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First published online 28 February 2007
doi: 10.1242/dev.02823

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Antagonistic and cooperative actions of the EGFR and Dpp pathways on the iroquois genes regulate Drosophila mesothorax specification and patterning

Annalisa Letizia^*, Rosa Barrio and Sonsoles Campuzano

Centro de Biología Molecular Severo Ochoa, CSIC and UAM, Cantoblanco, 28049 Madrid, Spain.

View larger version (88K): [in this window] [in a new window]   Fig. 1. Search for cis-regulatory elements in the Iro-C. (A) Expression of ara/caup in a third instar wing disc. Ara/Caup accumulated at the prospective alula (Al), dorsal radius (DR), lateral notum (LN), longitudinal veins L1, L3 and L5, pleura (Pl) and tegula (Tg) but not at the medial notum (MN). (B) Physical map of the Iro-C locus (Gomez-Skarmeta et al., 1996). Genomic DNA is shown as a thick black bar. Transcription units are shown as red arrows; location of quil is approximated. Blue arrows indicate positions of breakpoints associated with the iroDFM2 and iro1 mutations. The green triangle represents the P insertion irorF209. Small vertical magenta bars delimit fragments (horizontal black and green bars) tested for their enhancer ability. Fragments 1-5 (green bars) show enhancer activity. (C-H'') lacZ expression patterns (green) driven by the indicated REs and endogenous expression of ara/caup (red) in late third (C-G') and early third (H-H'') instar wing discs. Differences in the expression domains of IroRE2-lacZ and ara/caup are indicated by an arrowhead in D-D''. The wavy red lines in E' and F' mark the proximal limit of caup expression. Wing discs are oriented ventral side up and anterior to the left.

View larger version (78K): [in this window] [in a new window]   Fig. 2. EGFR signalling activates IroRE2-lacZ. (A-B') Expression of lacZ (red) is activated in clones of cells (green) expressing activated forms of Ras (A,A') or Raf (B,B') (arrows) except in the wing pouch (arrowheads). Insets in B,B' show enlarged and enhanced pictures of the central wing pouch. Clones were induced at 48-72 (A,A') or 72-96 hours AEL (B,B'). (C,C') lacZ expression is lost (arrows) in clones of cells expressing a dominant negative form of Raf (green, induced at 24-48 hours AEL).

View larger version (87K): [in this window] [in a new window]   Fig. 3. Dpp signalling represses IroRE2-lacZ. (A-C') tkva12 mutant cells (absence of green), upregulate IroRE2-lacZ (red, arrows) in the prospective medial notum (A,A'), lateral notum (B,B') and hinge (C,C'). (D,D') Overexpression of UAS-tkvQD in clones of cells (green) abolishes expression of IroRE2-lacZ (arrows).

View larger version (59K): [in this window] [in a new window]   Fig. 4. Delimiting a minimal notum enhancer. (A) VISTA plot comparing the D. melanogaster IroRE2-B sequence with those of D. pseudoobscura, D. virilis and D. mojavensis (window size: 100 bp). Pink peaks represent regions with more than 70% of identity (the most conserved one is marked by an asterisk). The light blue peak indicates the putative linc coding region. (B) Subfragments of IroRE2-B tested for transcription enhancing activity. Only the blue regions activate transcription. Putative binding sites for ETS proteins, Ttk, GATA proteins and Mad are indicated. Filled symbols, binding sites matching the consensus sequence; open symbols, one or two mismatches compared to the consensus sequence. (C-F) lacZ expressions mediated by the indicated fragments. Note in C that IroRE2B drives a weak expression in the prospective intervein regions, suggesting that shortening has removed some wing-pouch repressor elements. (G,G') Expression mediated by IroRE2-B2GATAm extends into the prospective medial notum, proximal to the wg (blue) and ara/caup (green) domains. The dotted lines in E and G' outline the proximal region of the discs. (H,H') Mutation of the ETS binding site of IroRE2-B2 that most closely matches the consensus strongly reduces enhancer activity in the wing disc. (I-I'') Up-regulation of IroRE2-B2GATAm-lacZ expression in tkva12 clones, some of which are indicated by arrows.

View larger version (89K): [in this window] [in a new window]   Fig. 5. Pnt activates IroRE2-lacZ and ara/caup expression. (A,A') Overexpression of pnt in clones of cells (green) activates lacZ (red, arrowheads). (B,B') In pnt88 null clones (absence of green) IroRE2-lacZ is downregulated (red, arrowhead). (C,C') ara/caup expression (red) is similarly lost in pnt88clones (arrowhead).

View larger version (85K): [in this window] [in a new window]   Fig. 6. Regulation of IroRE2-lacZ by Pnr. (A-B') pnrVX6 mutant cells (absence of green) show ectopic expression of IroRE2-lacZ (red) when located in the medial notum (arrows in A,A') but abolish expression in the wg notal domain (shown in blue, arrows in B,B'). In this region, expression of pnr (visualized in pnrGal4/UAS-GFP larvae) overlaps with that of IroRE2-lacZ (not shown). (C,C') Overexpression of UAS-pnr (green), activates IroRE2-lacZ at the hinge and alula domains (arrowhead) and alter the disc epithelium of the prospective lateral notum forming a fold around some clones (arrows). lacZ ectopic activation was stronger at the hinge, alula and pleura than at the wing pouch, consistent with a weak activation of ush by overexpressed pnr at this domain (A.L. and S.C., unpublished).

View larger version (133K): [in this window] [in a new window]   Fig. 7. IroRE2 mediates repression of ara/caup by Ush. (A-A'') In ushVX22 null clones (absence of green) IroRE2-lacZ (red) and ara/caup (blue) (arrows) are derepressed. Loss of ush at the hinge has no effect (arrowheads). (B-C') Overexpression of ush in clones (green) downregulates IroRE2-lacZ (B,B') and ara/caup (C,C') expression at the proximal-most lateral notum (arrows). (D-D'') Downregulation of IroRE2-lacZ (red) by overexpression of UAS-tkvQD is still evident in clones of ushVX22 mutant cells (green) (arrows).

View larger version (83K): [in this window] [in a new window]   Fig. 8. Dpp and EGFR pathways activate IroRE1-lacZ expression. Cells expressing activated forms of Tkv (A,A') or Raf (C,C') (green) activate IroRE1-lacZ (red) at the prospective lateral notum (arrows) but not at the wing hinge, wing pouch or proximal notum (arrowheads). lacZ expression (red) strongly decreased (arrows) in tkva12 (B,B') or UAS-RafDN (D,D') clones.

View larger version (35K): [in this window] [in a new window]   Fig. 9. Proposed regulatory mechanism that controls the expression of ara/caup in the prospective notum. Regulation of the activity of Iro-RE2 (A) and Iro-RE1 (B) by the EGFR and Dpp pathways is shown (green arrows, positive regulatory interactions; red T-shaped bars, negative regulatory interactions). Downstream effectors of the pathways are indicated. (C) Addition of the actions of both REs largely explains the expression of ara/caup in the prospective notum region of the third instar wing disc. Dpp expression was monitored with dpp-Gal4, UAS-GFP (A,B) and dpp-lacZ (C).