Regulation of the Drosophila ID protein Extra macrochaetae by proneural dimerization partners
- Albert Einstein College of Medicine, United States
Figures
Figure 1
Da and Emc are broadly expressed proteins that are modulated in proneural regions.
Panels show immunofluorescence labeling of Drosophila eye imaginal discs (A) and wing imaginal discs (B). Yellow arrowheads indicate the morphogenetic furrow of the eye disc (A) and future anterior …
Figure 2 with 1 supplement
Both Da and Emc protein levels depend on da gene dose.
(A) GFP signals in wing imaginal disc mosaic for the ubi-GFP transgene detected simultaneously by native GFP fluorescence (green) and by anti-GFP antibody (magenta). (B) Quantification of native GFP …
Figure 2—figure supplement 1
Panels A and B show gene dose mosaic experiments like those of Figure 2 except that the dose of only da (A) or emc (B) is varied.
Homyzygous mutant cells are negatively marked by GFP or βGal (green). (A) Very little Emc (blue) is detected in da-/- cells. da+/+ cells have more Da (red) and Emc (blue) proteins. (B) Da goes up in …
Figure 3
Emc is unstable alone but stabilized in the presence of Da.
In panels (A-F) proteins from S2 cells were analyzed by western blot following a time course after cycloheximide (CHX) addition. (-) indicates absence of CHX treatment. αTubulin is used as a loading …
Figure 4 with 1 supplement
Proneural proteins are necessary for modulating Da and Emc levels.
(A) Cartoon of Ato protein showing sequence changes in ato1 and ato3 mutants. ‘B’ indicating the basic domain and ‘HLH’ indicating the helix-loop-helix domain of wild type Ato. (B–E) Homozygous ato1 …
Figure 4—figure supplement 1
(A–B) Homozygous ato1 or ato3 mutant clones are marked by the absence of βGal (green). (A) Ato1 protein (magenta) is detected by an anti-Ato antibody in the ato1 clones. Initial expression of Ato …
Figure 5
Proneural genes regulate Emc and Da in the notum.
(A) In sc10-1 wing discs, fewer cells are labeled by Sens (green) in the wing margin (yellow arrowheads). Emc (red) and Da (blue) levels are not affected as strongly as in the wild type (compare Figu…
Figure 6 with 1 supplement
Atonal regulates Emc expression post-transcriptionally.
(A) emc enhancer trap expression in the eye disc in the emc-GFPYB0067 line. Downregulation in the morphogenetic furrow (arrow) is broader and less complete than seen fpr the Emc protein (compare Figu…
Figure 6—figure supplement 1
ato1 mutant clones are marked by the absence of βGal (green) and emc enhancer trap is detected by GFP (magenta).
emc enhancer trap shows similar downregulation in the furrow in ato1 clones. Genotype: eyFLP; emc-GFPYB0067, FRT82 arm-lacZ/FRT82 ato1.
Figure 7 with 2 supplements
Ectopic Ato or Sc does not reduce Emc levels.
Flip-on clones expressing ato or sc using act-Gal4 and UAS-ato or UAS-sc lines are marked by GFP (green). (A) Ectopic Ato expression from UAS-ato.ORF-3HA had little effect on Emc levels (red) but …
Figure 7—figure supplement 1
Flip-on clones expressing ato using act-Gal4 and various UAS-ato lines marked by GFP (green).
(A) Ectopic Ato induced from the UAS-ato.ORF-3HA allele slightly upregulates Da (blue) in wing disc but has little effect on Emc (red). Ato-expressing nuclei often localize more basally than their …
Figure 7—figure supplement 2
Flip-on clones expressing sc in eye discs using act-Gal4 and a UAS-sc line are marked by GFP (green).
Ectopic Sc induced from UAS-sc.39 slightly upregulated Da (red), especially posterior to the furrow, but had little effect on Emc (blue) expression. Genotypes: (A) hsFLP; UAS-sc.39/+; act > CD2>Gal4,…
Figure 8 with 1 supplement
Ubiquitous emc transcription confers normal expression pattern and neurogenesis.
(A) Wild type adult eye showing ommatidia and interommatidial bristles. (B) Actin-Gal4-mediated ubiquitous transcription of emc at 18C in the absence of the endogenous locus gives rise to normal …
Figure 8—figure supplement 1
(A–F) Gal4-driven ubiquitous emc transcription at 25C in emc mutant.
(A) Rescued pharate adults have essentially normal eye with interommatidial bristles. (B) Eye imaginal discs from (A) display broad Emc expression and numerous cells with higher levels, but always …
Figure 9
Model for HLH protein regulation inside and outside proneural regions.
The top part compares gene transcription in most imaginal disc cells (non-proneural) with that in cells in proneural regions. The bottom part compares the protein species active in these cells. In …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| gene (Drosophila melanogaster) | emc | FlyBase: FBgn0000575 | ||
| gene (D. melanogaster) | da | FlyBase: FBgn0267821 | ||
| gene (D. melanogaster) | ato | FlyBase: FBgn0010433 | ||
| gene (D. melanogaster) | sc | FlyBase: FBgn0004170 | ||
| genetic reagent (D. melanogaster) | da[3] | PMID: 3802198 | ||
| genetic reagent (D. melanogaster) | emc[AP6] | PMID: 7947322 | ||
| genetic reagent (D. melanogaster) | emc[Δ1] | this study | ||
| genetic reagent (D. melanogaster) | act > CD2>Gal4, UAS-GFP | PMID: 9053304 | ||
| genetic reagent (D. melanogaster) | ato[1] | PMID: 8196767 | ||
| genetic reagent (D. melanogaster) | ato[3] | PMID: 7635049 | ||
| genetic reagent (D. melanogaster) | emc-GFP[YB0040] | PMID: 17179094 | ||
| genetic reagent (D. melanogaster) | emc-GFP[YB0067] | PMID: 17179094 | ||
| genetic reagent (D. melanogaster) | P{PZ}emc[04322] | PMID: 9529525 | ||
| genetic reagent (D. melanogaster) | Df(1)sc[B57] | PMID: 2510998 | ||
| genetic reagent (D. melanogaster) | Df(1)sc[10-1] | PMID: 3111716 | ||
| genetic reagent (D. melanogaster) | UAS-HA-da | PMID: 25579975 | ||
| genetic reagent (D. melanogaster) | UAS-ato.ORF-3HA | PMID: 23637332 | ||
| genetic reagent (D. melanogaster) | UAS-sc | PMID: 8978666 | ||
| genetic reagent (D. melanogaster) | UAS-ato-4 | PMID: 8324823 | ||
| genetic reagent (D. melanogaster) | UAS-emc5.3 | PMID: 10804180 | ||
| cell line (D. melanogaster) | S2 | DGRC | Stock Number: 6 | |
| antibody | anti-βGal (mouse) | DSHB | 40-1a | (1:100) |
| antibody | anti-ElaV (rabbit) | DSHB | 7E8A10 | (1:50) |
| antibody | anti-Da (mouse) | PMID: 3802198 | (1:200) | |
| antibody | anti-Emc (rabbit) | Y.N. Jan | (1:8000) | |
| antibody | anti-Ato (rabbit) | PMID: 8196767 | (1: 50000) | |
| antibody | anti-Sca (mouse) | PMID: 8622662 | (1:200) | |
| antibody | anti-GFP (rat) | Nacalai Tesque | GF090R | (1:1000) |
| antibody | anti-Sens (guinea pig) | PMID: 10975525 | (1:50) | |
| antibody | anti-V5 (mouse) | Invitrogen | 46–0706 | (1:5000) |
| antibody | anti-Flag (mouse) | Sigma | F3165 | (1:8000) |
| antibody | anti-Tubulin (mouse) | Abcam | ab18251 | (1:5000) |
| antibody | anti-Tubulin (rabbit) | Abcam | ab7291 | (1:5000) |
| antibody | anti-HA (rabbit) | Cell Signaling Tech | C29F4 | (1:1000) |
| antibody | anti-HA (mouse) | Roche | 12CA5 | (1:1000) |
| recombinant DNA reagent | Emc-V5 (plasmid) | this study | ||
| recombinant DNA reagent | Da-Flag (plasmid) | PMID: 25694512 | ||
| recombinant DNA reagent | GFP (plasmid) | PMID: 25694512 |
Additional files
-
Transparent reporting form
- https://doi.org/10.7554/eLife.33967.017
