Figure 7.

Treatment with Gdf7 or BMP7 restores mATH1 and LH2A expression in Gdf7 mutant neural tissue. (A–F) Expression of mATH1 (red) in explants of mouse forelimb level dorsal neural tube ([d+rp] explants) isolated from wild-type (A,C,E) and Gdf7^m1 homozygous mutant (B,D,F) embryos. Msx1⁺ roof plate cells are green. Gdf7 mutant [d+rp] explants isolated at E11.5 (44 somites) (B) and cultured in COS (control) supernatant for 60 hr generate <10% as many mATH1⁺ cells as explants from age-matched wild-type siblings (A,G). Wild-type explants respond to added GDF7 with the generation of ∼5-fold more mATH1⁺ cells (C,G). Culture with GDF7 increases the number of mATH1⁺ cells in mutant explants to a level similar to that in treated wild-type explants (D,G). Treatment with BMP7 similarly increases mATH1 cell number in wild-type and mutant explants (E,F,G). In these experiments, explants were treated with equivalent doses of GDF7 and BMP7, as judged by their activity in inducing LH2A⁺/LH2B⁺ neurons in chick neural plate assays (data not shown). In untreated wild-type and mutant [d+rp] explants, mATH1⁺ cells are restricted to the region adjacent to the roof plate (A,B). After treatment with GDF7 or BMP7, mATH1⁺ cells are found at all dorsoventral positions within the explants, independent of genotype (C–F). Thus, at E11.5, cells more distant from the roof plate respond to GDF7 or BMP7 with the expression of mATH1. GDF7 treatment of [d+rp] explants isolated from E12.5 wild-type or mutant embryos did not increase mATH1⁺ cell number (data not shown), suggesting that GDF7 signaling normally acts prior to E12.5 to induce or maintain mATH1⁺ progenitors. (G) Histograms show mean ± s.d. of results from four to six explants of each genotype. (H) RT-PCR analysis of LH2A and ribosomal protein S12 gene (control) expression in E11.5 wild-type and Gdf7 mutant [d+rp] explants cultured for 60 hr with COS cell (control) supernatant or supernatant containing GDF7 or BMP7. Treatment of mutant explants with GDF7 or BMP7 restores LH2A expression to wild-type levels.