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Procedures for whole-mount in situ hybridization, immunohistochemistry, and cuticle preparations were as described (27). DNA probes for hb and lacZ were generated using the 2.4-kb insert from ph-bXba2.4 (11) and a 3.6-kb fragment from pCHABΔSal (27). Rabbit antibody to β-galactosidase (Cappel) was used at 1:5000 dilution. For Fig. 3, fully developed hb mutant embryos were selected under a Leica MZ12 stereomicroscope for green fluorescent protein (GFP) expression before cuticle preparations.
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The hb transgenes were constructed as follows: A 4-kb genomic Eco RI fragment containing the hb upstream sequences including the P1 promoter was isolated from phbXho10.5 (11) and cloned into the Eco RI site of pCHABΔXba (27), thereby creating pChbP1. The complete open reading frame for hb was excised by Not I and partial Eco RI digests from the cDNA clone J3, which was derived from a transcript originating at the P1 promoter (11). The cDNA fragment was inserted into pChbP1 that was opened by a complete Not I and partial Eco RI digest to fuse the genomic and cDNA at the Eco RI site within the 5′ untranslated leader, resulting in pChbP1only. To assess the expression pattern of the transgene, we replaced the hb coding region (including its 3′ untranslated region) by the lacZ gene. pChbP1only was opened by an Xba I digest and religated to generate pChbP1ΔXba, into which a 3.6-kb Xba I fragment containing the AUG-β-gal (AB) gene derived from pCHABΔSal (27) was cloned to create pChbP1AB. A pUAST-HB construct was generated by cloning the 2.4-kb Xba I fragment containing the hb coding region from phbXba2.4 (11) into pUAST (28). These constructs were introduced into the fly genome as described (27) to generate several fly strains carrying the transgenes hbP1only, hbP1AB, and UAS-HB.
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E1 allele was used and third chromosomal UAS-HB and hbP1AB transgenes were recombined. The GAL4 driver lines were nos-GAL4GCN4-3′bcd (29) and wg-GAL4.
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We thank P. Beaufils for UAS-GFP strains; M. Boyle, N. Dostatni, U. Gaul, F. Janody, and J. Posakony for materials; and V. Schaeffer, I. Brun, J. Burr, and the members of the Desplan, DiNardo, and Gaul labs for helpful discussions. E.A.W. was the recipient of a Human Frontier Science Program long-term fellowship. Supported by the Howard Hughes Medical Institute at Rockefeller University and by NSF grant IBN-9817981.
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