Growth factor signaling in mice lacking MC HS. Phospho-PDGFR-β protein expression levels as well as downstream signaling molecules p-Erk1/ 2 and p-SHP-2 are reduced in skin samples of several EXT1^MCko, whereas p-Akt signaling is unaffected (A). Comparison of pPDGFR-β levels in skin and CNS of EXT1^MCko confirmed that PDGFR-β signaling is unchanged in the CNS of EXT1^MCko mice (A) correlating with the observed phenotype; illustration of the percentage of pPDGFR-β normalized to ribosomal protein S6, internal loading control in the skin and brain of protein samples. *P < .05. Control and EXT1^MCko; n ≥ 3. Values are mean ± SEM. Immunoprecipitation of VEGFR2 followed by immunoblotting to detect phosphorylated VEGFR2 (B) revealed no differences in pVEGFR2 expression levels in the skin of EXT1^MCko; n = 2. Expression studies of pSMAD2 and pSMAD3 molecules by Western blotting, downstream effectors of the TGF-β pathway revealed a significant down regulation in EXT1^MCko embryos (C). The reduction in pSMADs is restricted to EXT1^MCko skin samples, whereas pSMAD levels in the CNS of mice lacking HS in MCs are unchanged compared with control littermates (C). To access whether reduced pSMAD levels are a result of MC loss in the skin of EXT1^MCko embryos, we analyzed pSMAD expression in Pdgfrb null mice lacking MC investment (C). (D) Summarization of densitometric analyses, illustrating the expression levels of pSMAD2 and pSMAD3 normalized to β-tubulin, internal loading control. *P < .05. ns indicates not significant. EXT1^MCko and control embryos (n = 4), Pdgfrβ^+/− and Pdgfrβ^−/− (n = 2). Values are mean ± SEM. Reduced endothelial pSMAD levels could be confirmed by immunohistochemical staining with a pSMAD3 antibody on embryonic skin sections (E-H). Whereas most ECs of blood vessels in control mice are stained positively for pSMAD3 (arrows; E-F), only few ECs in EXT1^MCko express pSMAD3 expression (arrows, G-H). Counterstaining with endothelial marker endomucin (red) confirmed endothelial-specific loss of pSMAD3 (green) in EXT1^MCko embryos (arrows, G-H), reflecting the reduction of pSMAD3 protein levels in skin (D); n > 3; illustration of different modes of MC recruitment in skin and CNS (I).