Fig. 3. Phospho-SMAD1 immunolabeling and Msx2 gene expression demonstrate loss of BMPR-IA signaling between 9.75 and 10.0 dpc in limb ectoderm. (A) Transverse section of a 9.75 dpc normal embryo demonstrating phospho-SMAD1 immunolabeling in the lateral mesoderm (lm) and the overlying ectoderm in the hindlimb field. Strong immunolabeling is detected on the ventral side of the coelom (co), and an overall gradient of labeling is detected with the highest labeling in the most ventral region of the embryo. (B) A higher magnification view of A. (C) Phospho-SMAD1 labeling is detected preferentially in the dorsal neural tube of normal embryos adjacent to the roofplate, which is a rich source of BMP factors. (D) Mutant embryo section corresponding to the normal embryos shown in A. (E) A higher magnification view of D. At this stage, only an occasional ectodermal cell shows reduced levels of phospho-SMAD1 immunolabeling, as indicated by the arrowhead. (F) Phospho-SMAD1 labeling is detected preferentially in the dorsal neural tube of mutant embryos, suggesting functional redundancy of BMP receptor function in the neural tube of these embryos. (G) Transverse section through hindlimb bud of a 10.0 dpc normal embryo demonstrating phospho-SMAD1 immunolabeling in the lateral mesoderm and the overlying ectoderm. Inset demonstrates that phospho-SMAD1 immunolabeling is low or undetectable in the dorsal ectoderm (see arrowhead). (H) Higher magnification view of G, showing the predominantly nuclear immunolabeling. (I) Phospho-SMAD1 immunolabeling in dorsal neural tube of 10.0 dpc normal embryo. (J) Phospho-SMAD1 labeling in a section of mutant embryo demonstrates that immunolabeling is not detected in most ectoderm cells, but robust immunolabeling is detected in the underlying mesoderm. Section comparable with that shown in G. (K) Higher magnification view of J demonstrates that only an occasional cell demonstrates phospho-SMAD1 immunolabeling in the mutant ectoderm (arrow), whereas the vast majority of ectodermal cells are not immunolabeled at 10.0 dpc (arrowhead). (L)Phospho-SMAD1 immunolabeling in the dorsal neural tube of mutant 10.0 dpc embryo. (M) Phospho-SMAD1 immunolabeling is detected in neural retina of 9.75 dpc eye. (N) Phospho-SMAD1 immunolabeling in dorsal hindbrain of 9.75 dpc embryo. (O) In situ hybridization demonstrates that expression of Msx2 at 10.0 dpc is detected in ventral hindlimb (bracket, transverse plane of section). Arrow indicates the border between lateral and paraxial mesoderm; arrowhead indicates Msx2 expression in the dorsal midline. (P) Expression of Msx2 in the mutant is abolished in the ventral hindlimb indicating that BMP signaling has been abrogated (bracket corresponds to region that normally expresses Msx2; arrowhead indicates Msx2 expression in the dorsal midline).
