Fig. 6. Nkx3.2 represses Runx2 expression. (A) Nkx3.2 and Runx2 are expressed in a reciprocal pattern in the developing cartilage elements of the limb. ISH for the indicated probes is displayed for E8 chick limbs. In the cartilage element, Runx2 expression is most intense where Nkx3.2 expression is diminished (marked by arrowheads in a,b). (B) Forced expression of Nkx3.2 represses Runx2 expression in vivo. The right wing buds of E3 chick embryos were infected with either RCAS(A)-Nkx3.2 (b,d) or RCAS(A)-Nkx3.2-C-VP16 (f,h) viruses. Embryos were harvested at either E8.5 (a-d) or E8 (e-h), and sections of the infected or contralateral control wings were hybridized with the indicated probes. (C) The intensity of the Runx2 ISH signal displayed in B and in two other experiments was quantified, in both the peripheral (blue rectangles in B) and central regions (orange rectangles in B) of the cartilage in both control and infected wings, as indicated. (D) Forced expression of Nkx3.2 represses Runx2 expression in presomitic mesoderm explants in vitro. Explants of presomitic mesoderm were either cultured for 2 days in either Shh (lanes 1 and 3) or infected with RCAS-Nkx3.2 (lanes 2 and 4). The explants were cultured in Bmp4 for either an additional 6 (lanes 1 and 2) or 12 (lanes 3 and 4) days. Expression of indicated markers was assayed by RT-PCR.