Figure 3. BRAF is required for limb muscle precursor cell migration during mouse embryogenesis.
(A) Pax3 whole mount in situ hybridization of WT, germ line Brafdel/del and Pax3-Cre//Brafdel/del mutant embryos at E10.5. (B) Transverse sections of WT and Pax3-Cre//Brafdel/del mutant embryos at E10.5 after Pax3 whole mount in situ hybridization. Inactivation of Braf results in loss of PAX3+ cells in forelimbs. NT: neural tube; dm: dermomyotome; fl: forelimb. (C) Whole mount in situ hybridization of E10.5 WT and Pax3-Cre//Brafdel/del mutant embryos using Lbx1, Myf5, Myod, and Myogenin probes. (D) Immunofluorescence staining of forelimbs from WT and Pax3-Cre//Brafdel/del mutants for myosin heavy chain (MHC) at E14.5. (E) Hematoxylin and eosin staining of forelimbs from WT and Pax3-Cre//Brafdel/del mutants at E14.5.
Figure 3—figure supplement 1. Inactivation of Braf in Pax3 expressing cells impairs limb muscle precursor cell but not endothelial cell migration during mouse embryogenesis.
(A, B, E, F) Immunofluorescence staining of E10.5 Pax3-Cre (A, B) and Pax3-Cre//Brafdel/del embryos (E, F) for BRAF (red). White arrows in (A, B) indicate the presence of BRAF in the dermomyotome. (C, D, G, H) Immunofluorescence staining of E10.5 Pax3-Cre (C, D) and Pax3-Cre//Brafdel/del embryos (G, H) for PAX3. White arrows in (C) indicate the delamination of PAX3+ cells from the dermomyotome in Pax3-Cre control embryos whereas delamination of PAX3+ cells is compromised in Pax3-Cre//Brafdel/del embryos (White arrows in (G)). Inserts in (D) and (H) represent magnification of boxed areas. (I, J, M, N) TUNEL staining of E10.5 Pax3-Cre (I) and Pax3-Cre//Brafdel/del embryos (M) to detect apoptotic cells. DAPI stained sections of Pax3-Cre (J) and Pax3-Cre//Brafdel/del embryos (N) without the TUNEL fluorescence channel are shown for comparison. Inactivation of Braf in Pax3-expressing cells does not lead to an increase of apoptotic cells. (K, L, O, P) Immunofluorescence staining of E10.5 Pax3-Cre (K, L) and Pax3-Cre//Brafdel/del embryos (O, P) for CD31 to visualize endothelial cells. Inactivation of Braf in Pax3-expressing cells does not compromise formation of the vascular network in limb buds.