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Fig. 6. Larval cuticle defects in CrebA and SPCG mutants. (A) Dark-field images are shown of cuticles prepared from wild-type, CrebA, srpRß, sec13, sec23 and sar1 first instar larvae. The cuticles from each of the mutants were smaller and fainter than the cuticle prepared from the wild-type larva. (B) High-magnification images of the ventral, lateral and dorsal cuticular structures of wild-type larvae (top panels), and ventral and dorsal cuticular structures of CrebA, srpRß, sec13, sec23 and sar1 first instar larvae (lower panels). The ventral cuticles of the mutants showed a loss of denticles and the denticles that were present have very light pigmentation, more consistent with the pigmentation seen in more lateral denticles of wild-type larvae [1]. The dorsal surfaces of the mutant larvae typically consisted of thin hairs and naked cuticle, an arrangement more typical of dorsal-lateral regions of wild-type cuticles (top panel, [2]). (C) High-power phase images are shown of the mouthparts (mp; left panels) and filzkörper (fk; right panels) from cuticles prepared from wild-type (wt), CrebA, srpRß, sec13, sec23 and sar1 first instar larvae. Both the mouthparts and filzkörper are less robust and have reduced pigmentation than observed in the mouthparts and filzkörper of wild-type embryos. (D) DIC images of stage 15 embryos stained with -DSC73 in combination with -ß-gal and -CrebA to distinguish CrebA mutant embryos (bottom two panels) from their heterozygous (wild-type) siblings (top two panels). Left panels show lower magnification images of the same embryo shown in the middle and right panels to reveal CrebA staining in the salivary glands of the heterozygotes (wild type) and the loss of CrebA staining in the salivary glands of the CrebA-mutant embryos. DSC73 staining intensity in the denticle-producing cells was reduced in the CrebA mutants compared with that of wild-type embryos. Staining of dorsal hair-producing cells, however, was only mildly decreased in the CrebA mutants compared with wild-type embryos. The process of dorsal closure appeared to be lagging in the CrebA mutants compared with wild type; the epidermal cells that meet and fuse at the dorsal midline were relatively closer in the wild-type embryos compared with the CrebA mutants. This defect is probably linked to the dorsal holes observed in the cuticles of CrebA mutant larvae. Embryos were staged based on extent of head involution, gut invaginations and elongation of the proventriculus.

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