Fig. 4. One copy of either Gli2 or Gli3 is required for tissue autonomous Shh responsiveness. (A) Shh expression was assessed by whole-mount in situ hybridization at E9.5. Transverse sections through the trunk reveal dramatically reduced Shh in the notochord and floorplate of Gli2^–/–Gli3^–/– embryos (compare arrowheads in A). (B) PSM tissue was isolated from E9.5 wild-type, Gli3^–/–, Gli2^–/–, Gli2^–/–Gli3^+/– and Gli2^–/–Gli3^–/– embryos and cultured in the presence (+) or absence (–) of Shh-N for 24 hours. Induction of target genes was assessed by RT-PCR. ß-Actin serves as a control for normalization. Compared with wild-type PSM, Gli3^–/– PSM shows Shh-responsiveness with an increase in Pax9 expression in the absence of Shh-N (n=4). Gli2^–/– PSM shows reduced responsiveness in activating Ptch and Pax9, but normal induction of other targets (n=4). In Gli2^–/–Gli3^+/– PSM, all targets are induced with some reduction in Pax9 induction (n=2). In Gli2^–/– Gli3^–/– PSM, all targets tested are expressed identically in the presence and absence of Shh-N. There is increased basal expression of Ptch and Pax9 in Gli2^–/–Gli3^–/– PSM independent of Shh-N (n=3).