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Fig. S1. All BAC constructs containing the distal enhancer are able to rescue sna mutant gastrulation defects. (A-F) Florescent in situ hybridizations with sim (green) and gfp (red) on sna1/snaIIG05 embryos expressing the indicated BAC show that sim expression during and after gastrulation is essentially normal in both the squish and the D to P constructs. Antibody staining using an anti-Eve antibody demonstrates that Eve expression (red, G-I) is also normal, showing 11 clusters of Eve positive cells on the lateral side of the embryo, indicating that mesoderm spreading was normal in these embryos.
Fig. S2. Even at high temperatures the distal enhancer is sufficient to rescue gastrulation in sna mutants. In situ hybridization for gfp (green) and antibody staining to detect Eve (red) show that even at 29°C the phenotype of the delete proximal and delete distal CRM constructs are very different. The delete proximal construct appears largely normal, with the mesodermal Eve cells present at the dorsal side of the embryo. The delete distal construct exhibits major defects, with only 4 clusters of Eve+ cells at the dorsal side of the embryo.
Fig. S3. Repressors function predominantly through the distal CRM, whereas expansion towards the anterior pole requires the proximal CRM. Embryos containing two intersecting dorsal-ventral patterning axes supported by expression of activated Toll receptor at the anterior of wild-type embryos accomplished through a transgene, F10 (Huang et al., 1997). (A) F10-expressing embryos were stained using an anti-Dorsal (green) and anti-Histone H3 (red) antibodies to identify dorsal-ventral patterning axes and nuclei (for image contrast), respectively. (B,C) In situ hybridization using riboprobes to lacZ (red) and sna (green) of embryos in which either the distal 2.0 kb CRM (B) or the proximal 2.2 kb CRM (C) lacZ reporter constructs were introduced into a background containing the F10 transgene. In B, lacZ and sna expression completely overlap, showing that the distal CRM is responsive to repressor X. In C, lacZ expression was found to extend well beyond the expression domain of sna expression into the posterior pole (large arrow) as well as into the domain of repressor X function (arrowheads).
