Fig. 3. The shared sequence motifs correspond to essential cis-regulatory elements. The shared sequence motifs in the vnd (A-C, 743 bp) and brk (E-H, 498 bp) enhancers were mutated as indicated, and the effects on enhancer activity were assayed by in situ hybridization as described in Fig. 1. Ventrolateral views of embryos oriented with anterior to the left and dorsal up are shown. All of the embryos (except D) are undergoing cellularization. (AC). A larger, more robust vnd enhancer than shown in Fig. 2 was used. The wild-type vnd enhancer directs lateral stripes of lacZ reporter gene expression (A). By contrast, point mutations that eliminate each of the two CACATGT motifs disrupt the activities of an otherwise normal vnd-lacZ fusion gene (B). Staining is restricted to the ventral-most regions of the neurogenic ectoderm, similar to the normal sim expression pattern (see Fig. 4). Mutations in the three CTGWCCY motifs in the vnd enhancer cause subtle changes in the lacZ staining pattern, including a slight narrowing and some irregularity in expression (C). (E-H). The embryos express different brk-lacZ fusion genes. The wild-type brk enhancer directs a staining pattern that is similar to the one produced by the vnd enhancer (E, compare with A). Mutations in the two CACATGT motifs disrupt the activities of the brk enhancer and cause a loss of lacZ staining, especially in the posterior half of the embryo (F, compare with E). Point mutations in the CTGWCCY motifs nearly abolish expression from an otherwise normal brk-lacZ fusion gene (G). Finally, mutations in the two Su(H)-binding sites cause a loss of expression in the posterior half of the embryo (H), similar to the altered pattern obtained with mutations in the Twist (CACATGT) binding sites (F). The transgenic embryo in D expresses a stripe2-Notch^IC fusion gene that causes constitutive activation of Notch signaling in the stripe 2 region. The embryo was hybridized with a digoxigenin-labeled rho antisense RNA probe. Expression is slightly expanded in the region where the stripe2-Notch^IC transgene is active (arrow).