Identification of Lb target genes by expression profiling and ChEST strategies. (A). Muscle- and heart-specific expression of lb and the two drivers (24B > GFP and tin > GFP) used for targeted transcriptional profiling. Three different embryonic stages are presented to illustrate Lb and 24B-GAL4 expressions in biological samples used for microarray experiments. In the first column (5–9 h AEL) are shown embryos at stage 11 or early stage 12 (lateral views). In the middle column (9–12 h AEL) are embryos at stage 13, and in the third column (12–16 h AEL) are shown embryos at stage 15–16. In the middle and right column, muscle (M) expression is illustrated by lateral views and heart (H) expression by dorsal views of embryos. Note that tin-GAL4 driver expression starts later, from stage 13, and persists until the late embryogenesis. To obtain wild-type biological samples for microarray and ChEST experiments, three collections of embryos aged from 5 to 16 h AEL have been used. In a similar manner, F1 embryos from the cross of 24B-GAL4 with UAS-lbe and with UAS-lbRNAi lines were collected and used as H + M GOF and H + M LOF samples, respectively. Embryos from the cross of tin-GAL4 with UAS-lbe or UAS-lbRNAi lines were aged according to tin-GAL4 expression and later labeled H GOF and H LOF, respectively. (B) Venn diagrams showing populations of candidate genes identified by comparison of GOF versus wild-type, LOF versus wild-type, and GOF versus LOF context. Note that in H conditions more candidate genes are common for these three candidate gene populations than in H + M context. (C) Main categories of Lb targets identified by targeted expression profiling. GO-based statistical distribution of genes identified in H + M targeting conditions and H targeting conditions. In both contexts we find an important enrichment for ECM and cytoskeleton components, signaling molecules, proteins involved in proteolysis, ATP binding, and factors carrying a DNA- or RNA-binding domain. Note that >50% of identified genes have no GO annotations and ∼17% have a GO, which does not fit into any of the five main categories. (D) Genome scanning strategy for Lb-binding CRMs. Different heart (H) targeting and muscle (M) targeting genome scanning conditions were used (asterisk; see Supplemental Material). A pool of predicted cardiac and muscular Lb-dependent CRMs was filtered with respect of distance to adjacent genes and their annotations (asterisk; see Supplemental Material). Selected CRMs were spotted to produce a computed Lb-CRM array. In parallel, a pool of DNA fragments bound in vivo by Lb was isolated by ChIP and used to probe the computed array. Among 74 CRMs spotted, 16 were found enriched in ChIP material. (E) Identification of the in vitro consensus binding site for Ladybird. Random 10-nt-long oligos flanked on 5′ and 3′ by 20-nt primer-compatible sequences were radiolabeled and used for the SELEX approach. Four cycles of incubation with 6xHis-Lb homeodomain fusion protein followed by the amplification of a selected subset of sequences were applied to select motifs bound preferentially by Lb. As shown by shift assays, Lb homeodomain (top panels) and both Lbe and Lbl proteins (bottom panel) recognize sequences containing TAAT and TAAC core motifs, and the deduced consensus sequence is RVYTAAYHAG.

bab2 expression and regulation during cardiac development. (A–D) Bab2 expression in cardiac precursors and the dorsal vessel. (A) At early stage 12 Bab2 is expressed in Eve-positive but not in Lb-positive cardiac precursors. (B) At the end of stage 12, a weak Bab2 expression starts to appear in cardioblasts including Lb-positive cells (arrows). (C) At stage 13, Bab2 is expressed in all cardiac cells. (D) After fusion of cardiac primordia, at stage 15, Bab2 protein is particularly well seen in the heart proper. (E–L) bab2 GOF and LOF influences cell fate specification within the heart and leads to the altered positioning of cardiac precursors (shown in E,I). Wild-type stage 12 and stage 15 embryos stained for Tin, Eve, and Lbe. Note that Lbe and Eve are coexpressed with Tin but mark distinct subset of cardioblasts and pericardial cells. Arrowheads in I point to the two Lbe/Tin cardioblasts present in each hemisegment. (F,J) In bab2 mutant embryos Lbe expression is enlarged (arrowheads in J) and appears in Eve-positive cells (arrows). (G,K) In contrast, bab2 GOF leads to the repression of Lbe within the cardiac primordium (arrows in G; arrowheads in K). (K) Asterisks indicate lacking cardioblasts and arrows point to a supernumerary Eve-positive pericardial cell. (H,L) Triple (β3-Tubulin, Lbe, Eve) staining of stage 15 wild-type (H) and bab2 mutant (L) embryos. Note irregular β3-Tubulin pattern (arrowheads in L) and abnormal, cardioblast-like position (arrow in L) of some Eve-positive cells in bab2 mutants.

CG8698, a ChEST-identified Lb target gene is specifically expressed in differentiating muscle fibers and required for muscle contraction. Lateral views of stage 14 (A,B) and stage 16 (C,D) embryos stained for CG8698 RNA (red), Lbe (green), and dMef2 protein (blue). (B,D) CG8698 is expressed specifically in differentiating somatic muscle fibers (coexpression with dMef2. Notice that CG8698 is also expressed in Lb-positive SBM (arrows in A). (E, top panel) A chart representing changes in the SBM size recorded in Mhc-tauGFP embryos injected with dsRNA against the lacZ gene (green plot) or against the CG8698 gene (red plot). Notice the significantly reduced fluctuations in the SBM size in embryos with RNAi-attenuated expression of CG8698. (Bottom panel) A chart representing mean changes in the SBM size recorded from six SBM muscles in the control condition (lacZ-RNAi) and after attenuation of CG8698 (CG8698-RNAi) (see Materials and Methods for details). Muscle size changes were recorded from time-lapse movies (see Supplementary Movie Fig. S3) and analyzed using Volocity Measurement software (Improvision). Deviations from the mean size changes are represented by the error bars.

Validation of selected Lb targets identified by expression profiling and ChEST approaches. (A,B) RT–PCR analyses of transcript levels in wild type and in lb GOF or LOF conditions for candidates identified in H targeting (A) and H + M targeting (B) conditions. At least two candidate genes from each of main categories have been analyzed (see also Table 1A,B). (A′) Real-time RT–PCR validation of additional candidates. Relative quantity of gene transcripts in GOF H + M, LOF H + M, or GOF H contexts versus wild type are presented (see Materials and Methods and Supplementary Table S8 for details). Alterations in candidate gene expression observed in RT–PCR and real-time RT–PCR experiments are consistent with expression changes identified by microarrays. Lateral views of stage 14 (C,D,G–J) and stage 13 (E,F) embryos. lb is able to repress (C–F) certain target genes and to activate (G–J) others. (C–F) Expression of muscle identity gene slou (C) and nau (E) are repressed by lb in 24B > lbe embryos (arrows in D,F). (G–J) Expression of Sls, a component of sarcomeric cytoskeleton (G) and a dystrophin related Msp300 (I) are ectopically activated (arrows in H,J) in lbe GOF conditions. Arrowheads and arrow indicate increased Sls (H) and Msp300 (J) level in the SBM and the ventral muscles, respectively. bab2 (K–O) and if (P–R) CRMs act as Lb-dependent regulatory regions in vivo. (K,P) A scheme showing the position and organization of Lb-dependent CRMs located within the first bab2 (K) and if (P) intron. Positions of Lb, Tin, Twi, Dmef2, and dTCF-binding sites are indicated. (L) Wild-type expression of bab2-GFP line (carrying Lb-dependent CRM) showing that GFP expression is initiated in Lbe cardiac cells at stage 13. Notice that initially in each hemisegment only one out of two Lbe-positive cardioblasts express GFP. GFP expression also coincides with endogenous Bab2 expression. (M) Wild-type bab2-GFP expression in stage 14 embryo showing that during later steps of cardiac development the CRM drives expression in an enlarged population of cardiac cells including Lbe-negative cardioblasts. GFP is coexpressed with endogenous Bab2. (N) bab2-GFP expression is not seen at stage 13 in embryos overexpressing Lbe in mesodermal cells, suggesting that during early stages of heart development lb is able to repress bab2. (O) In stage 14 embryo overexpressing Lbe, bab2-GFP is no longer repressed, indicating that the repressive influence of Lbe on this CRM is transient. (Q) The ChEST-identified if CRM drives LacZ expression in the SBM (arrows). (R) In embryos expressing Lbe ectopically in all muscle cells, expression of LacZ is enlarged (yellow arrow). (S) A scheme showing a model based on identity gene-dependent fine-tuning of muscle gene expression. Both direct (if case) and indirect (sls case) regulatory mechanisms are depicted. Black boxes represent exons, gray boxes represent the upstream and downstream noncoding sequences, and the light-gray boxes correspond to introns. Positions of dMef2 (Junion et al. 2005) and Lb-dependent CRMs are indicated. In pink is labeled a CRM to which binds hypothetical Lb-regulated factor X. if and sls transcription is regulated by generic dMef2-dependent modules. In the SBM context (in the presence of Lb protein) Lb binds to its intronic if CRM and contributes to the regulation of if transcription. In the case of sls, Lb action is indirect via an as-yet-unknown factor X. This factor most probably contributes to the activity of dMef2-dependent CRM1, which drives expression in a subset of muscles including SBM (Junion et al. 2005).

RNAi-based attenuation of Lb target genes leads to BM phenotypes similar to those observed in lb RNAi embryos. All panels represent lateral views of three abdominal segments from stage 15/16 embryos with focus on SBM and lateral transverse (LT) muscles. Muscles shapes are revealed with anti-β3-Tubulin antibody. Nuclei in the SBM fiber are labeled with anti-Lbe. (A,B) Wild-type views showing shapes, insertions, and positioning of Lbe-positive nuclei in SBM muscles. Note that the SBM founder cell nuclei (the biggest Lb-expressing nuclei seen the within SBM; arrows in A,B) occupy the most dorsal positions (see also schematic). Three types of SBM phenotypes observed in embryos with muscletargeted RNAi-based attenuation of lb expression (C,E,G) and in embryos in which Lb target genes were down-regulated (D,F,H). (C,D) A similar founder migration phenotype is observed in embryos with attenuated lb expression and in embryos with attenuated Msp300 expression SBM founder (arrowheads) is no longer located dorsally (cf. A,B) leading to the altered SBM shape and to the ventral accumulation of myoblast nuclei (arrows, see also corresponding scheme). (E,F) Attenuation of lb function and down-regulation of Lb target gene Mp20 affects myoblast fusion. Nonfused myoblasts (arrowheads) are detected around the SBM, and no myoblast nuclei are seen in the central, narrowed part of the muscle fiber (arrow, see also scheme). (G,H) In a portion of lb RNAi and in vkg RNAi embryos SBM fibers display abnormal shapes and attachments (arrowheads, see corresponding scheme). Attachment phenotypes are often accompanied by the presence of ectopic fibers located close to SBM (arrows).

A genome-wide revised view of Lb-governed cell fate specification. (A) A scheme showing that Lb acts at three different levels. Initially, it contributes to setting the so-called identity code by regulating negatively or positively other muscle- and heart-specific transcription factors. In the next step, Lb regulates genes required for the acquisition of cell-type-specific properties such as motility, shape, size, position, and cell–cell interactions. Lastly, Lb appears involved in regulation of genes required for terminal differentiation and the functional properties of muscle and cardiac cells. (B) A scheme showing that Lb targets are involved in all major steps of muscle and cardiac cell development. Note that the action of Lb at multiple levels and over a long time period is consistent with its expression during all steps of myogenesis and cardiogenesis. The direct Lb target genes identified by ChEST are labeled in red, and the pink-labeled genes correspond to microarray targets tested functionally by RNAi-based down-regulation.