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A common translational control mechanism functions in axial patterning and neuroendocrine signaling in Drosophila

Ira E. Clark^*, Krista C. Dobi^*, Heather K. Duchow^*, Anna N. Vlasak and Elizabeth R. Gavis

Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA
^* These authors contributed equally to this work
Present address: Department of Biology, University of Pennsylvania, Philadelphia, PA 19104

View larger version (14K): [in a new window]   Fig. 1. Nos reporter transgenes for ectopic expression. (A) The nos-coding region (shaded) and 5'UTR sequences (hatched) were fused to yeast UAS sequences to permit transcriptional activation of nos by GAL4. In the UAS-nos-tub3'UTR transgene (top), the nos 3'UTR is replaced by the -tubulin 3'UTR (open box). In the UAS-nos-tub:nos+2 transgene (bottom), the -tubulin 3'UTR sequences contain an insertion of the nos 3'UTR +2 element (black box), which includes the nos TCE and an adjacent region with weak translational repression function. The UAS-nos-tub:TCE transgene (not shown), which carries an insertion of the TCE alone, behaves similarly. (B) Transgenes are identical to those in A, except for the presence of a stop codon mutation (*) in the nos-coding region.

View larger version (58K): [in a new window]   Fig. 2. Ectopic expression of nos by GAL4109-68. Adult flies, carrying both GAL4109-68 and UAS-nos-tub3'UTR transgenes, 2-3 days post-eclosion. A small percentage of these flies appear wild type (A), whereas the majority show the adolescent phenotype (B).

View larger version (45K): [in a new window]   Fig. 3. GAL4 drivers that produce a TCE-sensitive adolescent phenotype are expressed in the CNS. Confocal images of CNS dissected from wandering third instar larvae carrying both UAS-GFP and (A) GAL4109-68; (B) HD44A-GAL4; (C) HD23A-GAL4; and (D) KF42A-GAL4 transgenes. The expression domains of each driver are revealed by direct GFP fluorescence.

View larger version (48K): [in a new window]   Fig. 4. Expression of nos in EH cells. (A) Adult fly carrying the EHups-GAL4 and UAS-nos-tub3'UTR transgenes displays the adolescent phenotype. The same phenotype is produced by the UAS-nos-tub:TCE transgene. (B) Confocal image of fixed larval CNS tissue with EH cells (arrow) revealed by direct GFP fluorescence. In this experiment, animals carry the EHups-GAL4, UAS-GFP and UAS-nos-tub3'UTR transgenes. GFP fluorescence and bright field image are visualized simultaneously for orientation (BL, brain lobe; VC, ventral nerve cord). (C) Confocal projection of larval CNS of an animal from experiment in B shown at high magnification to illustrate the EH cell processes. The morphology of the EH cells and their processes is not obviously different from that of animals carrying only the EHups-GAL4 and UAS-GFP transgenes (not shown).

View larger version (113K): [in a new window]   Fig. 5. EH cells are not the sites of ectopic nos expression. (A,C) Confocal images of CNS from third instar larvae carrying a UAS-GFP reporter transgene and either the GAL4109-68 (A) or HD44A-GAL4 (C) driver, immunostained with anti-EH antibody. The merge of anti-GFP immunofluorescence (green) and anti-EH antibody staining (red) is shown. In both cases, cells that lie below the EH cells express GFP (not readily visible in these sections) but the EH cells (arrow) do not. (B,D) Confocal images of CNS from third instar larvae carrying UAS-nos-tub3'UTR transgene and either GAL4109-68 (B) or HD44A-GAL4 (D) transgenes, immunostained with anti-EH antibody (red). EH cells, their processes and production of EH are not affected when nos is ectopically expressed by these drivers.

View larger version (11K): [in a new window]   Fig. 6. Suppression of ectopic nos expression by GAL80. (A) GAL4109-68 activates UAS-nos-tub3'UTR in a subset of cells in the CNS (dark shading). GAL4109-68 may also activate UAS-nos-tub3'UTR at low levels or at unexamined times in EH cells (light shading with ‘?’). Ectopic synthesis of Nos in one or more cells of the GAL4109-68 expression domain produces the adolescent phenotype. (B) Ubiquitous expression of GAL80 under control of the tubulin promoter (tubP-GAL80) blocks the ability of GAL4109-68 to activate transcription of the UAS-nos-tub3'UTR transgene in all cells and suppresses the adolescent phenotype (Table 3). (C) The ability of GAL80 to block transcriptional activation by GAL4109-68, as shown in B, was used to test whether GAL4109-68 is indeed active in EH cells. Expression of GAL80 specifically in EH cells by EHups-GAL80 should block GAL4 activation of UAS-nos-tub3'UTR and suppress the adolescent phenotype only if GAL4109-68 drives expression of UAS-nos-tub3'UTR in EH cells (Table 3).

View larger version (71K): [in a new window]   Fig. 7. GAL80 blocks GAL4-dependent activation in EH cells. (A) GFP fluorescence in CNS of third instar larva carrying EHups-GAL4 and UAS-GFP transgenes. Bright field image is visualized simultaneously for orientation. Arrow indicates one of the two EH cells, one brain lobe (BL) is indicated for point of reference. (B) Animal as in A but carrying the EHups-GAL80 transgene as well. No GFP fluorescence is detected.

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