Members of the T-Box gene family of transcription factors are important players in regulatory circuits that generate myogenic and cardiogenic lineage diversities in vertebrates. We show that during somatic myogenesis in Drosophila, the single ortholog of vertebrate Tbx1, optomotor-blind-related-gene-1 (org-1), is expressed in a small subset of muscle progenitors, founder cells and adult muscle precursors, where it overlaps with the products of the muscle identity genes ladybird (lb) and slouch (slou). In addition, org-1 is expressed in the lineage of the heart-associated alary muscles. org-1 null mutant embryos lack Lb and Slou expression within the muscle lineages that normally co-express org-1. As a consequence, the respective muscle fibers and adult muscle precursors are either severely malformed or missing, as are the alary muscles. To address the mechanisms that mediate these regulatory interactions between Org-1, Lb and Slou, we characterized distinct enhancers associated with somatic muscle expression of lb and slou. We demonstrate that these lineage- and stage-specific cis-regulatory modules (CRMs) bind Org-1 in vivo, respond to org-1 genetically and require T-box domain binding sites for their activation. In summary, we propose that org-1 is a common and direct upstream regulator of slou and lb in the developmental pathway of these two neighboring muscle lineages. Cross-repression between slou and lb and combinatorial activation of lineage-specific targets by Org-1–Slou and Org-1–Lb, respectively, then leads to the distinction between the two lineages. These findings provide new insights into the regulatory circuits that control the proper pattering of the larval somatic musculature in Drosophila.

INTRODUCTION

The musculature and the central nervous system are two examples of tissues featuring a high diversity of cell types. In each hemisegment of the Drosophila embryo, about 30 distinct body wall muscles and six adult muscle precursors are formed and in the CNS the number of neuronal and glial cell types generated is an order of magnitude higher. Currently, we have only a limited picture of the regulatory processes that generate these great cellular diversities, but we know that several steps of muscular and neuronal development in Drosophila are accomplished by closely related processes. For example, the earliest progenitor cells, termed muscle progenitors and neuroblasts, respectively, seem to be defined by intersecting anterior-posterior and dorsoventral upstream regulators. In both tissues, these progenitors are preceded by larger clusters of promuscular or proneural cells, respectively, that are characterized by their expression of proneural genes from the Achaete-Scute complex. Lateral inhibition mediated by Delta/Notch signals singles out individual progenitor cells from these clusters (reviewed by Beckett and Baylies, 2006; Technau et al., 2006; Tixier et al., 2010). In the case of the muscle progenitors, receptor tyrosine kinase (RTK) signaling, particularly via epidermal growth factor (EGF)- and fibroblast growth factor (FGF)-receptors (Egfr and Htl, respectively), is required as a positive input to antagonize Notch (Carmena et al., 2002). An additional similarity is the occurrence of asymmetric cell divisions that involve the unequal segregation of the Notch-inhibitor Numb (reviewed by Karcavich, 2005; Beckett and Baylies, 2006; Tixier et al., 2010). In the case of muscle progenitors, these asymmetric divisions typically generate two distinct muscle founder cells that go on to form two different larval muscle fibers. In some instances, one sibling of a muscle founder cell gives rise to certain heart precursors or to a stem cell-like adult muscle progenitor that will contribute to the adult body wall musculature (reviewed by Figeac et al., 2007; Speicher et al., 2008). Subsequently, muscle founder myoblasts fuse with surrounding fusion-competent myoblasts to form distinct body wall muscle fibers. During both muscle and neuronal development, the increasing diversity of the newly generated cells is reflected in the distinct combinations of transcription factors expressed in them. Several of these factors are known to have key roles in defining the particular cell identities and have been called muscle identity factors (reviewed by Beckett and Baylies, 2006; Tixier et al., 2010).

Although the broad picture of these diversification events is known, there are major gaps in knowledge with regard to the specific design of the regulatory networks involved. In the case of Drosophila muscle development, a few lineages of muscle progenitors have been characterized in some detail. The first are those marked by the expression of the identity factor Even-skipped (Eve, a homeodomain protein) and have been best characterized with regard to the essential early-acting inputs. These particular muscle progenitors arise in the dorsal portion of the mesoderm and give rise to somatic muscle fibers as well as to specific cells of the dorsal vessel (Frasch et al., 1987). One of the two adjacent muscle progenitors marked by Eve generates the founder of muscle 1 (DA1), which requires eve function (Fujioka et al., 2005), whereas the other generates the founder of muscle 10 (DO2) (Speicher et al., 2008). The activation of eve in these muscle progenitors requires combinatorial signals from ectodermal Decapentaplegic (Dpp) and Wingless (Wg), the mesodermal competence factor Tinman, which itself is downstream of Dpp, as well as RTK signals via Egfr and Htl (Azpiazu and Frasch, 1993; Bodmer, 1993; Frasch, 1995; Wu et al., 1995; Carmena et al., 1998). All these combinatorial signaling and transcription factor inputs are directly integrated at the level of a mesoderm-specific enhancer element at the eve locus (Halfon et al., 2000; Knirr and Frasch, 2001; Han et al., 2002).

The step-wise specification of muscle identities has been addressed for muscle founders expressing the identity factor Collier (Col, a COE transcription factor; Knot – FlyBase). In abdominal segments, Col is expressed in the two adjacent dorsolateral muscle progenitors of muscles 3 (DA3) and 20 (DO5) as well as 18 (DT1) and 19 (DO4) and the corresponding promuscular clusters. The expression is only maintained in the founder and precursor of muscle 3/DA3, which requires Col for its development (Crozatier and Vincent, 1999). This dynamic expression is reflected in the activities of at least two cis-regulatory modules (CRMs) of col. An intronic CRM activates expression within both Col+ promuscular clusters and the progenitors singled out from them, whereas an upstream CRM activates and maintains expression specifically in the muscle founder 3/DA3 and its muscle. The early-acting intronic CRM responds to positional and mesodermal inputs, which might involve binding by Twist and Tinman, whereas activation of the later-acting CRM in the M3/DA3 founder relies on the combinatorial binding of autoregulatory Col, Nautilus (Nau, also known as MyoD), and the Hox factors Antp and Ubx. In this manner, the early patterning and tissue-specific inputs are integrated with the axial inputs of the Hox genes that modulate the muscle pattern and fiber size in different body parts (Dubois et al., 2007; Enriquez et al., 2010).

The muscle lineages that are the focus of the present study are positioned laterally in the somatic mesoderm and are marked by the expression of the homeodomain factors Slouch (Slou) and Ladybird (Lb), which function as identity genes in these lineages [lb refers herein to the paralogous ladybird early (lbe) and ladybird late (lbl) genes]. Within this area of interest, Slou is expressed in the progenitor and sibling founders of muscles 5 (LO1) and 25 (VT1) and is crucial for the formation of these muscles (Dohrmann et al., 1990; Knirr et al., 1999). Lbe is expressed directly adjacent to Slou in a promuscular cluster and then two muscle progenitors. One of these progenitors forms muscle 8 (SBM) and a lateral adult muscle precursor (lAMP), which continue to express Lb, whereas the other forms a second Lb+ lAMP and a sibling that probably undergoes apoptosis (Jagla et al., 1998; Knirr et al., 1999; Figeac et al., 2010). Genetic and genomic analyses indicate that Lb has important functions in the specification and/or differentiation of muscle 8 (Junion et al., 2007; Bataille et al., 2010).

The expression of slou and lbe appears to be controlled by an overlapping set of upstream regulators, because their mutually exclusive expression in the neighboring progenitors of muscles 5/25 and muscle 8/lAMP requires cross-repression by their respective gene products (Knirr et al., 1999; Junion et al., 2007). However, the nature of these shared regulatory inputs has been unknown. In the present study, we identify org-1, a Drosophila Tbx1 ortholog that had not been characterized extensively prior to this work (Porsch et al., 1998; Lee et al., 2003), as a key activator of both slou and lbe in this pathway. org-1 expression in the mesodermal areas and progenitors that will form muscles 5, 25, 8 and the Lb+ lAMPs precedes that of slou and lbe, and the activation of these identity genes requires org-1 activity. Consequently, the development of these muscles and the lAMPs is disrupted in org-1 mutant embryos. We demonstrate that the activation of slou and lbe by org-1 requires T-box binding motifs in their respective founder-specific enhancer elements and show in vivo occupancy of these elements by Org-1. Hence, slou and lbe appear to be direct target genes of org-1 in the developmental pathway of these neighboring muscle lineages. In addition, org-1 is expressed in the progenitor of the alary muscles and its function is needed for the development of these muscles, which form segmental anchors of the dorsal vessel.

MATERIALS AND METHODS

Drosophila strains

We used lbl-SBM-lacZ (Philippakis et al., 2006) (from A. M. Michelson, NIH/NHLBI, Bethesda, MD, USA), RRHS59-lacZ (Knirr et al., 1999), Dp(1;3)sn13a1, w1118,Mi{ET1}org-1MB01466 and w1118;nocSco/SM6a,P{hsILMiT}2.4 (Bloomington Stock Center, Indiana University). Forced pan-mesodermal expression of UAS-org-1 with 2xPE-GAL4;how24B-GAL4 (Wang et al., 2005) was carried out at 28°C.

Generation of org-1 mutations

The viable insertion Mi{ET1}org-1MB01466 was used in a imprecise excision screen with Minos transposase as described by Metaxakis et al. (Metaxakis et al., 2005). Balanced Pax6-EGFP-negative females in F2 were crossed individually with FM7c males. Of the 591 excision lines screened for X-chromosomal lethality, the molecular lesions in two lines, org-1OJ423 and org-1OJ487 were defined by genomic PCR and sequencing of the shortened bands obtained.

Generation of an org-1 cDNA, UAS-org-1 and Org-1-specific antibodies

The 5′ portion (222 bp upstream of initiator ATG to NsiI site) of the cDNA was generated by RACE (SMART RACE Kit, Clontech) from cDNA of 8- to 12-hour-old embryos and was combined with a PCR-derived fragment of the 3′ portion (NsiI to 24 nucleotides downstream of stop codon) using an cDNA library (from 8- to 12-hour-old embryos) as a template. The combined and sequenced org-1 cDNA#1 (2385 bp) was cloned as an EcoRI (5′)/HindIII (3′) fragment into pBluescript SK+ and as an EcoRI/SalI fragment into EcoRI/XhoI of pUAST (Brand and Perrimon, 1993). The pUAST construct was injected into yw (Rubin and Spradling, 1982; Spradling and Rubin, 1982).

A fragment from a PCR product of org-1 cDNA#1 (using primers introducing BamHI and EcoRI sites directly upstream of the first ATG and at position 861 of the ORF, respectively) was cloned into BamHI/EcoRI cut pET30a (Novagen). Bacterially expressed recombinant protein corresponding to the N-terminal portion of Org-1 including the first six residues of the T-box was purified by His affinity chromatography and injected into rats (Covance, Denver, PA, USA) for antibody generation.

Construction of reporter and rescue constructs

Supplementary material Table S1 shows the genomic regions that were PCR-isolated from yw genomic DNA and used for reporter constructs. HN39, SK10 and SK16 were cloned into EcoRI/BamHI of pCaSpeR-hs-43-βgal (Thummel and Pirrotta, 1992). For SK16-GFP, the genomic fragment was cloned into EcoRI/BamHI of pGreen H-Pelican (Barolo et al., 2000). The slou-SK19 and lbl-SBMs fragments were cloned into EcoRI/KpnI of a modified pH-Stinger vector, which has attB sequences inserted into its AvrII site (pH-Stinger-attB; H.J. and M.F., unpublished). For the analogous creation of SK16 OrgI-VIImut-GFP, SK19 OrgI-IIImut-GFP and lbl-SBMs OrgI-VImut-GFP reporters the Org-1 binding sites within the SK19, SK16 and lbl-SBMs sequences were predicted using a positional weight matrix generated by SELEX with Org-1-GST [H.J. and M.F., unpublished; Target Explorer (Sosinsky et al., 2003)] and mutated via site-directed mutagenesis or de novo DNA synthesis (MrGene, Regensburg, Germany) (supplementary material Table S1). For org-1-HN39-GFP, the HN39 fragment was cloned into EcoRI/BamHI of pH-Stinger-attB. For org-1-HN39-org-1, org-1-HN39-slou and org-1-HN39-lbe, org1 cDNA (this study), slou cDNA (Dohrmann et al., 1990) and lbe cDNA (Jagla et al., 1998) with added C-terminal Myc-tags were cloned into AgeI/HpaI of org-1-HN39-pH-Stinger-attB, substituting the EGFP-cassette. pH-Stinger constructs were transformed into yw and the pH-Stinger-AttB constructs into strains with the landing sites ZH-35B (Bischof et al., 2007) or AttP2 (Groth et al., 2004). At least four independent transformed lines were tested for transgene activity.

Chromatin immunoprecipitation (ChIP) assays

Chromatin preparation using ChIP was performed using staged 2xPE-GAL4; how24B-GAL4xUAS-org1;UAS-org1;UAS-org1 embryo collections and two different Org-1 antibodies as described (Sandmann et al., 2006). The precipitated DNA was purified with MinElute PCR purification kit (Qiagen, Hilden) and quantified by qPCR using the Brilliant II SYBR Green QPCR Master Mix (Agilent) on an Mx3000P system (Stratagene). Three independent precipitations per amplicon were analyzed (supplementary material Table S1).

Immunohistochemistry

Embryo staining and in situ hybridization were performed as described previously (Azpiazu and Frasch, 1993; Knirr et al., 1999) with the following antibodies and probes: rat anti-Org-1 (1:100, this study), mouse anti-Lb monoclonal (1:10) (Jagla et al., 1998), rabbit anti-S59 (1:100) (Dohrmann et al., 1990), rabbit anti-β3-Tubulin (1:3000; gift from R. Renkawitz-Pohl, University of Marburg, Germany), mouse anti-Ubx monoclonal (1:50; gift from R. White, University of Cambridge, UK), chick anti-Twist (1:200, affinity-purified; Dominik Müller and M.F., unpublished), rat anti-Tropomyosin (1:200; Babraham, Cambridge, UK), rabbit anti-GFP (1:2000; Molecular Probes), mouse anti-GFP (1:2000; Molecular Probes), rabbit anti-βGal (1:1500; Promega), mouse anti-βGal (40-1a, 1:200; Developmental Studies Hybridoma Bank, University of Iowa, USA) and digoxygenin-labeled antisense transcripts from caup cDNA (Gomez-Skarmeta et al., 1996). All mutant lines were balanced with lacZ or GFP balancers to identify mutant embryos. Pictures were taken with a Zeiss Axio Imager equipped with a Zeiss ApoTome (20×/0.8 Plan-Apochromat; 40×/1.3 Plan-Apochromat Oil) or a Leica SP5 II (63×/1.3 PL APO Glycerol). Projections were created using Axiovision 4.8 or Leica LAS AF.

RESULTS

org-1 is expressed in two characterized somatic muscle lineages and in alary muscles

org-1, the Drosophila ortholog of vertebrate Tbx1 (see Fig. 1 for details on the gene locus), showed a striking and dynamic pattern of expression in the developing mesoderm. During early embryonic stage 9, Org-1 protein started to be expressed in narrow segmental stripes within the somatic mesoderm and by stage 10, Org-1 expression also appeared in the 11 segmental clusters of trunk visceral mesoderm precursors (Fig. 2A,B). As previously reported for org-1 mRNA (Lee et al., 2003), during stages 10-11 Org-1 protein expression in the visceral mesoderm became restricted to the visceral muscle progenitor and founder cells (Fig. 2B-D). Importantly, somatic mesodermal expression also refined during stage 11 and remained active in a few individual cells per hemisegment, which we identified as somatic muscle progenitors (Fig. 2D). During stage 12, these muscle progenitors divided into Org-1+ somatic muscle founder cells (Fig. 2E). After myoblast fusion, the abdominal expression of Org-1 could be assigned to somatic muscles 5 (M5, also known as LO1) and 25 (M25/VT1), which are derived from sibling founder cells, muscle 8 (M8/SBM) and two lateral adult muscle precursors (lAMPs), one of which is a sibling of the M8 founder. In addition, the alary muscle expressed Org-1 and was formed from one of the Org-1+ founder cells located in dorsolateral mesodermal areas (Fig. 2F,G). These assignments were supported further by the expression analysis of an org-1 reporter construct driven by a downstream enhancer, HN39, which faithfully recapitulated org-1 expression in the progenitors, founders, lAMPs, and fibers of the somatic muscles (Fig. 1, Fig. 2H-J). During stage 14, org-1 mRNA and protein expression became extinct in M5 and M8, but owing to the longer half-life of β-galactosidase (βGal), reporter signals perdured in these muscles until the end of embryogenesis (Fig. 2G-I′; C.S. and M.F., unpublished).

Fig. 1.

org-1gene locus and imprecise excision-induced deletion alleles.org-1 gene map (genome annotation R5.19) showing transcribed regions in black, regions coding for the T-Box DNA binding motif in blue and deleted regions in the imprecise excision alleles org-1OJ423 and org-1OJ487 underneath. The HN39 genomic region used for org-1-lacZ, org-1-GFP and the rescue constructs is shown in green.

Fig. 1.

org-1gene locus and imprecise excision-induced deletion alleles.org-1 gene map (genome annotation R5.19) showing transcribed regions in black, regions coding for the T-Box DNA binding motif in blue and deleted regions in the imprecise excision alleles org-1OJ423 and org-1OJ487 underneath. The HN39 genomic region used for org-1-lacZ, org-1-GFP and the rescue constructs is shown in green.

The expression of Org-1 during somatic muscle development was examined in more detail by double staining with org-1-HN39-lacZ and probes reflecting the expression of two known muscle identity genes, slouch (slou) and ladybird (lb). As shown in Fig. 3A,A′, by stage 11 org-1 expression became restricted to two neighboring somatic muscle progenitors per hemisegment, which were also positive for org-1-βGal. Shortly thereafter, the appearance of a third Org-1+ progenitor gave rise to a three-cell cluster at this position (Fig. 3A-B′). Double staining for βGal and Lb demonstrated that the two dorsal cells within this cluster correspond to the Lb+ muscle progenitors (Fig. 3F,F′). They divide into four Org-1+ (and Lb+) cells (Fig. 3C,C′,F,F′), although one of these rapidly became undetectable. The observation of about one extra Lb+ cell in this area upon blocking cell death (Figeac et al., 2010) suggests to us that this fourth cell normally undergoes apoptosis. Consistent with previous findings with Lb as a marker, one of the three Org-1+ and Lb+ cells was seen to form M8, whereas the other two formed the two lAMPs (Fig. 3G,G′). M8 and the lAMPs maintained βGal signals and Lb expression but M8 rapidly lost Org-1 expression after myoblast fusion (Fig. 3D-E′,G,G′).

The ventral muscle progenitor shown in Fig. 3A in each hemisegment became positive for Slouch (Slou; data not shown) and for βGal driven by the RRHS59 enhancer upstream of slou (Fig. 3H,H′) (Knirr et al., 1999). This progenitor divides into the founder cells of M5 and M25 (Fig. 3C-D′) (Dohrmann et al., 1990). Whereas M25 continued expressing Org-1 until the end of embryogenesis, Org-1 expression in M5 was gradually lost (Fig. 3E,E′,I,I′).

Fig. 2.

Org-1 protein is expressed in visceral and a subset of somatic muscle progenitor cells. (A,B) Org-1 protein is found during stage 10 (A, lateral view; B, ventral view) in all cells of the trunk visceral mesoderm (VM) and in narrow stripes of cells in the somatic mesoderm (SM). (C,D) At stage 11 (C, lateral view; D, ventral view), Org-1 expression domains in the segmented mesoderm become confined to progenitor cells of the visceral musculature (vPC) as well as to a specific subset of somatic muscle progenitor cells (sPC). (E,F) During germ band retraction (E), Org-1 is present in visceral (vFC) and somatic (sFC) founder cells, derived from the progenitors, and during stage 13 (F) neighboring nuclei become positive after being incorporated into Org-1+ muscle precursors upon myoblast fusion. (G-J) At stage 14, nuclear Org-1 (G) and cytoplasmic β-Gal (H-J) expression driven by org-1-HN39-lacZ are present in muscles 5 (LO1), 25 (VT1) and 8 (SBM), alary muscles (AM) and lateral adult muscle precursors (lAMPs). The muscle pattern is visualized by Tropomyosin (TM1) or β3-tubulin antibody staining.

Fig. 2.

Org-1 protein is expressed in visceral and a subset of somatic muscle progenitor cells. (A,B) Org-1 protein is found during stage 10 (A, lateral view; B, ventral view) in all cells of the trunk visceral mesoderm (VM) and in narrow stripes of cells in the somatic mesoderm (SM). (C,D) At stage 11 (C, lateral view; D, ventral view), Org-1 expression domains in the segmented mesoderm become confined to progenitor cells of the visceral musculature (vPC) as well as to a specific subset of somatic muscle progenitor cells (sPC). (E,F) During germ band retraction (E), Org-1 is present in visceral (vFC) and somatic (sFC) founder cells, derived from the progenitors, and during stage 13 (F) neighboring nuclei become positive after being incorporated into Org-1+ muscle precursors upon myoblast fusion. (G-J) At stage 14, nuclear Org-1 (G) and cytoplasmic β-Gal (H-J) expression driven by org-1-HN39-lacZ are present in muscles 5 (LO1), 25 (VT1) and 8 (SBM), alary muscles (AM) and lateral adult muscle precursors (lAMPs). The muscle pattern is visualized by Tropomyosin (TM1) or β3-tubulin antibody staining.

During early stage 12, a new muscle progenitor located dorsally to the Org-1+ and Lb+ muscle 8 founder started expressing Org-1 and divided into two Org-1+ and org-1-βGal+ muscle founders (Fig. 3C,C′). The dorsal sibling corresponds to the founder of the alary muscle. It fuses with dorsally adjacent fusion-competent myoblasts, which showed gradually increasing Org-1 signals (Fig. 3D,D′, see also Fig. 2F,G), and elongated towards the dorsal vessel. Owing to its rapid loss of Org-1 expression, the fate of the sibling of the alary muscle founder remains unknown.

In summary, org-1 shows a highly restricted pattern of expression in the somatic mesoderm, which precedes and overlaps with the expression of the previously characterized muscle identity genes slou and lb. In addition, org-1 represents the first clear marker for the founders and fibers of the alary muscles, which attach segmentally to the dorsal vessel.

org-1 functions as a muscle identity gene in all org-1+ lineages

The function of org-1 during somatic muscle development was analyzed with two lethal alleles made by imprecise Minos-element excisions (Fig. 1), which had identical phenotypes. Both alleles showed complementation of hemizygous lethality by Dp(1;3)sn13a1, a duplication covering org-1, and appeared to be null mutants. org-1OJ487 had the translation initiation codon as well as the first six exons deleted and did not show any detectable Org-1 protein, whereas org-1OJ423 caused a C-terminal truncation of the protein including a portion of the T-Box DNA-binding domain (Fig. 1). In hemizygous org-1 mutant embryos, M5 and M8 were not identifiable. Instead, there were small syncytia with variable shapes located near the normal ventral attachment sites of these two muscles, which did express differentiation markers such as β3-Tubulin. This phenotype exhibited 100% penetrance. Fibers resembling M25 appeared to be present, although they were highly variable in shape and often connected to incorrect attachment sites (Fig. 4B-B″′, compare with 4A-A″′). The formation of the alary muscles was also disrupted in org-1 mutant embryos, in which these muscles were completely missing (Fig. 4D, compare with 4C).

Although org-1-HN39-lacZ expression was activated in the progenitor and founder cells of M5, M8, M25 and the alary muscles, this expression rapidly disappeared upon myoblast fusion in the org-1 mutant background, as did the truncated protein detected in org-1OJ423 mutant embryos (Fig. 4F, compare with 4E; data not shown). Hence, normally there is direct or indirect autoregulation.

Because org-1 expression overlaps with that of slou and lb in the somatic mesoderm and org-1 mutations affect slou+ and lb+ muscles, we tested whether org-1 is required for the expression of these two muscle identity genes. Indeed, we found that lb expression was never activated in the somatic mesoderm of org-1 mutants and the Lb protein could be detected neither in the progenitors, founders and fibers of M8 nor in the lAMPs (Fig. 5B,D, compare with 5A,C). Staining for Twist, which marks all adult muscle precursors, showed that, unlike adult muscle precursors at other positions, the number of lAMPs was strongly reduced. In ∼50% of the hemisegments no lAMPs were detectable and in the remaining ones only one instead of two lAMPs was seen, which failed to express Lb (Fig. 5C,D). Thus, org-1 is required for proper formation of lAMPs.

Fig. 3.

The expression patterns oforg-1andorg-1-lacZoverlap with those of the muscle identity genesladybird(lb) andslouch(slou). (A,A′) During stage 11, Org-1 protein as well as org-1-lacZ reporter (β-Gal) activity are present in the progenitor cells (arrows) of muscles 5/25 (L01/VT1) and M8 (SBM)/lAMP in each abdominal segment. (B,B′) During stage 12, the Org-1+org-1-lacZ+ progenitors cells divide to generate muscle founder cells and lAMPs (arrowheads). Arrows indicate progenitor cells. (C,C′) At late stage 12, Org-1 and org-1-lacZ are present in the founder cells of muscles 5, 25 and 8 and the lAMPs, and are newly expressed in alary muscle founders (FAM). (D,D′) Org-1 and org-1-lacZ in muscle precursors during myoblast fusion and in lAMPs at stage 13. AM, alary muscle. (E,E′) At stage 14, Org-1 expression remains in M25 and lAMPs (and alary muscles; not depicted) but recedes in M5 and M8. (F,F′) In early stage 11 embryos, Ladybird protein (Lb) can be detected in two (left and middle) and in more advanced (right) segments in four of the org-1-lacZ+ muscle founders. (G,G′) At stage 15, these founders have given rise to M8 and the lateral adult muscle precursors (lAMPs). (H-I′) At stage 11, Org-1 protein expression is also observed in one slou-RRHS59-lacZ+ muscle progenitor (H,H′) and at stage 15 Org-1 overlaps in muscle 5 and 25 with the slou-lacZ signals (I,I′).

Fig. 3.

The expression patterns oforg-1andorg-1-lacZoverlap with those of the muscle identity genesladybird(lb) andslouch(slou). (A,A′) During stage 11, Org-1 protein as well as org-1-lacZ reporter (β-Gal) activity are present in the progenitor cells (arrows) of muscles 5/25 (L01/VT1) and M8 (SBM)/lAMP in each abdominal segment. (B,B′) During stage 12, the Org-1+org-1-lacZ+ progenitors cells divide to generate muscle founder cells and lAMPs (arrowheads). Arrows indicate progenitor cells. (C,C′) At late stage 12, Org-1 and org-1-lacZ are present in the founder cells of muscles 5, 25 and 8 and the lAMPs, and are newly expressed in alary muscle founders (FAM). (D,D′) Org-1 and org-1-lacZ in muscle precursors during myoblast fusion and in lAMPs at stage 13. AM, alary muscle. (E,E′) At stage 14, Org-1 expression remains in M25 and lAMPs (and alary muscles; not depicted) but recedes in M5 and M8. (F,F′) In early stage 11 embryos, Ladybird protein (Lb) can be detected in two (left and middle) and in more advanced (right) segments in four of the org-1-lacZ+ muscle founders. (G,G′) At stage 15, these founders have given rise to M8 and the lateral adult muscle precursors (lAMPs). (H-I′) At stage 11, Org-1 protein expression is also observed in one slou-RRHS59-lacZ+ muscle progenitor (H,H′) and at stage 15 Org-1 overlaps in muscle 5 and 25 with the slou-lacZ signals (I,I′).

As observed for Lb, the expression of the Iroquois homeobox genes caupolican and araucan is specifically missing at the normal positions of M8 in org-1 mutants, showing that the Iroquois genes are also expressed downstream of org-1 in M8 and confirming that M8 is not properly specified in the absence of Org-1 (Fig. 5E,F; data not shown). caup expression was unaffected (i.e. neither reduced nor expanded) in the org-1-independent lateral transverse muscles, in which the Iroquois genes were recently shown to act as muscle identity genes (Carrasco-Rando et al., 2011).

In the lineages of M5 and M25, the expression of Slou and slou-RRHS59-lacZ was absent during all phases of their (aberrant) development (Fig. 5H,J, compare with 5G,I; data not shown). As expected, the expression in M18 and M27, which lack Org-1 also during normal development, was unaffected. Together, these observations define org-1 as a crucial upstream regulator of the identity genes lb, slou and caup in the developmental pathway giving rise to M5, M8, M25 and the lAMPs.

Additional experiments addressed the question of whether org-1 is simply required to activate lb and slou in the precursor cells or whether it plays additional essential roles in the development of the respective muscles. Specifically, we tested whether restoring the expression of lbe or slou in org-1 mutant backgrounds would rescue the observed defects in the development of M8 and M5/M25, respectively. Whereas the expression of org-1 in org-1 mutant backgrounds under the control of the HN39-org-1 enhancer was able to rescue these muscles to their normal morphology, albeit at a low frequency (supplementary material Fig. S1D, compare with S1B,C), neither expression of lb (supplementary material Fig. S1F, compare with S1B,D,E) nor of slou (supplementary material Fig. S1H, compare with S1B,D,G) under the same conditions was ever seen to provide any rescue. This strongly suggests a role for org-1 in activating other essential target(s) in parallel to lb and slou during the development of these muscles.

In addition to Slou or Lb expression, the Org-1+ founder cells were also positive for Ultrabithorax (Ubx) protein during stage 12 in abdominal segments (Fig. 5K). In org-1 mutant embryos, not only Slou and Lb but also Ubx expression was abolished in these founder cells (Fig. 5L). Hence, Ubx is another example of an org-1 downstream gene within this subset of somatic founder cells and probably acts in the regional modulation of the respective muscle identities.

Fig. 4.

Loss oforg-1function leads to muscle specification defects. (A-B″′) Comparison of the segmental muscle patterns of stage 15 WT and org-1OJ487 mutant embryos (visualized with anti-β3-tubulin) reveals lost or mis-shapen M8, M5, M25 and alary muscles in mutants. In A″ and B″ the phenotypic differences between the WT (A″) and the org-1OJ487 null mutant (B″) situations are highlighted. Projections of confocal slices containing wild-type M8, M5, M25 (A″′) and their mutant counterparts (B″′) are shown. (C) In the wild type (WT), the alary muscles (AM), marked by Org-1 and β3-Tubulin, extend from the tip of M8 towards the dorsal vessel. (D) In org-1OJ487 mutant, alary muscles are absent (asterisks). (E) org-1-GFP expression marking all Org-1+ muscle precursors and lAMPs in stage 14 wild-type background (TM1, Tropomyosin). (F) Severely reduced org-1-GFP expression in stage 14 org-1OJ487 mutant background.

Fig. 4.

Loss oforg-1function leads to muscle specification defects. (A-B″′) Comparison of the segmental muscle patterns of stage 15 WT and org-1OJ487 mutant embryos (visualized with anti-β3-tubulin) reveals lost or mis-shapen M8, M5, M25 and alary muscles in mutants. In A″ and B″ the phenotypic differences between the WT (A″) and the org-1OJ487 null mutant (B″) situations are highlighted. Projections of confocal slices containing wild-type M8, M5, M25 (A″′) and their mutant counterparts (B″′) are shown. (C) In the wild type (WT), the alary muscles (AM), marked by Org-1 and β3-Tubulin, extend from the tip of M8 towards the dorsal vessel. (D) In org-1OJ487 mutant, alary muscles are absent (asterisks). (E) org-1-GFP expression marking all Org-1+ muscle precursors and lAMPs in stage 14 wild-type background (TM1, Tropomyosin). (F) Severely reduced org-1-GFP expression in stage 14 org-1OJ487 mutant background.

The potency of org-1 in muscle specification was also evident from experiments in which we forced its ectopic expression. Pan-mesodermal expression of Org-1 caused severe disruptions of the somatic muscle pattern in 100% of scored segments (Fig. 6A-B′). This effect was accompanied by a dramatic expansion of lb expression (Fig. 6D,D′, compare with 6C,C′). Slou expression was expanded to a lesser degree with pan-mesodermal Org-1 and the expression domains of Lb and Slou remained mutually exclusive (data not shown), indicating that cross-repression between Slou and Lb (Knirr et al., 1999; Junion et al., 2007) remains intact in this situation. Together with the observed specific increase of Twist-marked lAMPs upon ectopic expression of Org-1 (Fig. 6F, compare with 6E), these data suggest that ectopic Org-1 can shift the identities of other muscle founders and muscles towards those of M8, lAMPs and (to a lesser degree) M5 and M25.

Org-1 is a direct upstream regulator of slou and lb in the somatic mesoderm

To determine whether Org-1 is a direct activator of slou and lb expression, we examined relevant cis-regulatory modules of these two genes. The previously defined lbl-SBM enhancer element (Philippakis et al., 2006) and a 925 bp subfragment, lbl-SBMs, defined by us (Fig. 7A) showed reporter activity in the Lb+ muscle founder cells as well as in M8 and the lAMPs (Fig. 7B,D,F), thus recapitulating the somatic mesodermal Lb expression pattern. As shown for the Lb protein, lbl-SBM enhancer activity depends on org-1 (Fig. 7C). Conversely, it is dramatically expanded upon ectopic pan-mesodermal org-1 expression (data not shown).

Our dissection of the slou-RRHS59 element revealed two separate genomic regions that were active in the slou+ and org-1+ muscle lineage. A 622 bp element, slou-SK19, and a slightly shorter version of it, slou-SK10 (Fig. 7H), were already active in muscle progenitors and muscle founders of all Slou+ muscles (Fig. 7I,K). In addition, a non-overlapping 745 bp element located proximally to slou-SK19, termed slou-SK16 (Fig. 7H,M), initiated expression only in founder cells and remained active in muscles. Apart from the slou+ M5 and M25, SK16 also drove ectopic expression in M8, apparently because it lacks sequences that are subject to Lb-dependent repression (Fig. 7N,P; there is also weak ectopic activity in M21 and M23). In org-1 mutant backgrounds, the activity of the two distinct slou enhancers SK10 and SK16 was specifically absent in the lineages that normally express org-1, namely the progenitors and founders of M5 and M25 (SK10; Fig. 7J) and the founders and fibers of M5/M25 and M8/LaP (SK16; Fig. 7O). As expected, native and ectopic enhancer activity in other muscles and their precursors that normally are independent of org-1 was unaffected by org-1 mutation (M18, M27, M21, M23; Fig. 7J,O). The observed dependency of lb and slou enhancer activities on org-1 suggested that these enhancers could be transcriptional targets of Org-1.

Sequence analysis of these lb and slou enhancer elements in combination with data from SELEX experiments with Org-1 fusion proteins identified six putative Org-1 binding motifs within lbl-SBMs, three within slou-SK19 and seven within slou-SK16 (Fig. 7A,H,M,R). Anti-Org-1 ChIP analysis with chromatin from stage 11-12 embryos (2xPE; how24B>>UAS-org-1) clearly confirmed in vivo binding of Org-1 to the binding sites in the SK19 and lbl-SBMs enhancer fragments, whereas chromatin including the binding sites within the SK16 fragment was enriched less strongly (Fig. 7R).

Fig. 5.

The expression of the muscle identity genesladybird,caupolicanandslouchis regulated byorg-1. (A) In stage 11 wild-type embryos, Lb is detected in two muscle progenitor cells in each abdominal segment (arrowhead). (B) In the org-1 mutant background, Lb expression in the progenitors is absent. (C) Stage 14 wild-type embryo showing Lb expression in M8 and the lAMPs (identified by Twist). (D) In an org-1 mutant embryo, Lb expression in M8 and the lAMPs is abolished. In ∼50% of the segments one weakly Twist+ lateral cell remains (asterisks). (E) Stage 14 wild-type embryo showing mesodermal caupolican (caup) mRNA expression (green) in M8 (arrows) and in M21-24 (asterisks) (blue, β3-Tubulin; red, Org-1). (F) In org-1 loss-of-function embryos, caup expression at the normal positions of M8 is absent (arrows). (G) In a stage 12 wild-type embryo carrying slou-lacZ, β-Gal protein is observed in one Slou+ muscle progenitor (P5/25; asterisk) but not yet in a second progenitor (P26/27; arrowhead) per hemisegment. (H) In the org-1OJ487 background, both Slou protein and slou-lacZ activity in P5/25 and the two corresponding muscle founders are absent. Arrowhead indicates progenitor of M26/27. (I) Stage 15 embryo with Slou and slou-lacZ signals in four distinct muscles per hemisegment. (J) In the org-1OJ487 background at stage 15, Slou expression and slou-lacZ signals in M5 and M25 are missing. (K) In a stage 12 wild-type embryo, high levels of Ultrabithorax protein (Ubx) are found in the Org-1+ muscle founders, where it shows co-expression (insets) with either Slou (arrowheads) or Lb (asterisks). (L) Loss of Org-1 function causes loss of Ubx as well as Slou and Lb expression in these founder cells.

Fig. 5.

The expression of the muscle identity genesladybird,caupolicanandslouchis regulated byorg-1. (A) In stage 11 wild-type embryos, Lb is detected in two muscle progenitor cells in each abdominal segment (arrowhead). (B) In the org-1 mutant background, Lb expression in the progenitors is absent. (C) Stage 14 wild-type embryo showing Lb expression in M8 and the lAMPs (identified by Twist). (D) In an org-1 mutant embryo, Lb expression in M8 and the lAMPs is abolished. In ∼50% of the segments one weakly Twist+ lateral cell remains (asterisks). (E) Stage 14 wild-type embryo showing mesodermal caupolican (caup) mRNA expression (green) in M8 (arrows) and in M21-24 (asterisks) (blue, β3-Tubulin; red, Org-1). (F) In org-1 loss-of-function embryos, caup expression at the normal positions of M8 is absent (arrows). (G) In a stage 12 wild-type embryo carrying slou-lacZ, β-Gal protein is observed in one Slou+ muscle progenitor (P5/25; asterisk) but not yet in a second progenitor (P26/27; arrowhead) per hemisegment. (H) In the org-1OJ487 background, both Slou protein and slou-lacZ activity in P5/25 and the two corresponding muscle founders are absent. Arrowhead indicates progenitor of M26/27. (I) Stage 15 embryo with Slou and slou-lacZ signals in four distinct muscles per hemisegment. (J) In the org-1OJ487 background at stage 15, Slou expression and slou-lacZ signals in M5 and M25 are missing. (K) In a stage 12 wild-type embryo, high levels of Ultrabithorax protein (Ubx) are found in the Org-1+ muscle founders, where it shows co-expression (insets) with either Slou (arrowheads) or Lb (asterisks). (L) Loss of Org-1 function causes loss of Ubx as well as Slou and Lb expression in these founder cells.

When we introduced two to three base pair changes into each of these motifs at positions known to be important for T-box protein binding (Müller and Herrmann, 1997), we observed dramatic changes in all reporter expression patterns. The lbl-SBMs OrgI-VImut reporter showed a total loss of activity in the Lb+ muscle progenitor and founder cells (Fig. 7E) as well as strong downregulation in M8 and the lAMPs (Fig. 7G). Reporter activity of slou-SK19 OrgI-IIImut in the Org-1+ precursor cells was completely abolished (Fig. 7L), and also slou-SK16 OrgI-VIImut was specifically inactivated in all Org-1+ muscles, namely M5, M8 and M25 (Fig. 7Q). By contrast, the activity in the Slou+/Org-1 muscle M18 and the ectopic activity in the Slou/Org-1 muscles M21 and M23 was unaffected, thus providing an internal control for the specificity of the effects (Fig. 7Q). Altogether, these data strongly suggest that the normal activation of lb and slou in the founder cells and muscles of these org-1+ muscle lineages requires binding of Org-1 to some or all of the Org-1 binding motifs within the lbl-SBMs, slou-SK19 and slou-SK16 enhancer sequences.

DISCUSSION

Our analysis of the expression and function of org-1 in somatic muscle development has established this gene as a new and crucial representative of muscle identity genes in Drosophila. The data have provided new insights into developmental controls in two well-defined muscle lineages and somatic muscle development in general. These lineages include one dependent on the homeobox gene slou, which gives rise to muscles M5 and M25 (also known as ‘cluster I’) (Dohrmann et al., 1990), and another dependent on the lb homeobox genes, which gives rise to the segment border muscle M8 and lateral adult muscle precursors (lAMPs) (Fig. 8). They arise from promuscular clusters and muscle progenitors abutting each other in the ventrolateral somatic mesoderm. Until now, it was assumed that slou and lb are positioned at the top of the regulatory hierarchy set up within each of these lineages, as their expression and function is already detected at the progenitor cell stage prior to asymmetric divisions into different founder cells (Dohrmann et al., 1990; Jagla et al., 1998; Knirr et al., 1999). As in the model proposed for the even-skipped-expressing lineage in the dorsal somatic mesoderm, the progenitor-specific expression of slou and lb was assumed to be determined directly by the antagonistic actions of various more broadly active activators and negative influences through lateral inhibition via Notch (Bate et al., 1993; Jagla et al., 1998). Based on genetic assays using the expression of slou and lb, and the formation of the respective muscles as outputs, good candidates for such activators included localized receptor tyrosine kinase (RTK)/Ras/MAPK signals (Baylies and Michelson, 2001; Bidet et al., 2003) (H.N. and M.F., unpublished) in conjunction with the relatively broadly expressed mesodermal transcription factors Tinman (Azpiazu and Frasch, 1993; Jagla et al., 1998), Six4 (in the case of lb) (Clark et al., 2006), Twist (Cox and Baylies, 2005) and Sloppy paired (Lee and Frasch, 2000). Ectodermal Wg signals were also found to regulate these Slou+ and Lb+ lineages (Baylies et al., 1995; Jagla et al., 1998), although at least in the case of slou these apparently act indirectly by upregulating twi and slp expression (Baylies et al., 1995; Lee and Frasch, 2000; Cox and Baylies, 2005).

Fig. 6.

Ectopic Org-1 expression induces founder cell fate transformations. (A-B′) Severe disruption of the normal somatic muscle pattern (A,A′) marked by b3-Tubulin (green) as a result of pan-mesodermal overexpression (B,B′) of Org-1 (red in A,B). (C-D′) Lb expression (red), normally restricted to one M8 and two lAMPs per hemisegment (C,C′), strongly expands upon pan-mesodermal Org-1 expression (D,D′). (E,F) Ectopic Org-1 expression leads to increased formation of Twi+ lAMPs (F, arrows) in comparison with the wild type (E, arrows).

Fig. 6.

Ectopic Org-1 expression induces founder cell fate transformations. (A-B′) Severe disruption of the normal somatic muscle pattern (A,A′) marked by b3-Tubulin (green) as a result of pan-mesodermal overexpression (B,B′) of Org-1 (red in A,B). (C-D′) Lb expression (red), normally restricted to one M8 and two lAMPs per hemisegment (C,C′), strongly expands upon pan-mesodermal Org-1 expression (D,D′). (E,F) Ectopic Org-1 expression leads to increased formation of Twi+ lAMPs (F, arrows) in comparison with the wild type (E, arrows).

Our current findings have uncovered an additional layer of regulation upstream of slou and lb within the M5/M25 and M8/lAMP lineages that involves org-1 (Fig. 8). The initial expression of org-1 occurs in segmented areas of the lateral somatic mesoderm, from which the org-1+ M5/M25 and M8/lAMP progenitors emerge. These areas probably delineate the two abutting promuscular clusters from which these progenitors are singled out (as well as a third one dorsolaterally, from which the progenitor and founder of the alary muscle is formed). Although this early expression of org-1 is not required for progenitor formation per se (based upon the normal pattern of org-1:HN39-lacZ in progenitors of org-1 mutants; C.S. and M.F., unpublished), org-1 is crucial either during this phase or during early phases of progenitor formation for activating slou and lb in the respective progenitors. Hence, we propose that the previously identified regulators of slou and lb, including Notch, RTKs, tin, wg, twi and slp, act predominantly in establishing progenitor-specific expression of org-1, which in turn activates slou and lb. However, a plausible alternative to this linear model of regulation would be a feed-forward model, in which some of the above upstream regulators are re-employed during the second step to activate slou and lb together with mandatory Org-1. Whether org-1 activates slou or lb in any given progenitor would depend on the outcome of the previously reported mutual inhibition between slou and lb. We think it is unlikely that the outcome of this process is completely random; instead, we favor a mechanism involving an initial bias towards one or the other. Such biases could, for example, arise through slight differences in the spatial activities of the EGF and FGF receptors (Egfr and Htl), or of transcription factors such as Six4, coupled with differential responses of slou and lb to these regulators. Regardless of the specific mechanism, this principle of joint activation followed by mutual repression allows for the differential specification of directly neighboring cells, in this case of P5/25 and P8/lAMP, that are under the influence of common upstream regulators.

The combination of genetic data and functional enhancer analysis provides convincing evidence that both slou and lb are direct transcriptional targets of Org-1. This conclusion also fits nicely with the observation that a slou enhancer fragment used herein, SK16, is active ectopically in M8 and that Org-1 and the putative Org-1 binding sites are required for its activity not only in M5 and M25, but also in M8. By contrast, an adjacent enhancer fragment, SK10, is not active in M8 and thus reflects the endogenous Org-1-dependent pattern of slou. Presumably unlike SK16, this element still includes lb-dependent repression elements that block its activation by Org-1 in M8.

The lbl-SBM enhancer (1.36 kb) was identified initially in a bioinformatics approach for sequences near founder cell-expressed genes that are enriched with binding motifs for Twi, Tin, dTCF (Pan – FlyBase; Wg signaling effector), Pointed (RTK signal effector) and Mad (Dpp signaling effector), and also showed increased evolutionary conservation (Philippakis et al., 2006). However, this enhancer had a relatively low score and, furthermore, the Tin and Twi motifs with decent matches to bona fide binding sites for these factors lie outside of our shortened version, lbl-SBMs. Additionally, Smad proteins are not expected to be active in this ventrolateral region. It is possible that this enhancer was picked up with this algorithm largely because of the adjacent high-scoring cardiac enhancer. Nevertheless, some of the remaining binding motifs within lbl-SBMs, e.g. those for dTCF and Pnt, might contribute to the enhancer activity in progenitors and founders together with the essential Org-1 binding sites. The residual activation of the lbl-SBMs enhancer with mutated Org-1 binding motifs in the differentiated M8 shows that the maintenance of enhancer activity at later stages depends on regulators other than Org-1, which agrees with the observation that Org-1 is no longer expressed in M8 during these stages. This maintenance might be regulated by inputs shared with two separate later-acting enhancer elements upstream of lbe (termed LAMPE) and lbl (termed LME). Both of these elements include homeodomain binding motifs that probably mediate autoregulation by Lb, in addition to ETS domain and Mef2-binding motifs, respectively, that also contribute to full activity in M8 (Figeac et al., 2010). In org-1 mutants, the lbl-SBM enhancer is inactive not only early but also late, possibly because Lb protein is not available for autoregulation.

Fig. 7.

The Org-1 binding sites in somatic mesodermalladybirdandslouchCRMs are essential for lineage-specific enhancer activity. (A) Schematic of the genomic region used for the generation of the lbl-SBM (boxed, black) and lbl-SBMs (green) reporters. Predicted T-Box binding sites are indicated in detailed view below. (B) lbl-SBM-driven lacZ expression in M8 and lAMPs in stage 15 wild-type embryo. (C) Stage 15 org-1 mutant embryo carrying lbl-SBM-lacZ showing complete absence of β-Gal expression. (D) lbl-SBMs-GFP reporter recapitulates expression of Lb protein in somatic muscle founders (arrowhead). (E) The lbl-SBMs OrgI-VImut-GFP enhancer derivative with all predicted Org-1 binding sites mutated shows loss of reporter activity in somatic muscle founders (arrowhead) (stage 12, anti-GFP, anti-Lb). (F) lbl-SBMs reporter expression in M8 and lAMPs at stage 15. (G) The lbl-SBMs OrgI-VImut-GFP enhancer construct displays reduced reporter activity in M8 and the lAMPs (stage 15, anti-GFP, anti-TM1). (H) Schematic of the genomic region used for the SK10 (boxed) and SK19 (solid blue) reporter constructs with predicted T-Box binding sites. (I) In a stage 10 embryo, the slou-SK10-lacZ reporter includes activity in the Org-1+ P5/25 progenitors. (J) In org-1 mutant backgrounds, the slou-SK10-lacZ signals are absent in P5/25 progenitors. (K) The slou-SK19-GFP enhancer element is active in Slou+ progenitor cells including the Org-1+ P5/25. (L) The slou-SK19 OrgI-IIImut-GFP reporter with mutated Org-1 binding sites in SK19 shows loss of activity in the Org-1+ progenitors (stage 12, anti-GFP, anti-Org-1). (M) Schematic of the genomic region used for the SK16 reporter construct (purple). Predicted T-Box binding sites are indicated. (N) slou-SK16-lacZ reporter activity in the Org-1+ muscles 5, 25 and 8 (arrowheads) as well as in M18 (plus weak ectopic activity in M21 and M23). (O) In org-1 mutant background, the slou-SK16-lacZ reporter fails to be activated in remnants of M5, M25 and M8 (arrowheads), but remains active in M18, M21 and M23. (P) slou-SK16-GFP reporter activity in Slou+ muscles 5, 25 (arrowheads) and 18 as well as in M8 (arrowhead), M22 and M24. (Q) The slou-SK16 OrgI-VIImut enhancer derivative with mutated Org-1 binding sites fails to activate GFP in muscles 5, 25 and M8 (arrowheads) (stage 15, anti-GFP, anti-TM1) whereas expression in M18, M21 and M23 remains. (R) Anti-Org-1 ChIP was assayed by qPCR using amplicons covering T-Box binding sites (color-coded in A,H,M) and an ey exonic amplicon as negative control. Each bar represents the average of three independent biological replicates normalized to negative controls from the C15 gene (see supplementary material Table S1). The sequence logo shows the Org-1 consensus motif identified by SELEX (H.J. and M.F., unpublished).

Fig. 7.

The Org-1 binding sites in somatic mesodermalladybirdandslouchCRMs are essential for lineage-specific enhancer activity. (A) Schematic of the genomic region used for the generation of the lbl-SBM (boxed, black) and lbl-SBMs (green) reporters. Predicted T-Box binding sites are indicated in detailed view below. (B) lbl-SBM-driven lacZ expression in M8 and lAMPs in stage 15 wild-type embryo. (C) Stage 15 org-1 mutant embryo carrying lbl-SBM-lacZ showing complete absence of β-Gal expression. (D) lbl-SBMs-GFP reporter recapitulates expression of Lb protein in somatic muscle founders (arrowhead). (E) The lbl-SBMs OrgI-VImut-GFP enhancer derivative with all predicted Org-1 binding sites mutated shows loss of reporter activity in somatic muscle founders (arrowhead) (stage 12, anti-GFP, anti-Lb). (F) lbl-SBMs reporter expression in M8 and lAMPs at stage 15. (G) The lbl-SBMs OrgI-VImut-GFP enhancer construct displays reduced reporter activity in M8 and the lAMPs (stage 15, anti-GFP, anti-TM1). (H) Schematic of the genomic region used for the SK10 (boxed) and SK19 (solid blue) reporter constructs with predicted T-Box binding sites. (I) In a stage 10 embryo, the slou-SK10-lacZ reporter includes activity in the Org-1+ P5/25 progenitors. (J) In org-1 mutant backgrounds, the slou-SK10-lacZ signals are absent in P5/25 progenitors. (K) The slou-SK19-GFP enhancer element is active in Slou+ progenitor cells including the Org-1+ P5/25. (L) The slou-SK19 OrgI-IIImut-GFP reporter with mutated Org-1 binding sites in SK19 shows loss of activity in the Org-1+ progenitors (stage 12, anti-GFP, anti-Org-1). (M) Schematic of the genomic region used for the SK16 reporter construct (purple). Predicted T-Box binding sites are indicated. (N) slou-SK16-lacZ reporter activity in the Org-1+ muscles 5, 25 and 8 (arrowheads) as well as in M18 (plus weak ectopic activity in M21 and M23). (O) In org-1 mutant background, the slou-SK16-lacZ reporter fails to be activated in remnants of M5, M25 and M8 (arrowheads), but remains active in M18, M21 and M23. (P) slou-SK16-GFP reporter activity in Slou+ muscles 5, 25 (arrowheads) and 18 as well as in M8 (arrowhead), M22 and M24. (Q) The slou-SK16 OrgI-VIImut enhancer derivative with mutated Org-1 binding sites fails to activate GFP in muscles 5, 25 and M8 (arrowheads) (stage 15, anti-GFP, anti-TM1) whereas expression in M18, M21 and M23 remains. (R) Anti-Org-1 ChIP was assayed by qPCR using amplicons covering T-Box binding sites (color-coded in A,H,M) and an ey exonic amplicon as negative control. Each bar represents the average of three independent biological replicates normalized to negative controls from the C15 gene (see supplementary material Table S1). The sequence logo shows the Org-1 consensus motif identified by SELEX (H.J. and M.F., unpublished).

Fig. 8.

Org-1is a direct regulator ofladybirdandslouchand is required for cell fate specification and differentiation in distinct lateral muscle cell lineages. (A) During early development, cell-specific org-1 expression leads to the direct activation of either slouch or ladybird in the corresponding progenitor cells in combination with mutual repression between slouch and ladybird. (B) After asymmetric cell division, org-1 activity together with slou or lb expression is required for cell fate specification of muscles 5 and 25 (slou) as well as the Lb+ cell lineages. This leads to identity-determining target activation in each founder cell and lAMP specification. (C) Following myoblast fusion, activation of lineage-specific expression profiles in all nuclei of the syncytia induces muscle-specific differentiation events and formation of the mature muscles.

Fig. 8.

Org-1is a direct regulator ofladybirdandslouchand is required for cell fate specification and differentiation in distinct lateral muscle cell lineages. (A) During early development, cell-specific org-1 expression leads to the direct activation of either slouch or ladybird in the corresponding progenitor cells in combination with mutual repression between slouch and ladybird. (B) After asymmetric cell division, org-1 activity together with slou or lb expression is required for cell fate specification of muscles 5 and 25 (slou) as well as the Lb+ cell lineages. This leads to identity-determining target activation in each founder cell and lAMP specification. (C) Following myoblast fusion, activation of lineage-specific expression profiles in all nuclei of the syncytia induces muscle-specific differentiation events and formation of the mature muscles.

Unlike in slou mutants or embryos with ectopic lb expression, in org-1 mutants the muscles derived from the progenitors of M5/25 and M8/lAMP are not transformed into other muscles with recognizable identities. Instead, they form syncytia with variable shapes that do not appear to be properly attached to tendon cells. This phenotype is explained by the requirement of org-1 for the activation of both slou and lb. Whereas in slou mutants the progenitor of M5/M25 expresses ectopic lb, which transforms it into a second progenitor of M8/lAMP (Knirr et al., 1999), in org-1 mutants de-repression of lb is not possible as org-1 is also needed for the activation of lb. Hence, neither of the two progenitors is able to express any of these identity genes that would allow them to form identifiable muscle fibers, although they still divide into founder cells that form syncytia. Only the founder of M25 forms a syncytium that resembles M25 to some extent, perhaps because it still expresses an unknown, org-1-independent identity gene that can establish some of the characteristics of M25 in the absence of slou and org-1 function.

Vertebrate Tbx1 is also expressed prominently in mesodermal areas that form muscles, although in this case not in the trunk region but rather in the core mesoderm of the pharyngeal arches that form branchiomeric muscles of the head (Kelly et al., 2004; Sambasivan et al., 2009). Notably, Tbx1 is also expressed in pharyngeal arch core mesoderm derived from anterior portions of the second heart field, which makes prominent contributions to the outflow tract and right ventricle of the heart (Huynh et al., 2007; reviewed by Vincent and Buckingham, 2010). All of these muscle and heart tissues are disrupted upon mutation of Tbx1 in the mouse and are also affected in patients suffering from the DiGeorge (or velo-cardio-facial) syndrome, in which mutations and deletions cause haploinsufficiency of TBX1 (reviewed by Baldini, 2004). We do not see any obvious connections between Drosophila org-1 function and the cardiogenic roles of vertebrate Tbx1, as org-1-expressing cells do not contribute to the dorsal vessel proper and the org-1+ alary muscles are specialized skeletal muscle fibers rather than genuine cardiac muscles. However, conceivably some of the regulatory interactions upstream or downstream of Tbx1/org-1 during the development of vertebrate head muscles and Drosophila trunk muscles have been conserved. This could, for example, be the case during tongue muscle development, in which the lb ortholog Lbx1 is co-expressed with Tbx1 (Brohmann et al., 2000; Zoupa et al., 2006; Shih et al., 2008). The identification of larger numbers of upstream regulators and targets of both org-1 and Tbx1 will be required in order to obtain a clearer picture of the evolutionary conservation and divergence of the developmental functions of these genes.

Acknowledgements

We thank Hsiu-Hsiang Lee for making org-1 cDNA and UAS-org-1,Juan Modolell for caup cDNA, and Katrin Domsch for injection assistance. We are grateful for receiving fly stocks from Alan Michelson and the Bloomington Stock Center (Indiana, USA), and antibodies from Krzyzstof Jagla, Renate Renkawitz-Pohl, Rob White and the Developmental Studies Hybridoma Bank (University of Iowa, USA).

Funding

This work was funded by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) [DK59406 to M.F.]; the National Institute of Child Health and Human Development (NICHD) [HD30832 to M.F.]; and Deutsche Forschungsgemeinschaft (DFG) [FR696/3-1 to M.F.]. Deposited in PMC for release after 12 months.

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Competing interests statement

The authors declare no competing financial interests.

Supplementary information