5/HH31. (D) Whole-mount preparations of PDGFB-implanted wings hybridized with MyoD show inhibition of MyoD expression around the bead. Sections of the PDGFB (E,I) or PBS (F,G,H) -treated wings were hybridized with the MyoD (E-G) or the Pdgfrß (H,I) probes. The expression of MyoD (E) and Pdgfrß (I) was inhibited around PDGFB beads, whereas PBS beads did not have any effect on MyoD (F,G) or Pdgfrß (H) expression. The PBS bead (H) is shown at a more proximal level than the PDGFB bead (I), explaining the difference of muscle pattern in ventral muscles between limbs. The two anterior and ventral muscles visualized in the control PBS limbs (H) are only observed in proximal region of the forearm. For all the sections (A-C,E-I), top is dorsal and left is posterior; u, ulna; r, radius.

 

Figure 8
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  		Fig. 8. PDGFB activates the expression of connective tissue markers. PDGFB or PBS beads were implanted into the dorsal regions of E5/HH26 chick wings and the embryos were fixed 2 days later, at E7/HH30. Consecutive sections of the PDGFB-treated wings (B,D) and of the corresponding control left wings (A,C) were hybridized with probes for the muscle connective tissue markers collagen I (A,B) and Tcf4 (C,D) and then incubated with the MF20 antibody. (E) Transverse section of an E6 wing showing the endogenous expression of Pdgfr{alpha} in muscle connective tissue. Pdgfr{alpha} transcripts (blue) are detected in the dorsal muscle mass (brown) in a future site of cleavage. (F) PDGFB-treated wings were hybridized with the Pdgfr{alpha} probe.

 

Figure 9
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  		Fig. 9. PDGFB increases cell density without modifying cell proliferation. Cell density around PBS (A) and PDGFB (B) beads were visualized with Hoechst-labeled nuclei at E7, 2 days after grafting into chick wings. (C,D) Adjacent sections to those shown in A,B, hybridized with the MyoD probe. There is an increase in cell density homogenously around PDGFB beads (B), where MyoD expression is inhibited (D), whereas PBS beads do not induce any cell accumulation (A) and do not modify muscle organization (C). It should be noted that cell density is higher in muscles compared with limb connective tissue at this stage (A-D). Transverse sections from PBS (E) or PDGFB (F,G) -treated wings incubated with the anti-BrdU antibody show that application of PDGFB does not modify cell proliferation around the bead. (G) The adjacent section to that shown in F, hybridized with the MyoD probe and then incubated with the MF20 antibody.

 

Figure 10
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  		Fig. 10. PDGFB acts on connective tissue cells before it acts on myogenic cells. PDGFB beads were implanted into the dorsal regions of E5/HH26 chick wings and the embryos were fixed at various times after grafting. Consecutive sections of the PDGFB-treated wings, 9.5 (A,B), 12 (C,D) and 24 (E,F) hours after grafting were hybridized with the MyoD (A,C,E) and collagen I (B,D,F) probes. As soon as 9.5 hours, an increase in collagen I expression was observed (B), whereas no obvious effect on MyoD expression was observed (A). An effect on MyoD expression (loss of MyoD expression around the bead) was observed 24 hours after grafting. (E). (G) Analysis of cell density, visualized with Hoechst-labeled nuclei, 24 hours after PDGFB bead implantation. (H) An adjacent section to G was hybridized with the MyoD probe.

 

Figure 11
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  		Fig. 11. Model for the effect of the vasculature on muscle mass separation. (A) The endothelial cells (blue) delineate the future cleavage site in the muscle mass, which is composed of myogenic cells (red) and muscle connective tissue cells (green). (B) At a later stage, the muscle masses are separated. (C) The PDGFB secreted by the endothelial cells acts in a paracrine manner on muscle connective tissue cells, which express PDGFR{alpha}. In response to PDGFB, connective tissue cells (expressing PDGFR{alpha}) increase the secretion of extracellular matrix by producing collagen I. This promotes the accumulation of connective tissue cells and the formation of a new muscle membrane, allowing muscle mass separation. Concomitantly, muscle differentiation will be inhibited as a consequence of the accumulation of muscle connective tissue and/or directly by PDGFB acting on muscle cells, which express PDGFRß.

 

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© The Company of Biologists Ltd 2007