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Fig. 5 Pbx is required downstream of Shh signaling to induce fast-muscle, but not slow-muscle, gene expression. (A-L) RNA in situ expression of (A-F) smyhc1 (blue) and myod (red) at the 14-somite (14s) stage or (G-L) mylz2 (blue) and myod (red) at 16s in embryos injected with (A,G) pbx2-MO mismatch control and gfp mRNA, (B,H) pbx2-MO mismatch control and shh mRNA, (C,I) myod-MO and gfp mRNA, (D,J) myod-MO and shh mRNA, (E,K) pbx2-MO; pbx4-MO and gfp mRNA, or (F,L) pbx2-MO; pbx4-MO and shh mRNA. The most-anterior 8-10 somites of each embryo are shown, in dorsal view, anterior towards the left. Control embryos injected with shh mRNA (control+shh) show increased expression of smyhc1 (B, 106/135 embryos; 29/135 show normal smyhc1 expression) or increased expression of mylz2 (H, 88/121; 33/121 show normal mylz2 expression). myod-MO+shh embryos show less-frequent and less-extensive induction of smyhc1 compared with control+shh (46/90 embryos resemble D; 44/90 show normal smyhc1 expression) and fail to upregulate mylz2 expression beyond that seen in myod-MO+gfp embryos (84/88 embryos resemble J; 4/88 show slightly increased mylz2 expression). pbx2-MO; pbx4-MO+shh embryos show strongly increased expression of smyhc1 (77/79 embryos resemble F; 2/79 have normal smyhc1 expression). Approximately half (43/102; L) of pbx2-MO; pbx4-MO+shh embryos show expansion of weak levels of mylz2 across the somites; the other half (59/102) resemble pbx2-MO; pbx4-MO+gfp embryos. Similar results were observed in multiple experiments.