Left hand panels: Colorimetric in situ hybridization (black and white images). (A–I) In situ hybridization of wild-type embryos with probes specific for (A) βTub60D, (B) act57B, (C) CG5080, (D) blow, (E) sug, (F) sns, (G) CG14687, (H) CG9416 and (I) gol, detecting specific expression in the mesoderm. No specific staining was observed in the ectoderm (red brackets). An engrailed-Gal4 driver line was used to ectopically express (A′–I′) UAS-Lmd, (A″–I″) UAS-Mef2-HA or (A′″–I′″) both UAS-Mef2-HA and UAS-Lmd in ectodermal stripes. Lmd and Mef2 show differential ability to activate ectopic target gene expression (red arrows). Expression of 8 of 9 genes can be induced in the ectoderm when both factors are present (A′″–H′″), while gol expression was never observed in the ectoderm (I′″). Right hand panels: Double fluorescent in situ hybridization of the same genotypes with probes specific for the corresponding genes (green) and wingless (red). A high magnification of the ectodermal wingless staining reveals adjacent ectopic staining in the engrailed domain (white arrows), not detected in wild-type embryos. Full embryos are shown in Figure S2.

In situ hybridization of GFP-reporter mRNA in (A–F) wt embryos, (A′–F′) homozygous lmd¹ mutant embryos and (A″–F″) homozygous Mef2^P544 mutant embryos. The intronic βTub60D enhancer requires Lmd enhancer activity (A′), but remains active in Mef2 mutants (A″). Similarly, activity of the act57B enhancer is strongly dependent on Lmd (B′), but only mildly reduced in Mef2 mutants (B″). CG5080 reporter expression is clearly reduced in both background (C′,C″), while activity of the blow enhancer is not detectable in the absence of either Lmd or Mef2 (D′,D″). The CG14687 enhancer show different requirements in different muscle types: while expression in somatic muscles requires Lmd (E′), expression in the visceral muscle is not affected in neither Lmd nor Mef2 mutants (E″). The gol enhancer requires Lmd activity (F′) but is active normally in Mef2 mutants (F″).

(A) Schematic overview of a genomic region within an intron of the Mef2 locus: An exon (located on the antisense strand) is shown in orange. Genomic fragments on the tiling arrays are indicated as stacks of two horizontal bars in their corresponding genomic position. The top bar represents the results from the 6–8 hour (stages 10–11) ChIP-on-chip time point, the lower one corresponds to the 8–10 hour (stages 12–13) time point. Significantly enriched regions are indicated in blue for Lmd (top) and in green for Mef2 (bottom). The black bar indicates the location of the previously identified Lmd-binding site. Both Lmd and Mef2 are co-bound to genomic regions overlapping the known Lmd binding site at the 6–8 hr time point, as well as to other sites in this area. (B) Schematic overview of the genomic region upstream of the sns locus (exons show in orange): The known sns enhancer (black bar) partially overlaps with tiling array probes bound by Lmd (at both time points, blue bars) and Mef2 (at 8–10 hour time point, green bar). Additionally, Mef2 binds to other locations upstream and intronic of the sns locus. (C) Lmd and Mef2 co-occupy many genomic locations: Venn diagram displaying the number of non-overlapping regions significantly enriched in Lmd ChIPs (blue) and significantly enriched in Mef2 ChIPs at the same stages of development (green). Both factors co-occupy a large number of regions (overlap). (D) Co-regulation of common direct target genes by Lmd and Mef2: The majority of Lmd target genes (blue) are co-regulated by Mef2 (overlap), while Mef2 regulates a large number of additional genes (green). (E) Differential gene expression in lmd and Mef2 loss-of-function mutants [log₂]: differences in expression between mutant and wt embryos were recorded in a timecourse for lmd (left) or Mef2 (right) mutant embryos. Shown are all direct target genes of Lmd that are significantly misregulated at one or more time points in either mutant background (fold change >1.6, q<1%). K-means clustering was used to highlight similar downregulation (top) in both mutants, similar upregulation (centre) or divergent expression changes (bottom). Color scale indicates fold changes [log₂]. Genes studied in more detail are marked in grey.

(A–D) Schematic overviews of the tiling array probes covering the (A) blow, (B) CG5080, (C) gol and (D) ttk loci. Regions significantly enriched in Lmd ChIPs (blue) and Mef2 (green) were selected and intergenic sequences (black bars) assayed for regulatory activity in vivo. (A′–D″) Immuno-histochemistry using an anti-GFP antibody to detect reporter gene expression. All four enriched sequences were able to specifically activate GFP expression in transgenic embryos in the mesoderm as early as (A′–D′) stage 11, with persistent GFP-presence at (A″–D″) stage 13.

Minimal regions required for Firefly luciferase reporter activity in vitro were identified in the (A) CG14687, (B) CG5080, (C) gol, (D) ttk, (E) blow, (F) βTub60D, (G) CG9416 and (H) CG30035 enhancers which were cloned and assayed for activity in vitro. Expression plasmids encoding for Lmd or Mef2 proteins were co-transfected with the reporter plasmids and a Renilla normalization control at different concentrations (1 ng/10 ng) alone or in combination (x-axis). Dual-luciferase readout was normalized to reporter-only controls and fold changes are indicated as mean +/− 1 standard error (at least three biological replicates, each done in triplicate). Informative combinations of transcription factor transfections are indicated (brackets). (A–C) Both Lmd and Mef2 can activate the CG14687, CG5080 and gol reporters. Co-transfection of both transcription factors leads to roughly additive fold changes (brackets). (D–F) Presence of both Lmd and Mef2 yields higher activity from the ttk, blow and Tub60D enhancers than expected by summing the individual fold changes (brackets), indicating cooperative regulation. (G, H) The CG9416 and CG30035 reporters can readily be activated by Mef2, but show reduced activity upon co-transfection with Lmd, revealing its inhibitory influence in this context. The regulatory interactions are highly significant (unpaired, two-tailed student′s t-test, (*) p<0.05, (**) p<0.01, (***) p<0.001).