Fig. 7. Mutational analysis of the early Pax2 enhancer. (A) Binding of homeodomain proteins to the early enhancer. In vitro translated proteins and a MHB extract were analyzed for binding to a 157 bp probe containing the wild-type enhancer (SX[wt], from –3833 to –3677) or the corresponding sequence mutated at all four TAAT sites (SX[TAAT]). Oct1 was analyzed at a 10-fold lower concentration. (B) Interaction of in vitro translated Oct1 and Oct3/4 proteins with a wild-type (wt) or mutated (M1) probe (see C) containing the distal two TAAT motifs. X, nonspecific DNA-binding activity. (C) Sequence of the early enhancer. Recognition sequences for homeo (HD) and POU-specific (POU_S) domains and binding sites for Otx and Sp1-like zinc finger (Zn) proteins are shown together with an alignment of the E2 element present in the MHB-specific enhancer of Pax5 (Pfeffer et al., 2000). (D-F) Inactivation of the early enhancer by mutation of the TAAT motifs in the context of transgene #1. The introduced mutations are indicated together with the statistical analysis (D) and representative examples of transgenic embryos (E,F). Posterior refers to ectopic expression in the posterior region of late gastrula embryos (D). The mid-hindbrain (mh) territory is indicated by a bracket (E,F).