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doi: 10.1242/10.1242/dev.00205

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EcR isoforms in Drosophila: testing tissue-specific requirements by targeted blockade and rescue

Lucy Cherbas1,{dagger}, Xiao Hu1,*, Igor Zhimulev2, Elena Belyaeva2 and Peter Cherbas1

1 Department of Biology, Indiana University, Bloomington, IN 47405, USA
2 Institute of Cytology and Genetics, Russian Academy of Sciences, Novosibirsk 630090, Russia
* Present address: Pharmacia, Chesterfield, MO 63017, USA



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  		Fig. 1. Domain structures of the ecdysone receptor components. The three isoforms of Drosophila EcR and the single Drosophila USP are shown, with the standard nuclear receptor regions indicated. Region C is the DNA-binding domain (DBD); region E, the ligand-binding domain (LBD). The three EcR isoforms are identical in sequence except in the A/B regions that are unrelated. The number of isoform-specific residues is shown for each EcR. The scale underneath (in residues) is aligned to EcR-B1. The residue mutated in EcR-F645A is indicated.

 


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  		Fig. 5. Effects of targeted expression of individual EcR isoforms in the presence of EcR-F645A. Flies homozygous for both the EcR-F645A responder and a responder corresponding to the indicated wild-type EcR were crossed to each driver stock. EcR-C was not tested with the act5C and EH drivers. All other combinations of drivers were tested; if no bar is visible, there were no progeny with wild-type phenotype. For the EH driver, wild-type phenotype is expanded wings; for the other drivers, wild-type phenotype is survival to adulthood. In the case of the GMR driver, wild-type EcR isoforms restored wild-type eye morphology as well as viability (see Fig. 5C). Data are shown for crosses performed at 25°C, but the results were independent of temperature (data not shown).

 


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  		Fig. 2. Properties of dominant-negative mutant EcRs used in this paper. Sequences are shown only for the region around helix 12 of the LBD; residue numbers are for isoform EcR-B1. Bold type indicates strongly conserved residues; mutated residues are boxed. Functional assays are described by Hu et al. (X. H., L. C. and P. C., unpublished).

 


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  		Fig. 3. Effects of EcR-F645A expression in targeted tissues. (A-C) Effects on eye development. Adult eyes are shown from animals containing a GMR driver and (A) no responder, (B) an EcR-F645A responder, and (C) responders for both EcR-F645A and EcR-B2. (D) Effects on larval epidermis. The two animals are of equivalent age, and both contain an Eip71CD657 driver; the pupa on the left has no responder, the animal on the right has an EcR-F645A responder. (E,F) Effects on glue secretion. The two puparia are of equivalent age, and both carry a transgene expressing fluorescent glue (Sgs-GFP) (Biyasheva et al., 2001Go). They have an Sgs3 driver and either no responder (E) or an EcR-F645A responder (F). Note that the green fluorescence in E is entirely external to the puparial case. (G,H) Effects on fat body dissociation. Pupae containing the Lsp2 driver, a GFP.nls responder and either no EcR responder (G) or an EcR-F645A responder (H) were dissected and viewed by fluorescence; only the fat body cells are visible. (I,J) Effects on border cell migration. Stage 9 egg chambers were dissected from adult females containing the slbo driver, a GFP responder, and either no EcR responder (I) or an EcR-F645A responder (J). Arrows indicate border cells; o, oocyte. A-D were photographed under bright field illumination. G,H were photographed with GFP-fluorescence optics. E, F, I and J were photographed with a mixture of bright-field and fluorescence optics.

 


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  		Fig. 4. PS1' chromosomes from an animal containing the Sgs3-GAL4 driver and UAS-F645A responder. Chromosomes 2R (A) and 3L (B) from a white prepupa are shown. An identical pattern occurs in animals throughout the period from mid-instar transition to white prepupa. See text for discussion of the labeled sites. Note that these chromosomes are of a normal size for white prepupal polytene chromosomes, but that their puffing pattern is similar to that seen in the much smaller PS1' chromosomes of wild-type early third-instar larvae.

 


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  		Fig. 6. Partial rescue of polytene puffing pattern by individual EcR isoforms. Each panel represents animals containing the Sgs3 driver and responders for EcR-F645A and for a wild-type EcR of the indicated isoform. Salivary glands were isolated from post-wandering larvae (black bars) and white prepupae (gray bars), and their developmental stage was confirmed by salivary gland duct morphology (Zhimulev and Belyaeva, 1999Go). The glands were squashed and puff stages of the chromosomes determined. White extensions of the bars indicate animals selected as white prepupae in which the puffing pattern showed minor deviations from the indicated puff stage. ? indicates animals with a puffing pattern too aberrant to permit assignment of a puff stage. Salivary glands from wild-type post-wandering larvae have PS7-9 polytene chromosomes, and those from wild-type white prepupae have PS10-11 chromosomes.

 


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  		Fig. 7. Polytene chromosomes from a white prepupa bearing an Sgs3 driver and responders for EcR-F645A and EcR-A. The three panels are different regions of a single chromosome spread. See text for a discussion of the marked sites.

 

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