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Spatial patterns of ecdysteroid receptor activation during the onset of Drosophila metamorphosis

Howard Hughes Medical Institute, Department of Human Genetics, University of Utah, 15 North 2030 East Room 5100, Salt Lake City, UT 84112-5331, USA

View larger version (12K): [in a new window]   Fig. 1. Schematic representation of the GAL4-LBD system. The upper diagram depicts a fusion gene consisting of the coding region for the yeast GAL4 DNA-binding domain (GAL4 DBD) fused in-frame with the ligand binding domain (LBD) of EcR or USP. This construct is introduced into the Drosophila genome under the control of the hsp70 promoter. Heat-induced GAL4-LBD protein can bind to GAL4 upstream activating sequences (UAS) that are present in a second transgenic construct. In the absence of ligand, the GAL4-LBD protein does not activate lacZ reporter gene transcription; however, in the presence of its ligand (black circle) it can switch into an active conformation and induce lacZ expression.

View larger version (86K): [in a new window]   Fig. 2. GAL4-EcR and GAL4-USP are widely activated by the late larval ecdysteroid pulse. hs-GAL4-EcR; UAS-nlacZ (GAL4-EcR) or hs-GAL4-USP; UAS-nlacZ (GAL4-USP) third instar larvae were heat treated, allowed to recover for several hours, staged at either 12-18 hours before puparium formation (–18 h; A-D) or approx. 2 hours before puparium formation (–2 h; E-L), and processed for histochemical staining. Blue precipitate indicates expression of nuclear ß-galactosidase. GAL4-EcR (A-D) and GAL4-USP (data not shown) are not activated in third instar larvae 12-18 hours before puparium formation. In contrast, both GAL4-EcR (E-H) and GAL4-USP (I-L) are widely activated in late third instar larvae, as depicted in the midgut, hindgut, muscles and fat body.

View larger version (117K): [in a new window]   Fig. 3. Distinct patterns of GAL4-EcR and GAL4-USP activation in the central nervous system at the onset of metamorphosis. hs-GAL4-EcR; UAS-nlacZ (GAL4-EcR) or hs-GAL4-USP; UAS-nlacZ (GAL4-USP) third instar larvae were heat treated, allowed to recover for several hours, staged at approx. 2 hours before puparium formation, and processed for histochemical staining. The CNS and attached leg imaginal discs are shown, with anterior to the right. (A) GAL4-EcR activation is prominent in the optic lobes of the brain. (B) GAL4-USP activation is weaker in this region, but strong in the ventral nerve cord. Neither fusion construct is activated in the leg imaginal discs at this stage (arrows).

View larger version (71K): [in a new window]   Fig. 4. GAL4-EcR and GAL4-USP are widely activated by 20E in cultured larval organs. Larval organs dissected from heat treated hs-GAL4-EcR; UAS-nlacZ (GAL4-EcR) or hs-GAL4-USP; UAS-nlacZ (GAL4-USP) third instar larvae 18-8 hours before puparium formation were cultured with 5x10–6 M 20E and processed for histochemical staining. GAL4-EcR and GAL4-USP are not activated in culture in the absence of 20E (data not shown). Both GAL4-EcR (A-D) and GAL4-USP (E-H) are widely activated by 20E in many larval tissues, including the midgut, hindgut, muscles and fat body.

View larger version (58K): [in a new window]   Fig. 5. GAL4-EcR and GAL4-USP can be activated by 20E in the ring gland and imaginal discs. Larval organs dissected from heat treated hs-GAL4-EcR; UAS-nlacZ (GAL4-EcR) or hs-GAL4-USP; UAS-nlacZ (GAL4-USP) third instar larvae 18-8 hours before puparium formation were cultured with 5x10–6 M 20E and processed for histochemical staining. Both GAL4-EcR and GAL4-USP are activated by 20E in the ring gland (B,C) and imaginal discs (E, wing imaginal disc is shown; F, leg imaginal disc is shown) but not in the absence of hormone (A, ring gland; D, both wing and leg imaginal discs are shown).

View larger version (109K): [in a new window]   Fig. 6. Stage-specific GAL4-USP activation by 20E in imaginal discs. Larval organs dissected from heat treated hs-GAL4-USP; UAS-nlacZ third instar larvae at 18 and 4 hours before puparium formation were cultured with 5x10–7 M 20E (+20E) or without hormone (–20E). No GAL4-USP activation is seen in the absence of 20E (A,C). In the presence of 20E, GAL4-USP activation is observed in imaginal discs from mid-third instar larvae (B) but not in discs from late third instar larvae (D).

View larger version (64K): [in a new window]   Fig. 7. GAL4-USP activation is significantly reduced in an EcR mutant background. GAL4-USP activation was assayed in yw; EcRM554fs/CyO, y+; hs-GAL4-USP/UAS-nlacZ or yw; EcRA483T/CyO, y+; hs-GAL4-USP/UAS-nlacZ animals (control) staged in parallel with yw; EcRA483T/EcRM554fs; hs-GAL4-USP/UAS-nlacZ animals (EcR-ts) at the onset of metamorphosis. The cross was shifted to 29°C for 24 hours before heat treatment and histochemical staining. GAL4-USP activation is greatly reduced in the midgut, muscles and fat body in the EcR-ts mutant (D-F) compared to controls (A-C).

View larger version (16K): [in a new window]   Fig. 8. GAL4-EcR and GAL4-USP expression leads to stage-specific lethality. The graph shows percentage viability after a single 30 minute heat treatment at 37°C of third instar larvae between 8-0 hours before puparium formation (late L3) or early pupae 12-20 hours after puparium formation. Five different stocks were tested: a w1118 control (gray bars), two hs-GAL4-EcR lines outcrossed to w1118 (blue bars), and two hs-GAL4-USP lines outcrossed to w1118 (red bars) (n=120-150 for each line).

View larger version (115K): [in a new window]   Fig. 9. Lethal phenotypes caused by GAL4-EcR overexpression are similar to EcR mutant phenotypes at the onset of metamorphosis. Control w1118 and homozygous hs-GAL4-EcR third instar larvae were subjected to two sequential heat treatments for 40 minutes at 38°C, separated by a 4 hour recovery period. (A) 98% of the control w1118 animals form tanned barrel-shaped pupal cases under these conditions. 62% of heat-treated hs-GAL4-EcR animals arrest their development as non-pupariating third instar larvae (B) and 28% form elongated untanned prepupae (C). The remaining 10% form partially tanned and misshapen pupal cases (data not shown) (n=120 for each line).