Persistent expression of Pax3 in the neural crest causes cleft palate and defective osteogenesis in mice
J. Clin. Invest. Meilin Wu, et al. 118:2076 doi:10.1172/JCI33715 [Go to this article.]

Figure 5
Persistent Pax3 expression impairs palate osteogenesis. (A) Frontal sections through the palatal regions of E15.5 (top panels) and E17.5 (bottom panels) control (Pax3^Cre/+, R26^+/+), heterozygous (Pax3^Cre/+, R26^Pax3/+), and homozygous (Pax3^Cre/+, R26^Pax3/Pax3) littermates persistently expressing Pax3 are stained for mineralized bone (green) by Goldner’s trichrome. Pax3^Cre/+, R26^Pax3/Pax3 embryos have less mineralized bone in the palate compared with control (arrows). At E17.5, Pax3^Cre/+, R26^Pax3/Pax3 embryos lacked the medial mineralizing centers (arrows) seen in control (Pax3^Cre/+, R26^+/+) and heterozygotes (Pax3^Cre/+, R26^Pax3/+). Original magnification, ×2.5. (B) Von Kossa–stained ex vivo cultures of primary palate cells grown for 9 days in mineralizing medium. Control cells (left wells) formed mineralized nodules with BMP-2 treatment, but cells derived from Pax3^Cre/+, R26^Pax3/Pax3 mice (right wells) failed to form nodules and mineralize. Replicate wells are shown for each genotype. (C) Primary palate cell cultures stained for ALP activity. Control cells (left panel) cultured in osteogenic medium containing BMP-2 respond with robust expression of ALP, but cells from Pax3^Cre/+, R26^Pax3/Pax3 (right panel) mice are unable to respond to BMP-2 with upregulated ALP expression. Original magnification, ×16. (D) Immunofluorescent detection of Pax3 demonstrates persistent expression in cells from mutant mice (right panels) and the absence of Pax3 protein in control (Pax3^Cre/+, R26^+/+) cells. (E) Western blot analysis for Pax3 expression of primary palate cell culture lysates from control (Pax3^Cre/+, R26^+/+), heterozygous (Pax3^Cre/+, R26^Pax3/+), and homozygous (Pax3^Cre/+, R26^Pax3/Pax3) mice persistently expressing Pax3. Scale bar: 10 μm.