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First published online 14 November 2007
doi: 10.1242/dev.008409

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collier transcription in a single Drosophila muscle lineage: the combinatorial control of muscle identity

Centre de Biologie du Développement, UMR 5547 CNRS/UPS, IFR 109, Institut d'Exploration Fonctionnelle des Génomes, 118 route de Narbonne, 31062 Toulouse cedex 9, France.

View larger version (39K): [in this window] [in a new window]   Fig. 1. Mapping of the col DA3 muscle CRM in Drosophila. (A). Schematic representation of the col genomic region. Coding exons and the 5' and 3' untranslated regions are indicated by black and white boxes, respectively. The positions of the immediately upstream and downstream predicted genes (http://flybase.bio.indiana.edu/), CG10200 and BEAF-32, are indicated by grey boxes and their direction of transcription by arrows. The extent of col upstream region present in each lacZ reporter gene, P9cl to P0.9cl is indicated by a black line. (B) Diagrammatic, colour-coded representation of the different col expression sites in stage 11 and 14 embryos. (C) In situ hybridisation showing expression of P2.6cl, P2.3cl and P1.6cl, compared to endogenous col, at embryonic stages 6, 11, 12 and 14. (D) Close-up view of the DA3 promuscular cluster and progenitor in the T2 and T3 segments of a P9cl embryo at stage 11, stained for Col (green) and β-gal (red). Unlike Col, lacZ expression is restricted to the progenitor cell. (E) Schematic representation of the modular organisation of the col cis-regulatory region, underlining the position of the DA3 muscle CRM.

View larger version (30K): [in this window] [in a new window]   Fig. 2. Conserved cis-regulatory elements and TF-binding sites in the DA3 muscle CRM. (A) Col expression in a stage 14 D. vir embryo (top left) and in situ hybridisation to lacZ transcripts showing expression of different D. vir and D. mel col reporter genes, as indicated in each panel. Note that P2.6-1.6cG is a Gal4/UAS-lacZ line. (B) Diagrammatic representation of the relative positions of conserved motifs, numbered from 1 to 10 and potential binding sites for Twi, Nau, Col and Mef2 in the DA3 muscle CRM (for details, see Fig. S2 in the supplementary material). (C,D) Ubiquitous hs-col driven Col expression specifically activates col-lacZ reporter genes in the VL1 muscle (white arrow), as shown here for P2.6cl (C). This is mediated by conserved cis-regulatory elements in the DA3 muscle CRM (D).

View larger version (84K): [in this window] [in a new window]   Fig. 3. col activation in FCM nuclei incorporated in the DA3 myofibre in Drosophila. (A-C) col transcription in wt DA3 muscle precursors, visualised by in situ hybridisation to col primary transcripts (red dots), immunostaining for Col (green) and nuclear staining (blue). (A'-C') Blue and red channels; (A''-C'') green channel. (A) Stage 14 embryo. The DA3 muscle precursor contains several nuclei; the two distalmost have already accumulated a high level of Col protein and activated col transcription. One central nucleus starts accumulating Col protein (lower inset) but does not yet transcribe col. Two other FCM have probably fused but not started to import Col protein (surrounded by a dashed line in A', upper inset). Another FCM has started engaging in the fusion process, (dashed notch in A'). (B) Stage 15 embryo. At this stage, each DA3 muscle nucleus contains high levels of Col protein and transcribes col. (C) Stage 16 embryo. All the DA3 muscle nuclei still contain high levels of Col protein but col transcription has almost completely ceased. (D,E) In situ hybridisation to col primary transcripts (red dots) in (D) wt and (E) col1 mutant embryos (two segments are shown). A membrane-targeted form of GFP expressed under control of the col promoter (P9cg construct) allows the visualisation of the DA3 muscle (green). Note the complete absence of col transcription in col mutant embryos (E). The white arrowhead points to a dorsal md neuron expressing Col. Scale bar: 5 µm.

View larger version (100K): [in this window] [in a new window]   Fig. 4. Nau-dependent col transcription during the DA3 muscle fusion process in Drosophila. (A-D) Double in situ hybridisation with intronic probes for col (green) and nau (red) nascent transcripts and Col immunostaining (blue) show that nau and col are co-expressed in (A) the DA3/DO5 progenitor cell, (B) the DA3 FC (outlined by a plain line) and (C), the DA3 muscle precursor when it contains two to three nuclei (outlined). nau remains transcribed in the DO5 FC (dashed outline in B), whereas col transcription is rapidly turned down. (E-H) col transcription (green dots) in (E,G) wt and (F,H) nau188 mutant embryos (two segments are shown); the DA3 muscle is visualised by immunostaining for Col (red) and MHC (blue in E,F). In stage 15 nau188 mutant embryos (F), the DA3 muscle is reduced, compared to wt (E) and most nuclei do not transcribe col. At stage 12, col expression in the DA3 muscle precursor (asterisk) when it contains two to three nuclei is similar in (H) nau188 and (G) wt embryos, although only one nucleus, probably the FC nucleus, expresses high levels of col transcripts in nau188 embryos. Arrowheads indicate col transcription in a dorsal multidendritic neuron. Scale bars: 5 µm.

View larger version (50K): [in this window] [in a new window]   Fig. 5. Nau and Col separately and synergistically activate ectopic col transcription in specific subsets of muscles. (A) P2.6cl expression in the DA3 muscle in stage 15 wt embryos, visualised by β-gal antibody staining. (B) rp298Gal4-driven Col expression of in all FCs activates P2.6cl in a subset of somatic muscles cells, activation being most robust in the VL1 muscle. Nau expression (C) is unable to activate ectopic P2.6cl expression, except for, sporadically, the DA2 muscle. (D) Together, Col and Nau activate P2.6cl expression in a large number of somatic muscles in addition to VL1. A schematic representation of the abdominal muscle pattern is shown of the right side of each panel to indicate the P2.6cl expressing muscles. The DA3, DA2 and VL1 muscles are designated by an arrowhead, a dot and an arrow, respectively.

View larger version (75K): [in this window] [in a new window]   Fig. 6. col transcription in the DA3 muscle precursor depends upon a Nau and a potential Col-binding site in the DA3 muscle CRM. (A) Col and P2.6cl expression in wt Drosophila embryos at stage 15, visualised by Col (red) and β-gal (green in the right and white in the left panel) antibody staining. (B,C) P2.6cl expression is lost when either the putative EBF/Col- (B) or Nau- (C) binding site present in the DA3 muscle CRM is mutated. Red dots in A and C correspond to Col expression in md neurons. (D) Col expression in all FCs (rp298-Gal4/UAS-col) induces ectopic P2.6cl expression in the VL1 (arrow) and DA2 (dot) muscles, as visualised by β-gal immunostaining; the arrowhead points to the DA3 muscle. (E) Ectopic expression of P2.6cl is not observed when the EBF/Col-binding site is mutated. (F) The consensus EBF- and MyoD-(Nau) binding sites (Huang et al., 1996; Travis et al., 1993) are represented above the predicted sites found within the DA3 muscle CRM. The mutated positions introduced in P2.6clcol and P2.6clnau are shown in red.

View larger version (23K): [in this window] [in a new window]   Fig. 7. A model for the combinatorial coding of DA3 muscle identity in Drosophila. (A) Col is activated in one (T1-T3 segments) and two (A1-A2 segments) promuscular clusters (Crozatier and Vincent, 1999), in response to positional and mesodermal cues. This first step is probably mediated by clusters of relevant TF-binding sites [light orange boxes (Philippakis et al., 2006)], including Twi-binding sites (+) (Sandmann et al., 2007) that are located within the col upstream region and introns. Col expression subsequently becomes restricted to the DA3/DO5 progenitor (orange cell) by lateral inhibition (Crozatier and Vincent, 1999). We postulate that positive inputs from TFs binding to the -2.6 to -2.3 fragment, including Twi, are sufficient to allow P2.6cl activation in the selected DA3/DO5 progenitor, upon relief of N repression. (B) Following division of the progenitor, restriction of Col expression to the DA3 FC involves positive auto-regulation in this FC and negative regulation by N in the sibling DO5 FC. From this stage, a combination of Nau and Col activity is required for col transcriptional activation in the FCM nuclei, which are recruited by the DA3 FC to form a myofibre, thereby ensuring that all nuclei in the DA3 muscle express the same identity programme.