Fig. 2.
Foxc1 inhibits Msx2 expression in the supraorbital ridge and in cultured 10T1/2 and O9-1 cranial neural crest cells. (A,A′,B,B′,E,E′,F,F′) Control and Foxc1ch/ch embryos at E12.5 or E13.5 were sectioned as in Fig. 1A and subjected to in situ hybridization for Msx2 and Foxc1 mRNAs simultaneously. Msx2 is in green; Foxc1 in red. (C-D′) Sections from the same embryo were stained for ALP. Boxed areas are shown at higher magnification on the right. (G) The area of Msx2 and Foxc1 co-expression, indicated by the yellow color in B and B′, is significantly larger (P<5.00×10–4) in Foxc1 mutants. (H) Effect of siRNA-mediated knockdown of Foxc1 on Msx2 mRNA levels in C3H10T1/2 cells and O9-1 cranial neural crest cells. mRNA levels of Foxc1 and Msx2 were measured by qPCR and normalized to Gapdh. siRNA against EGFP provided a negative control. (I) We assessed the influence of Foxc1 knockdown on the rate of osteogenic differentiation of cultured O9-1 cells. Cells were treated with siRNA against Foxc1, and qPCR was used to measure levels of Msx2, ALP, Runx2 and osteocalcin mRNAs. Significant (P<0.05) upregulation of each of these osteogenic differentiation markers was observed after 48 hours of Foxc1 siRNA treatment. Significance was assessed by Student’s t-test. Error bars indicate s.d.