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First published online 18 May 2005
doi: 10.1242/dev.01863

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CrebA regulates secretory activity in the Drosophila salivary gland and epidermis

Department of Cell Biology, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA

View larger version (22K): [in a new window]   Fig. 1. The early secretory pathway. Transcripts encoding secretory/transmembrane proteins are targeted to the ER through interaction of the N-terminal peptide (black line), as it emerges from the ribosome (blue), with the Srp complex (yellow oval). The Srp complex is composed of a 7S RNA and six RNA-binding proteins, each of which is conserved in Drosophila. The Srp/signal peptide complex interacts with the -subunit of the SR (brown). The ribosome is then transferred to the sec61 complex (light blue), which is composed of -ß- subunits (all are conserved in Drosophila). sec62, sec63, sec71 and sec72 are thought to be involved in Srp-independent (or post-translational) protein translocation. TRAM is required for efficient co-translational translocation and is important for incorporation of transmembrane proteins into the lipid bilayer of the ER. After the nascent peptide is translocated into the ER, the signal sequence is cleaved by the SPC. Four out of the five peptide subunits (red lines) are conserved in Drosophila, with one of the peptides showing homology to two of the mammalian proteins. The COPII coatamer is involved in the anterograde movement of vesicles from the ER to the Golgi (blue circles). It comprises sec13, sec16, sec23, sec24 and sec31 subunits. A sec16 homolog is not recognizable in the current annotated Drosophila genomic sequence. Sar1 (orange circle) is a small G-protein involved in the regulation of COPII assembly/disassembly. The p24 transmembrane family of proteins interact with soluble cargo destined to leave the ER. COPI-coated vesicles are involved in retrograde movement of secretory vesicles from the Golgi to the ER (green ovals). All of the COPI coatamer components, which include , , , -cop and ARF-1, as well as other proteins involved in the retrieval of escaped resident proteins back to the ER are conserved in Drosophila. The Srp region of this figure was adapted from Wild et al. (Wild et al., 2002); the COPII region from Shaywitz et al. (Shaywitz et al., 1997); the SPC region from Kalies et al. (Kalies et al., 1996); the translocon region from Romisch (Romisch, 1999); and the COPI region from Wieland and Harter (Wieland and Harter, 1999).

View larger version (147K): [in a new window]   Fig. 2. Transcriptional regulation of early SPCGs in the salivary gland. -Pasilla staining of stage 13-14 wild-type, fkh, CrebA and hkb mutant embryos reveals the morphology of the salivary gland (A). All embryos are ventral views, with anterior towards the left. This arrangement and positioning of wild-type and mutant embryos is also used for B and C. (D) Left panels are CrebA heterozygotes (lacZ expression in posterior regions), middle panels are CrebA homozygotes and the right panels are fkh homozygotes, all at early stages (stage 11), oriented with anterior towards left and showing lateral views. Salivary gland SPCG expression is controlled by fkh (late) and CrebA (B-D; see Figs S1, S2 in the supplementary material). With a small subset of the genes, including sec62 and TRAP, expression can still be detected in fkh mutants (even late stage), albeit at reduced levels (B). The salivary gland expression of all the tested SPCGs in hkb mutants is comparable with that of wild-type salivary glands (B,C; see Figs S1, S2 in the supplementary material). Arrows indicate salivary gland expression in the mutants.

View larger version (75K): [in a new window]   Fig. 3. Regulation of CrebA by Fkh. A map of the region upstream of the CrebA transcription unit showing the 2.8 kb enhancer fragment that drives reporter gene expression in the salivary gland (A). This fragment is just upstream of a P-element insertion (B204) that results in ß-gal expression in only the salivary gland and amnioserosa. Two subfragments of 1100 and 770 bp also resulted in salivary gland expression of a lacZ reporter gene, with the CrebA 770 fragment driving salivary gland expression slightly earlier than the CrebA 1100 fragment (B,C). The CrebA 770 fragment has four Fkh consensus binding sites (A, blue circles) as well as two consensus Scr/Exd/Hth-binding sites (A, blue circles with black edges). The Creb 1100 site has six Fkh consensus binding sites and no consensus Scr/Exd/Hth-binding sites (A). The CrebA 1100 lacZ construct is not expressed to high levels in the salivary glands of fkh mutant embryos (D), with only some very low level expression detectable at late embryonic stages (D, right-most embryo, arrows). CrebA 1100 constructs with all six Fkh consensus sites mutated also show a loss of reporter gene expression specifically in the salivary gland (E), again with only some very low level expression detectable in late embryonic stages (E, right-most embryo, arrows).

View larger version (76K): [in a new window]   Fig. 4. Characterization of SPCG salivary gland enhancers identifies at least three CrebA-dependent enhancers. Reporter gene constructs were built for six of the 34 SPCGs analyzed in this study (A-F, diagrams at the top). Yellow circles of each set of embryos indicate the consensus motif found in SPCGs by MEME analysis. Arrows indicate putative transcription start sites based on the longest sequenced 5' end cDNAs for each gene. Green lines indicate the open reading frames. Three independent lines carrying the srp68 lacZ construct did not have ß-gal expression in the salivary gland (A). Multiple independent lines carrying p24-1 lacZ, srpR lacZ and zcop lacZ constructs showed salivary gland ß-gal expression beginning during embryonic stage 13 and continuing through the rest of embryogenesis (B-D). Independent lines carrying the spase25 lacZ (F) and sec61ß lacZ (E) constructs had ß-gal salivary gland staining from early stage 12 through the rest of embryogenesis (E,F). The zcop and sec61ß lacZ constructs that gave salivary gland expression of ß-gal in wild-type embryos did not express ß-gal in the salivary glands of CrebA mutants (D,E, lower two rows). The spase25 lacZ constructs showed significantly reduced expression in the salivary glands of CrebA mutants (F, lower two rows).

View larger version (66K): [in a new window]   Fig. 5. CrebA mutations affect secretory activity in the salivary gland. (A,B) Stage 15 and 17 wild-type (CrebA/+) and CrebA mutant embryos were stained with -PH4SG1 (red) and -En PAb (green). (A) At stage 15, Engrailed-positive secretory granules can be detected in CrebA/+, while very few were seen in CrebA mutant salivary glands (arrows). (B) Significantly more secretory granules were detected in stage 17 CrebA/+ salivary glands than in the stage 17 CrebA mutant salivary glands (arrows and arrowheads). The amount of lumenal content increases significantly from stage 15 to 17 in both CrebA/+ and CrebA salivary glands, but is not different in the wild-type versus CrebA mutant glands. (C) Stage 17 sec13 and sec23 salivary glands appear relatively normal with respect to secretory granule levels. The abnormal salivary gland shape in the sec13 mutants is probably linked to other patterning defects observed when this P-element insertion is homozygous. Arrows indicate punctate staining near the apical surface. Arrowheads indicate larger vesiclar staining throughout the cell.

View larger version (160K): [in a new window]   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.

View larger version (22K): [in a new window]   Fig. 7. Model of transcriptional regulation of early SPCGs. Fkh and CrebA are directly regulated by the Scr/Exd/Hth complex. Late expression of CrebA requires Fkh and Fkh is autoregulated. The expression of most of the SPCGs is greatly reduced in CrebA mutants at all embryonic stages, whereas only late (beyond stage 13) expression of all tested SPCGs is affected by the loss of fkh function. As late expression of the SPCGs was lower in late fkh mutants than in CrebA mutants, we propose the existence of another early transcription factor gene in SPCG regulation. As with CrebA, late but not early expression of this putative transcription factor would require Fkh.

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