Figure 9.
Noggin and Shh function synergistically to activate the expression of sclerotome marker Pax1 in presomitic mesoderm. Mouse psm explants (9.5 dpc) were cultured in collagen gels for 24 hr in the presence or absence of purified recombinant Noggin, SHH-N (the amino-terminal 19-kD peptide), BMP2, or BMP4. The concentration of each of these is indicated. RNA samples from each culture (two pieces of psm per culture) were extracted and subjected to RT–PCR to assess marker gene expression. Expression of β-actin was assayed as a control for RNA recovery and cDNA synthesis (see Materials and Methods). (A) Psm explants were treated with a series of concentrations of SHH-N (as indicated) with (right) or without (left) 50 ng/ml Noggin. Expression of the sclerotome marker Pax1 and the β-actin control was assessed by RT–PCR. (B) Noggin alone can also activate the sclerotomal marker Pax1. (C) Noggin can induce Pax-1 expression in the presence of a SHH-N blocking antibody, IBMX, and forskolin, whereas all these reagents block SHH-N mediated induction of Pax-1 in psm explants. (D) Purified BMP2 and BMP4 protein can inhibit Pax-1 induction in response to SHH-N, Noggin, or a combination of each protein. (E) The psm and the five caudal-most somites (somI–V) were assayed immediately (0 hr) or after 24 hr of culture (24 hr) for expression of Bmp2 and Bmp4 (30 cycles). RT–PCR products were analyzed by Southern hybridization (see Materials and Methods). β-actin (13 cycles) expression was similar in all samples.