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First published online August 18, 2003
doi: 10.1242/10.1242/dev.00677

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senseless is necessary for the survival of embryonic salivary glands in Drosophila

Department of Molecular and Cell Biology, 401 Barker Hall, University of California, Berkeley, CA 94720, USA

View larger version (96K): [in a new window]   Fig. 1. sens expression in the embryonic salivary glands. All views are ventral and embryos are oriented with anterior to the left. (A-D) sens mRNA is expressed in the salivary placodes at stage 11 (arrowheads) and later in the salivary glands at stage 14 (arrows). (E,F) The salivary glands in sens mutant embryos at stage 14 are smaller than those of wild-type embryos.

View larger version (94K): [in a new window]   Fig. 2. Regulation of sens by daughterless (da) in salivary glands. (A,B) da10 embryos had small salivary glands compared with wild type. (C,D) sens mRNA is expressed in the salivary placodes of da10 embryos at stage 11. The PNS expression of sens is missing in da10 embryos (arrowhead). (E,F) sens expression is markedly reduced at later stages in the salivary glands of da10 embryos compared with wild-type embryos.

View larger version (72K): [in a new window]   Fig. 3. sage, a salivary-specific bHLH protein, regulates sens. (A,B) sage is first expressed in the salivary placodes at stage 10 and continues to be expressed in the salivary glands of stage 14 wild-type embryos. (C,D) The embryos injected with sage dsRNA showed smaller salivary glands compared with embryos injected with injection buffer. (E,F) sage mRNA is expressed at normal levels in the salivary placodes of sensE2 embryos but is reduced during later stages in the salivary glands of sensE2 embryos compared with wild-type embryos (compare F with B). (G,H) SENS levels, although present, are reduced in sage dsRNA-injected embryos when compared with embryos injected with injection buffer. (I) GST pulldown showing that DA can bind SAGE. Lane 1, in vitro transcription translation extract without the GST fusion proteins; lane 2, GST + DA; lane 3, GST-SAGE + DA. The arrowhead indicates the band corresponding to DA. DA shows strong binding to GST-SAGE but not to GST alone.

View larger version (45K): [in a new window]   Fig. 4. fork head is necessary for the initiation of sens expression in the placodes. (A-C) sens mRNA expression is observed in the salivary placodes of wild-type (A) and hkbAI72 (C) embryos but not in fkhXT6 (B) embryos. The arrowhead (B) indicates the continued expression of sens in the PNS neurons in fkhXT6 embryos.

View larger version (114K): [in a new window]   Fig. 5. sensE2 embryos have normal placodes and salivary ducts. (A,B) The salivary placodes in stage 11 sensE2 embryos are identical to those in wild-type embryos. (C,D) In addition, the salivary ducts (arrow) in sensE2 embryos as visualized by duct marker, dead ringer, are similar in length and diameter to wild-type embryos.

View larger version (131K): [in a new window]   Fig. 6. Apoptosis in salivary glands of sensE2 embryos. (A-C) TUNEL staining in the salivary glands showed no apoptotic cells in the salivary glands of wild-type embryos. However, sensE2 embryos showed presence of a large number of apoptotic cells that are absent in arm-GAL4: UAS-P35; sensE2 embryos. Note the longer salivary glands in the arm-GAL4: UAS-P35; sensE2 embryos compared with sensE2 embryos. (D-G) reaper mRNA is not expressed in the salivary glands of stage 13 wild-type embryos but is upregulated in sensE2 as well as arm-GAL4: UAS-P35; sensE2 embryos. In sensE2 embryos, reaper RNA disappears from the salivary glands at stage 14 (data not shown), whereas it continues to be expressed in the salivary glands of stage 15 arm-GAL4: UAS-P35; sensE2 embryos (G). (H,I) Expression of hid is also induced in the salivary glands of sensE2 embryos when compared with wild-type embryos.

View larger version (25K): [in a new window]   Fig. 8. Graphical representation of the rescue of sens phenotype by cell death genes. The graph shows the percentage of embryos with particular salivary gland lengths in the different genotypes and the table below shows the numbers represented in the graph. The lengths of the salivary glands are assessed by comparing them with the length of the ectodermal thoracic segments. The numbers in parenthesis are the number of embryos for each condition. Asterisk indicates the percentage of embryos with salivary glands that did not invaginate completely. Double asterisks indicate that all the embryos in this category had salivary glands that were two segments long.

View larger version (77K): [in a new window]   Fig. 7. The phenotype of sens is suppressed by cell death genes in Df(3L)H99. (B,C) The small salivary glands in sensE2 embryos (B) are rescued in embryos that are also mutant for Df(3L)H99 (C). Note that the salivary glands of Df(3L)H99 embryos are similar to wild type (compare A with D). (E,F) The small salivary glands in sensE2 embryos are partially rescued in embryos mutant for sensE2 and either reaper (Df(3L)XR38; E) or hid (W05014; F). (G,H) The salivary glands in fkhXT6 mutants are not rescued by overexpression of UAS-sens driven by arm-GAL4. (A-F) Salivary glands visualized using an antibody to FKH. (G,H) An antibody against ß-galactosidase is used to visualize the enhancer trap N33 that is expressed in the salivary glands.

View larger version (109K): [in a new window]   Fig. 9. sens represses the expression of a reaper reporter in the salivary glands. (A,A',A'') In wild type embryos, at stage 12 the Rpr-11-lacZ expression (green) is observed in the dorsal posterior cells of the salivary placodes. (C,C',C'') At later stages, expression of Rpr-11-lacZ in barely above background levels throughout the salivary glands. (B,B',B'',D,D',D'') By contrast, sensE2 embryos show marked upregulation of Rpr-11-lacZ expression in the salivary placodes and in the invaginated portion of the salivary glands at stage 12 and continue to show high expression at later stages in the salivary glands.

View larger version (14K): [in a new window]   Fig. 10. The epistatic relationships in the salivary glands and in the peripheral nervous system. In the salivary glands, Scr activates fkh and sage. fkh is necessary for the initiation (I) of sens expression. sage, together with da, is necessary for the maintenance (M) of sens expression. In turn, sens is necessary to maintain the expression of sage in the salivary glands via a positive feedback loop. sens represses reaper and hid and prevents cell death. By contrast, in the peripheral nervous system, achaete-scute complex genes (AS-C) and da are needed together to initiate and maintain sens expression (Nolo et al., 2000). By a feedback loop similar to that in the salivary glands, sens amplifies the expression of the AS-C genes. Downstream from sens, it represses E(spl) transcription and is necessary for neuronal cell fate determination.

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