Fig. 5. PTHrP signals are necessary to drive Nkx3.2 expression in the growth plate. (A) Infection of limbs with RCAS-Nkx3.2 results in the loss of PTHrP and endogenous Nkx3.2 expression. Arrowheads (part a) indicate strong PTHrP expression in the periarticular region of the control wing; arrowheads (part c) indicate normal expression of endogenous Nkx3.2 in control wing, whereas arrows (d) indicate the absence of endogenous Nkx3.2 expression in RCAS(A)Nkx3.2-infected cartilage. (B) Ectopic PTHrP delays chondrocyte maturation and maintains uniform Nkx3.2 expression throughout the cartilage. Wing buds of E3 chick embryos were infected with RCAS(A)-PTHrP virus. Embryos were harvested at E10, and RCAS(A)-PTHrP-infected wings and contralateral control wings were analyzed by Alcian Blue/Alizarin Red staining (AB/AR, parts a,b) or section ISH (c-l). Arrowheads (c,e,g) indicate the central region of the ulna in which expression of Sox9, Nkx3.2 and col II is downregulated in the control wing. Arrows (d,f,h) indicate the central region of the ulna in which expression of these markers is maintained in the RCAS-PTHrP infected wing. (C) Nkx3.2/Bapx1 expression is lost in PTHrP and PTHrP-receptor null animals. Bright field (a,d,f) or ISH (b,c,e,g) of E18.5 mouse tibiae with the indicated probes, taken from mice of indicated genotypes. (D) Loss of p57 expression rescues the absence of immature chondrocytes but not Nkx3.2/Bapx1 expression in the absence of PTHrP signals. ISH of E17.5 mouse ulnae, taken from mice of indicated genotypes, with indicated probes. (E) PTHrP signals induce the expression of Nkx3.2 in cultured chondrocytes. Upper sternal chondrocytes (USC) were isolated from the cephalic portion of 15-day-old chick embryo sternae and cultured for 3 days in either the absence (lane 1) or presence (lane 2) of 100nM PTHrP. Gene expression was assayed by RT-PCR.