DEV02867 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Representation of the sequence of divisions of the ventral muscle mass in the chick forearm. (A) The sequential splitting of the ventral muscle mass leads to six flexor muscles. The muscle splitting is a continuous process along the p-d axis, making it difficult to reproduce precisely in a diagram. The times (green) correspond to when the masses are separated along the p-d axis. At E10, when the process is over, six main ventral muscles can be visualized, running from posterior to anterior: FCU, flexor carpi ulnari; FDS, flexor digitorum superficialis; UMV, ulni metacarpalis ventralis; FDP, flexor digitorum profondus; PP, Pronator profondus; PS, pronator superficialis. (B,C) Transverse section of a forearm cut at the mid-diaphysis level from an E5 (B) and an E10 (C) chick embryo hybridized with VE-cadherin (blue) probe and then incubated with the MF20 antibody (brown). At E5, the vascular network surrounds the dorsal and the ventral muscle masses, whereas at E10, it surrounds the individual muscles. The names of the ventral muscles are indicated. The FDS and UMV are very small distal muscles, closely apposed to the large FCU and FDP muscles, respectively, and are not visible on this section. U, ulna; r, radius.

• Supplemental Figure S2 -

  Fig. S2. QH1-positive cells delineate the future cleavage zone of the FCU muscle. Wings from E7/HH30 and E7.5/HH31 quail embryos were hybridized with the MyoD probe (blue), followed by incubation with the QH1 antibody (brown). High magnifications are shown (B,D) of the ventral and posterior muscle (A) and of the FCU (C). The posterior ventral muscle mass will separate into three masses giving rise to the FCU and the FDS (see Fig. S1). The FDS is the smallest ventral muscle present in the distal region of the forearm of the wing. The FCU is a large posterior muscle running the full length of the ulna and is composed of two separate parts that are joined distally (Sullivan, 1962). We observed QH1-positive cells at the future site of separation of the two parts of the FCU (A,B). The separation of the two parts of the FCU is complete at E7.5 in quail embryos (C,D). u, ulna; r, radius; p, posterior; a, anterior; D, dorsal; V, ventral.

• Supplemental Figure S3 -

  Fig. S3. Ectopic blood vessels inhibit muscle formation. (A) VEGF/RCAS-expressing cells were grafted to the presumptive wing regions at E2/HH14 and the embryos fixed at E7/HH30. (B) The control left wings of manipulated embryos display the normal pattern of muscles visualized by the expression of the muscle markers Fgfr4 (blue, in situ hybridization) and myosin (brown, immunohistochemistry using the MF20 antibody). (C) Five days after grafting, the VEGF/RCAS virus had infected all the limb regions, the infection being visualized by in situ hybridization using the Vegf probe. Adjacent sections display an anarchic vascular network visualized by Hif2α expression (D) and an impairment of muscle formation, visualized by Fgfr4 and MF20 expression (E), compared with the control limb of the same embryo, sectioned at the same p-d level (B). (F) VEGF/RCAS-expressing cells were grafted to the presumptive wing regions at E2/HH14 and the embryos fixed at E5.5/HH27. Consecutive sections of the control left wing (G,H) and VEGF-infected wing (I,J,K) were hybridized with the Vegf (I), Hif2α (G,J) and MyoD (H,K) probes. (G,J) The in situ hybridization with the Hif2α probe was followed by immunohistochemistry using the MF20 antibody. At E.5.5/HH27, the VEGF/RCAS virus had infected all the limb regions (I), leading to ectopic vessels in dorsal and ventral muscle masses (J). Adjacent sections display a normal expression pattern of MyoD in muscle masses (K) compared with the control left wing (H). This shows that limb muscle masses are not affected by ectopic vessels at this stage of development. For all sections, top is dorsal and left is posterior; u, ulna; r, radius.

• Supplemental Figure S4 -

  Fig. S4. Blocking vessel formation leads to muscle fusion. Transverse sections of sFLT1-treated right wings (A-D) and control left wings (E-H) from E8 chick embryos (2 days after sFLT1 bead implantation) were hybridized with the MyoD probe in order to visualize muscle organization. Pictures at regular intervals, running from distal to proximal, show two dorsal muscles (EMU, extensor metacarpi ulnans and EDC, extensor digitorum communis), which are fused in the sFLT1-treated wings (A-D) and well separated in the control wing (E-H). The muscle fusion between EMU and EDC was observed to extend over 600 μm in length distal to the sFLT1 bead.