Fig 1.
Analysis of the intron-3 enhancer.
(A) Evolutionary conservation of the intron-3 enhancer. VISTA plot [41, 42] of the D. melanogaster intron-3 enhancer (ExIntron3) aligned with sequence from D. erecta, D. ananassae, D. pseudoobscura, D. mojavensis, D. virilis and D. grimshawl. A window size of 100 bp is used, and regions that are greater than 70% identical are indicated in pink. Regions A-F are defined on the basis of the distribution of sequence conservation among Drosophila. ABCDEF is 1501 bp. A is 1–183; B is 184–345; C is 346–723; D is 724–842; E is 843–1010; F is 1011–1501. Putative E-box and Scalloped (Sd) binding sites are indicated. (B) Deletional or mutational constructs are generated as indicated. Spatial wing expression of each construct is summarized in the table. +++: strong expression; ++: middle expression; +: weak expression; +/-: much weaker or partial expression; -: no expression. Region directing expression in the hinge of wing disc is indicated in pink; regions directing expression in the neuronal precursors of wing disc are pointed in brown; region driving expression in the wing pouch is indicated in blue. (C-K) Wing expression patterns of ex-LacZ and the indicated genomic sub-fragments of ExIntron3. Note the fragments BCDE, BCD, BC and CD drive GFP expression in neuronal precursors. Scale bars, 50 μm.
Fig 2.
Effect of bHLH protein on ExIntron3 enhancer.
(A) Third instar wing imaginal discs containing da3 mutant cells (RFP [red] negative) are visualized for BCD-GFP reporter expression (A’, green). Note the decreased levels of BCD-GFP in da mutant clones within the wing margin proneural region but not hinge. (B-G) GFP reporter expression driven by the indicated genomic sub-fragments in the third instar wing imaginal discs. (H) Summary of regions important for spatial expression. Note that + (red): sufficiency; +/-: partial sufficiency; + (green): requirement; -: no effect. Scale bars, 50 μm.
Fig 3.
SWH pathway requires Sd/Yki to regulate ex.
(A) Blockage of the SWH pathway by expressing an RNAi against yki (en>RFP+yki RNAi) leads to the downregulation of ExIntron3-GFP (green) in the posterior compartment (marked by RFP, red) of wing disc at 25°C. (B-C) Leg and wing discs of en>RFP+yki RNAi (red) staining for ExIntron3-GFP (green) at 30°C. Fly cross and culture was performed at 25°C. After 36–48 hr AEL, en>RFP+yki RNAi flies were incubated at 30°C until dissection at late third instar. Note the autonomous reduction of GFP was more obvious in flies that were shifted at 30°C. The altered en domain (marked by RFP, red) at 30°C suggest that Yki may regulate en expression. (D-E) Wing disc of en>RFP+sd RNAi (red) staining for ExIntron3-GFP (green) at 25°C and 30°C, respectively. Note the decrease of GFP was more obvious when animals were shifted and incubated at 30°C. (F) Wing disc of en>RFP+sd RNAi (red) staining for CD-GFP (green) at 30°C. Note that CD-GFP was decreased in a cell autonomous manner. Scale bar, 50 μm.
Fig 4.
Element E is required for Notch-dependent repression.
(A) Blockage of the Notch pathway by expressing an RNAi against Notch in wing pouch (nub>N RNAi) leads to the upregulation of ExIntron3-GFP (green). (B) GFP expression under the control of element BCDE in nub> N RNAi wing discs. Note the de-repression of BCDE-GFP in response to knockdown of Notch. (C) Wing disc of nub>N RNAi staining for BCD-GFP expression. Note that the BCD-GFP did not respond to downregulation of Notch pathway. (D-F) Wing imaginal discs of nub-Gal4 for ExIntron3-GFP (D), BCDE-GFP (E) and BCD-GFP (F), respectively. Scale bars, 50 μm.
Fig 5.
Sens inhibits Da-mediated ex expression in the wing pouch.
(A, B) Wing disc overexpressing Da homodimer (A, nub>Da-Da) or monomer (B, nub>Da) in the wing pouch and staining for ExIntron3-GFP (green) expression. Note the upregulation of ExIntron3-GFP caused by Da homodimer is attenuated in Sens-expressing cells (red). (C) Wing imaginal disc of dpp>Sens staining for ExIntron3-GFP expression. Note the inhibition of ExIntron3-GFP by high levels of Sens in the dorsal proximal wing (indicated by yellow arrow), while ventral wing is barely affected (white arrow). Some enlarged cells with GFP-positive staining are seen around the Dpp domain. (D) Wing disc of nub>Sens staining for ExIntron3-GFP expression. (E) Wing disc of nub>Da+Sens staining for ExIntron3-GFP expression. Note the GFP expression is slightly disrupted as seen in nub>Sens disc. (F-F”) A late third instar wing disc containing sens mutant cells (GFP negative) is visualized for ex-LacZ expression (red, F’). (G-G”) Third instar wing imaginal disc of sens mutant cells (arm-lacZ negative) visualized for ExIntron3-GFP expression (green, G”). Note no elevation of ex-LacZ and ExIntron3-GFP is detected in sens mutant clones. Scale bars, 50 μm.
Fig 6.
ex expression is independent of hairy.
(A) Schematic representation of the ABCDEF enhancer. Yellow bars denote putative Hairy binding sites. (B-B’) Clones of homozygous hairy mutant cells are labeled by the lack of LacZ expression (red). Note ExIntron3-GFP (green) is not changed in h clones. (C, D) Expression of ExIntron3-GFP in wing discs of dpp-GAL4 (C) and dpp>h (D) flies. Note ExIntron3-GFP is not affected in Dpp domain when h expression is manipulated. Scale bars, 50 μm.
Fig 7.
ex expression in the proneural regions.
Expression patterns of ExIntron3-GFP (green) and sca-LacZ (red) in notum (A-A”) and wing pouch (D-D”). (B-C) Higher magnification of the yellow box (B-B”) and white box (C-C”) in A. Note the anterior scutellar SOP showed higher GFP activity than nearby cells (yellow arrow), the posterior post-alar SOP expressed the GFP at levels similar to other proneural cells (white arrow), whereas the posterior dorsocentral SOP and anterior post-alar SOPs showed reduced levels of GFP activity (blue and red arrows, respectively).
Fig 8.
Model of differential regulation on ex.
In the wing margin proneural region (high SWH activity; low Yki activity), ex is negatively regulated by inputs acting through elements A and F. The E-box site #2 (E2) is required for expression in the wing margin proneural cells while Da acts through E1 and E3 to regulate ex transcription. Sd/Yki regulates ex transcription through element BCD in wing pouch and hinge. Notch acting through element E and other inputs acting through elements B and C repress ex transcription in wing pouch. Note the image of wing disc is adopted from Fig 7D”.