Fig. 6. Changes of other molecular pathways in Msx2 knockout mutant hairs. (A) Timecourse RNase protection assays. Expression analyses of molecules implicated in hair differentiation. A 1 cm² dorsal skin sample from indicated time points extracted for RNA. Examination of hair cortex differentiation markers by RNase protection assay revealed significantly lower levels of Foxn1 and its target gene Ha3 in Msx2 knockout mutants. At P13 (postnatal day; d on figure) Ha3 expression sharply decreases in the mutant skin and is barely detectable at P15 and P17 (A,C). This loss of Ha3 expression correlates with hair loss in Msx2 knockout mutants. Lef1 expression in the hair matrix cells and in the wild-type skin, increases from P7 to P11, which was not seen in Msx2 knockout mutants. Although the difference is not striking, the trend is consistent in different experiments. Expression of two other genes, Bmp4 and Tgfa (not shown) is not affected by the Msx2 mutation. Scale bar: 200 µm. (B) Quantitation of Foxn1 and Ha3 message levels at P11 revealed that Foxn1 and Ha3 mRNA is downregulated 50% compared with that in wild-type littermates. A much more dramatic 72% reduction in Ha3 expression was observed at P15. (C) Indirect immunohistochemistry with an affinity-purified Foxn1 antibody showed reduced Foxn1 protein in Msx2 knockout mutant cortex.