Fig. 5. A combination of the Zen and Smad activators and a repressor are necessary for establishing the C15 domain. Dorsal views of late stage5/early stage 6 embryos carrying the 650 bp C15 enhancer-lacZ transgene with mutations in the Smad- and Zen-binding sites. Embryos were hybridized with lacZ probes. Arrowheads indicate the limits of the expression domain. No effect on the lacZ expression pattern was observed when Smad sites were mutated in the M2 (A) or M5 (B) clusters. However, mutation of the Smad sites in M7 (C) caused a reduction in the intensity of lacZ expression, especially in lateral regions. Arrowheads denote faintly stained cells. Embryos carrying mutations in both the M2 and M5 clusters (D) or the M5 and M7 clusters (E) also showed weaker lacZ expression. Mutation of all Smad sites in M5, M6 and M7 caused severe loss of expression (F); however, the overall size of the domain appears unchanged (arrowheads). Mutation of the putative Zen-binding site in the Z1 sequence caused reduced expression (G). However, mutation of the Zen sites in the Z2 (H) or Z3 (I) sequences caused significant expansion of the lacZ domain, implicating those sites in repression of the C15-lacZ transgene. When the Zen-binding sites were eliminated, expression was drastically reduced (J). Mutation of Z2 and M5 had no effect (K); however, mutation of M2, M5 and Z2 caused lateral expression to be lost (L), presumably because low levels of Mad-P in lateral regions is insufficient to activate transcription even in the absence of the putative repressor.