gli2 MO injection rescues nk2.2 and myoD expression defects in yot/gli2 mutants and reveals a weak activator role for Gli2. (A) gli2 MO injection expands ptc1 expression ventrally in the diencephalon (arrowheads) and causes a minor but consistent overall expansion of ptc1 expression (compare to inset). (B) yot/gli2 mutants have significantly reduced ptc1 expression. (C) gli2MO injections rescue the ptc1 defects seen in yot/gli2 mutants and expand ptc1 expression ventrally (arrowhead). (D) Injection of gli2MOs into wild-type embryos has no effect on nk2.2 expression. (E,F) Injection of a gli2MOs into yot–/–mutant embryos can completely rescue yot-induced defects in nk2.2 expression (compare arrows). (G) gli2MO injection does not affect myoD expression in adaxial cells (arrowheads). (H,I) gli2MO injections partially rescue yot-induced defects in adaxial myoD expression (compare arrowheads). (J-M) Injection of 3-10 ng of gli2MO into embryos from a cross between dtr–/+heterozygous parents (25% dtr–/–mutants expected) resulted in an additional loss of nk2.2 expression in the tegmentum (compare arrows in J,K) and a reduction in adaxial myoD expression (compare arrowheads in L,M) in 60/206 embryos (29%), all of which were dtr–/–mutants as judged by forebrain and hindbrain nk2.2 expression defects. This suggests Gli2 may activate Hh signaling in a small area of the ventral midbrain and in adaxial cells. Control MO injections had no effect on nk2.2 expression in 85/85 embryos from a similar dtr–/+x dtr–/+cross, with 25 embryos (29%) showing the dtr–/–nk2.2 defects (J) and 60 embryos (71%) showing wild-type nk2.2 expression as expected for dtr–/+ and dtr+/+embryos. (A-F,J, and K) are lateral views of the head, eyes removed. (G-I,L, and M) are dorsal views of the tail region. All embryos are at the 20 somite (19 hour) stage. For yot/gli2, embryo genotypes were inferred by myoD expression in adaxial cells, then were verified by PCR (not shown, see Materials and Methods). D and G, E and H, F and I, J and L and K and M show the same individual labeled simultaneously with nk2.2 and myoD.