Intrinsic control of muscle attachment sites matching

Each muscle in the body has a unique size, shape and set of attachment points. Animals need all of their muscles to have the correct identity to help maintain posture and control movement. A specific set of proteins, called transcription factors, co-ordinate and regulate gene activity in cells so that each muscle develops in the right way.

To create a muscle, multiple precursor cells fuse together to form a muscle fibre, which then elongates and attaches to specific sites. Correct attachment is critical so that the fibre is properly oriented. When this process goes wrong, for example in disease, muscle fibres sometimes attach to the wrong site; they become branched and cannot work properly.

Collier is a transcription factor protein that controls muscle identity in the fruit fly Drosophila melanogaster. However, like many transcription factors, Collier also has several other roles throughout the body. This made it difficult to evaluate the effect of the protein on the formation of specific muscles.

Here, Carayon et al. managed to selectively deactivate Collier in just one muscle per body section in the larvae of fruit flies. This showed that the transcription factor is needed throughout muscle development; in particular, it is required for muscle fibres to select the correct attachment sites, and to be properly oriented. Affected muscles showed an altered orientation, with branched fibres attaching to the wrong site. Even minor changes, which only affect a single muscle from each body segment, greatly impaired the movement of the larvae.

The work by Carayon et al. offers a new approach to the study of muscular conditions. Branched muscles are seen in severe human illnesses such as Duchenne muscular dystrophy. Studying the impact of these changes in a living animal could help to understand how this disease progress, and how it can be prevented.

The musculature of each animal is composed of an array of body wall muscles allowing precision and stereotypy of movements. The somatic musculature of the Drosophila larva - about 30 distinct body wall muscles per each hemi-segment which are distributed in three layers, internal, median and external - is a model to study how a muscle pattern is specified and linked to locomotion behaviour. Each muscle is a single multinucleated fibre with a specific identity: size, shape, orientation relative to the dorso-ventral and antero-posterior body axes, motoneuron innervation and attachment sites to the exoskeleton via tendon cells located at specific positions. Intersegmental tendon cells, where a large fraction of muscles is attached, are distributed in three groups, dorsal, lateral and ventral (Bate, 1990; Volk and VijayRaghavan, 1994; Schweitzer et al., 2010; Armand et al., 1994). Internal muscles also attach to muscle(s) in the next segment, forming ‘indirect’ muscle attachment sites (iMAS) (Maartens and Brown, 2015).

Drosophila muscle development proceeds through fusion of a founder myoblast (founder cell, FC) with fusion-competent myoblasts (FCMs) (Deng et al., 2017). FCs originate from asymmetric division of progenitor cells (PCs) themselves selected from equivalence groups of myoblasts, called promuscular clusters (PMCs). Muscle identity ensues from the expression by each PC and FC of a specific combination of identity transcription factors (iTFs) (de Joussineau et al., 2012), established in three steps: First, different iTFs are activated in different PMCs, in response to positional information, and this expression is only maintained in PCs. Second, refinement of the iTF code occurs via cross-regulations between different iTFs in PCs and/or FCs and Notch signalling (Carmena et al., 1998; Carmena et al., 2002; Enriquez et al., 2010). Several PCs can be serially selected from a PMC and give rise to different muscle identities according to birth order, adding a temporal dimension to these regulations. A well-documented example is the distinction between DA2 and DA3 identities (Figure 1A; Dubois et al., 2016; Boukhatmi et al., 2012). Third, transcriptional activation of iTFs in syncytial nuclei after fusion correlates with the activation of identity ‘realisation’ genes acting downstream of some iTFs (Crozatier and Vincent, 1999; Knirr et al., 1999; Bataillé et al., 2010; Bataillé et al., 2017).

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The consequence of specific muscle identity defects on locomotion, a question of prime importance for progress on studying human myopathies which affect subsets of muscles, remains largely to be assessed. Genetically controlled muscle identity changes should, in principle, provide suitable models for studying locomotion deficits linked to muscle imbalance. However, mutations for known Drosophila iTFs are embryonic lethal and/or show pleiotropic phenotypes reflecting their multiple expression sites. Here, we took advantage of our previous characterisation of col expression in a single larval muscle, the DA3 muscle and of the two involved cis-regulatory modules (CRMs), early (E-CRM) and late (L-CRM), to generate muscle-specific mutants and circumvent lethality/pleiotropy of null mutants.

CRM deletions show that E-CRM and L-CRM act redundantly at the PC stage, emphasising that PC is a key stage in specification of muscle identity. L-CRM deletion results into loss of col transcription in DA3 FCs and morphological transformation of the DA3 into a DA2-like muscle. Removal of an auto-regulatory cis-module located in L-CRM specifically abolishes col activation in syncytial nuclei fusing with the DA3 FC. This leads to incomplete DA3 transformations and the formation of bifid/branched muscles of mixed DA3/DA2 morphology. In summary, our data show that i) the FC transcriptional program must be propagated to syncytial nuclei for a muscle to adopt a specific morphology; ii) the precise matching of muscle/muscle attachments over the intersegmental border, which leads to a staggered rows pattern, involves a process of selective adhesion controlled by iTFs; iii) branched muscles affect larval locomotion performance.

Branched muscles are typical of late, severe phases of human Duchenne Muscular Dystrophy (Chan and Head, 2011). Drosophila iTF CRM deletion could be an effective setting for creating muscle-specific transformations and branched muscles, as new paradigms to study myopathies specifically affecting subsets of muscles in humans.

Redundant CRMs at the PC step ensure robustness of iTF transcription col transcription in myogenic lineages is first observed in a dorso-lateral PMC from which are sequentially selected several PCs (Dubois et al., 2016). It is subsequently maintained in two PCs, then one FC, the DA3 FC, and is activated in FCM nuclei recruited into the DA3 growing fibre (Crozatier and Vincent, 1999; Figure 1A). Two col CRMs containing embryonic in vivo binding sites for the master myogenic TFs, Mef-2 and Twist (Twi) (Sandmann et al., 2007; Zinzen et al., 2009) were previously identified, which reproduce this sequence of expression in reporter assays: E-CRM, which is active in the PMC and the DA3/DO5 and DT1/DO3 PCs, and L-CRM which is active in these same 2 PCs, the DA3 FC and DA3 syncytial nuclei (Enriquez et al., 2010; Dubois et al., 2007; Figure 1A; Figure 1—figure supplement 1 and Figure 1—figure supplement 2). With the prospect of using CRM deletions to generate muscle-specific mutants and to exclude the possible existence of additional, redundant enhancers (Cannavò et al., 2016), we conducted a systematic analysis of Gal4 reporter lines (Manning et al., 2012; Kvon et al., 2014) covering 36 kb of the col genomic region. A single reporter, GMR13B08, reproduces PMC Col expression and it overlaps E-CRM, and two reporters, GMR12G07 and GMR12H01, display DA3 expression and they both partly overlap L-CRM (Figure 1B and data not shown), attesting to the existence of only two col muscle CRMs.

The activity of E-CRM and L-CRM at different phases of muscle development raised the question of their respective roles in defining muscle identity. To address this question, we separately deleted each of them from the genome using the CrispR/Cas9 technology. Deletion of a 2,4 kb fragment removed the E-CRM. Two deletions within the L-CRM were generated: deletion of a 1.3 kb fragment removes the entire core region (Dubois et al., 2007) and deletion of a 0.5 kb fragment removes the Col autoregulation site (de Taffin et al., 2015; Figure 1C; Figure 1—figure supplement 1 and Figure 1—figure supplement 2). The corresponding mutant Drosophila strains are designated col^ΔE, col^ΔL1.3 and col^ΔL0.5, respectively. col^ΔL strains are homozygous viable and fertile. col^ΔE strain is homozygous female sterile, a sterility unrelated to col activity, since fertility is restored by placing col^ΔE over a deficiency (Df(2L)BSC429) (abbreviated Df in the rest of the text and figures) removing the entire col locus (data not shown). As a first step to determine the consequences of deleting either E-CRM or L-CRM, we compared col transcription between wt, col¹ (a protein null mutant), col^ΔE and col^ΔL1.3 strains, using an intronic probe to detect nascent transcripts (Figure 1D). In col¹ homozygous mutant embryos, col transcription is detected at the PMC and PC stages and lost at the FC stage, showing the key role of autoregulation in the maintenance of col transcription (Crozatier and Vincent, 1999; de Taffin et al., 2015). In col^ΔE embryos, we found that col is not activated in PMC cells, as could be expected. Yet, col transcription is detected in the DA3/DO5 and DT1/DO3 PCs, showing that inheritance of Col protein synthesised under E-CRM control is not required for activation of col transcription in PCs. A normal pattern is observed in the developing DA3 muscle, at stage 14. Reciprocal to E-CRM deletion, col is transcribed in PMC cells and the DA3/DO5 and DT1/DO3 PCs in col^ΔL1.3 embryos, but no more at stage 14, showing that L-CRM is required for col transcription maintenance.

Both E-CRM and L-CRM activity are detected in PCs. In absence of E-CRM, no Col protein is inherited from the PMC. Therefore, L-CRM activity in PCs cannot be due to col autoregulation. Analysis of new col-lacZ^yi reporter genes (Perry et al., 2010) revealed the existence of a PC-specific CRM located within the −3.3 to −2.3 fragment of L-CRM, i.e., which is separate from the autoregulatory CRM defined by the in vivo Col binding site and contains the in vivo binding sites for Mef2 and Twist (Figure 1C; Figure 1—figure supplement 3; Zinzen et al., 2009). Activity of this PC-specific CRM is transient and lost upon removal of Mef2 and Twist binding sites (Figure 1—figure supplement 3). Overall, we conclude that two CRMs separately drive col transcription in muscle PCs, suggesting that robust iTF expression at the PC stage is critical to confer a muscle its identity.

col CRM deletions lead to muscle transformation and branched muscles

Having deleted separately each col CRM allowed to assess the respective roles of iTF transcription before or after the PC step. To compare the different CRM deletions, we placed each of them over the deficiency Df chromosome. We introduced the L-CRM-moeGFP reporter to visualise the DA3 morphology at stage 15 (Enriquez et al., 2012). Control (+/Df) embryos display reporter expression in the DA3/DO5 and DT1/DO3 PCs at stage 11 and the DA3 muscle at stage 15 (Figure 2A). The same pattern is observed in PCs for all col-CRM deletion strains, consistent with transcript analyses (Figure 1D). L-CRM-moeGFP expression at stage 15 shows that the DA3 morphology is normal in about 80% of segments in col^ΔE/Df embryos (Figure 2A–B), and we did not pursue the analysis of this deletion strain. Low level GFP expression in col^ΔL1.3/Df embryos, consistent with col transcription data (Figure 1D), shows that the DA3 muscle is most often (85.2% of segments) transformed into a DA2-like muscle (designated below as DA3^>DA2; Figure 2A–B), like in col null mutant embryos (Enriquez et al., 2012). In col^ΔL0.5/Df embryos, i.e, when only the autoregulation module has been deleted, a high number (29%) of branched muscles is observed (Figure 2A–B). Branched muscles correspond to incomplete transformations, with two stable anterior attachment sites, overlapping the DA3 and DA2 sites in wt embryos. The high ratios of either complete (DA3^>DA2) or incomplete (branched) transformations in L-CRM deletion mutants demonstrate that an iTF CRM deletion strategy is effective for creating viable muscle-specific identity mutants and explore branched muscle properties.

Figure 2

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Re-programming of syncytial nuclei is required for muscle morphological identity

Complete vs incomplete transformations in col^ΔL1.3 versus col^ΔL0.5 deletions suggest that proper level and/or maintenance of iTF expression is crucial for proper muscle development. This led us to compare the pattern of Col protein in growing DA3 syncytium between wt, col^ΔL1.3 and col^ΔL0.5 embryos. In either deletion strain, Col is detected in PCs at stage 11 but not in muscles at stage 15 (Figure 3A). However, at stage 14, some Col protein is still detected in muscle precursors in col^ΔL0.5, not in col^ΔL1.3 embryos (Figure 3B). To trace the origin of this difference, we examined col transcription in the DA3 PC, FC and stage 14 syncytium using Df/hemizygous embryos which display one hybridisation dot per active nucleus (Figure 3C). In control wt/Df embryos, a dot is systematically detected in the DA3/DO5 PC (20/20 segments; five embryos analysed), the DA3 FC (20/20) and 80% of DA3 nuclei at stage 14 (6 of 7–8 nuclei per fibre on average; 27 segments). A dot is detected as well in the DA3/DO5 PC, in either col^ΔL1.3 (21/21) or col^ΔL0.5 (18/18) embryos, reflecting E-CRM activity (Figure 1D). In col^ΔL0.5 embryos, a col hybridisation dot is detected in the DA3 FC (19/19) and in one nucleus, likely the FC nucleus (11/21 segments), sometimes two nuclei at stage 14. In col^ΔL1.3 embryos, however, col transcription is only detected in a minor fraction of FCs (4/15) and is completely lost at stage 14 (0/6–7 nuclei per fibre on average; 24 segments). Patterns similar to stage 14 are observed at stage 15, while at stage 16, col transcription is detected neither in L-CRM deletion strains nor in control (Figure 3—figure supplement 1). Since col transcription at, and from, the PC stage appears to be nodal to DA3 identity, we measured the level of col transcripts relative to nautilus (nau), the Drosophila MyoD-MRF serving as internal reference (Figure 3D and Figure 3—figure supplement 2). As expected, similar levels of col transcription are found in the DA3 PC, in control (Mean ± sem: 1.22 ± 0.06; n = 18), col^ΔL1.3 (1.15 ± 0.04; n = 26) and col^ΔL0.5 embryos (1.19 ± 0.04; n = 18), which confirms handling by the E-CRM, with only a minor contribution from the L-CRM (Figure 1D). On the contrary, high level of col transcription in the DA3 FC in wt embryos (2.20 ± 0.05; n = 28) is dependent upon L-CRM activity, since a drop is observed in col^ΔL1.3 FCs (1.10 ± 0.04; n = 24), p<0.001. More precisely, it is dependent upon the presence of Mef2 and Twist binding sites since a basal level of transcription is still observed when only the col autoregulation module is deleted (col^ΔL0.5: 1.41 ± 0.04; n = 25). Quantification of col and nau transcripts shows that the col/nau increase between the PC and FC stages in wt embryo is due to increased col transcription while the level of nau is relatively constant and is unaffected in col L-CRM mutants (Figure 3—figure supplement 2). Together, the data suggest that binding of Mef2 and Twist is required to prime col transcription in the FC nucleus before autoregulation takes off (Figure 3E). Taken with one another, Col immunostaining, FISH data and col transcription quantification indicate that sustained col transcription in the FC nucleus in col^ΔL0.5 embryos (Figure 3C–D), provides enough Col protein for some uptake by other DA3 nuclei at stage 14, and this leads to their partial reprogramming to DA3 identity. Partial reprogramming could, in turn, explain the formation of branched muscles retaining some DA3 morphological characters. Moreover, the col^ΔL1.3 and col^ΔL0.5 expression data and deletion phenotypes show that both iTF transcription in the FC and reprogramming of ‘naïve’ syncytial nuclei contribute to ensure robust muscle morphological identity (Figure 3E).

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Muscle attachment: tendon attraction and muscle-muscle matching

We next investigated in more detail how ectopic muscle attachment sites at the origin of transformed and branched muscles are selected and stabilised. Double staining of ready-to-hatch (st17) wt embryos for F-actin and Ilk-GFP, a component of muscle attachment sites (Zervas et al., 2011; Sarov et al., 2016) shows that the DA3 posterior edge anchors to dorsal, and anterior edge to lateral intersegmental tendon cells (Figure 4A), giving its final acute shape (Bate, 1990). Moreover, the DA3 and DA2, as well as the DA2 and DA1 muscles precisely align with each other over each intersegmental border, forming heterotypic muscle-muscle attachment (iMAS; [Maartens and Brown, 2015]) at the origin of the staggered rows disposition of DA muscles. No DA3/DA3 (homotypic) iMAS surface is observed (Figure 4A). On the contrary, in col^ΔL1.3 embryos, the anterior edge of DA3^>DA2 transformed muscles anchors to dorsal tendon cells instead of lateral intersegmental tendon cells, leading to a ‘dual DA3^>DA2 morphology’, DA2-like at the anterior, and DA3 at the posterior edge. This dual identity leads to iMASs between adjacent DA3^>DA2 muscles (Figure 4A). As a consequence, DA2 iMASs are shifted dorsally and the general pattern of DA muscles is affected.

Figure 4

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To explore the dynamics of DA3 muscle attachment, we live-imaged wt and col^ΔL1.3 embryos, starting at stage 12 (defined as t₀ in Videos 1 and 2). The DA3 muscle is visualised by L-CRM-moeGFP and tendon cell along the entire intersegmental border by stripe Gal4;UASmCD8RFP (Volohonsky et al., 2007). In wt embryos between stages 12 and 13 (Video 1; Figure 4B), the DA3 muscle precursor extends protrusions towards dorsal tendon cells, posteriorly, and both dorsal and lateral tendon cells, anteriorly, diverging from a bipolar extension scheme (Schnorrer and Dickson, 2004). At stage 14, the posterior DA3 edge makes contacts with dorsal tendon cells before the anterior edge(s) reaches the intersegmental border, a time gap previously observed during live-imaging of a ventro-lateral muscle (Gilsohn and Volk, 2010). In addition, numerous filopodia emanate from the DA3 dorsal surface and contact the DA2 ventral surface, contributing to give the DA3 muscle precursor its transient angled-shape. At late stage 14, the posterior DA3 attachment widens parallel to the intersegmental border. Its anterior attachment to lateral tendon cells in turn becomes stable, whereas dorsal anterior filipodia appear to be repelled from the intersegmental border where the DA3 muscle in the preceding segment is attached. Remaining filopodia are limited to the rims of DA3 anchoring, possibly suggesting a mechanism of homotypic repulsion. In col^ΔL1.3 embryos, the main axis of the DA3^>DA2 muscle precursor elongation is more longitudinal than wt, already at stage 12 (Video 2; Figure 4B). At stage 13 and like in wt, many protrusions emanate from the DA3 dorsal surface, but unlike in wt, protrusions contacting dorsal tendon cells do not retract at later stages when they contact the DA3^>DA2 muscle from the preceding segment. Rather, stabilisation of contacts made by these protrusions prefigures abnormal DA3^>DA2/DA3^>DA2 iMAS formation (Figure 4A). In some segments, contacts with lateral tendon cells are also stabilised, resulting into branched muscles. In summary, imaging of live wt embryos illustrates different steps involved in establishment of the acute DA3 orientation: posterior attachment to dorsal tendon cells at the same time as anterior exploration of dorsal and lateral tendon cell. This is followed by DA3 attachment to lateral, and retraction from dorsal tendon cells (Figure 4B, Video 1), cumulating into heterotypic DA3/DA2 iMAS stabilisation (Figure 4A and B). This retraction does not take place in col^ΔL1.3 embryos (Figure 4B and Video 2).

Video 1

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Video 2

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Muscle attachment: heterotypic adhesion

Further determining how DA3^>DA2/DA3^>DA2 homotypic attachment could interfere with the staggered rows pattern of DA muscles, required to visualise at the same time the DA3 and DA2 muscles and tendon cells contours. Vestigial (Vg) is expressed in DA muscles (Deng et al., 2010; Tixier et al., 2010). We screened the vg regulatory landscape and characterised one vg CRM active in the DA3 and DA2 (and VL1) muscles, VgM1 (Figure 5—figure supplement 1). Expressing together VgM1-mCD8GFP-H2bRFP, L-CRM-moeGFP and stripeGal4:UAS-mCD8RFP, and co-staining of stage 16 control embryos for α-Spectrin, a protein enriched at muscle attachment sites, shows that the posterior edge of DA3 precisely aligns with the anterior edge of DA2 (Figure 5A, Figure 5—figure supplement 1), suggesting heterotypic attractive cues. This attraction is already observed at stage 14, when the dorsal DA3 and ventral DA2 surfaces contact each other via numerous protrusions (Figure 5A and B; Figure 5—figure supplement 2). Strikingly, at stage 15, the contact zone gets away from intersegmental borders and the DA3/DA3 and DA2/DA2 homotypic connections disappear, to give way to heterotypic DA3/DA2 stable iMAS formation. In col^ΔL1.3 embryos, the initial steps, stages 14 and 15, are similar to wt, except for the absence of stable connection of DA3^>DA2 to lateral tendon cells, as seen by live imaging (Video 2). At stage 16, DA3^>DA2/DA3^>DA2 and DA3^>DA2/DA2 iMASs are privileged, while similar to control, no homotypic DA2/DA2 iMAS forms, suggesting homotypic repulsion (Figure 4; Figure 5A). Together, the data indicate that the precise pattern of iMASs and muscles is contributed by a combination of attractive and, possibly, repulsive cues downstream of muscle iTFs (Figure 5B).

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Muscle strength lines in larvae

To investigate the impact of embryonic muscle patterning defects on Drosophila larval crawling, we first recorded the fraction of DA3, DA3^>DA2 and branched muscles in 3^rd instar wt and col^ΔLCRM larvae expressing GFP under control of the Myosin heavy chain (Mhc) promotor region (Figure 6A). It conforms to the statistics in late embryos (Figure 2B), except for an increased proportion of branched muscles, which could reflect under-evaluation of their number in embryos, due to threshold detection limit of L-CRM-moeGFP expression in thin fibres (Figure 6B). We then examined the pattern of MASs, using scanning electron microscopy (SEM) of dissected larval filets (Figure 6C; Figure 6—figure supplement 1). In wt larvae, as seen in stage 17 embryos (Figure 4A), the anterior edge of the DA3 muscle is anchored to a nodal lateral attachment site shared with the LL1 muscle, while its posterior edge aligns with the anterior edge of the DA2 muscle in the next posterior segment. Precise heterotypic DA2/DA1 iMASs are also visible over each intersegmental border. The regular alignment of the DA1, DA2 and DA3 muscles both draws strength lines spanning three adjacent segments, and regular tension surfaces between segments (Figure 6C; Figure 6—figure supplement 1). In col^ΔL1.3 larvae, the anterior DA3 muscle attachment has shifted from lateral to dorsal to form a DA3^>DA2/DA3^>DA2 homotypic iMAS (Figure 6C). This leads in turn to the formation of narrow, ectopic DA2/DA2 contacts and the DA1-DA2-DA3 strength line is distorted (Figure 6—figure supplement 1). In case of branched DA3 muscles, two strength lines co-exist (Figure 6C and Figure 6—figure supplement 1). In conclusion, SEM analyses of larval muscles show that loss of DA3 identity leads to ectopic iMASs between several dorsal internal muscles, distortion of the staggered ends architecture of DA muscles and of their alignment between consecutive segments.

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Branched muscles result in subtle locomotion defects

Drosophila larval crawling relies upon ordered abdominal body wall muscle contractions (Heckscher et al., 2012). The distorted muscle patterns observed in col^ΔL1.3 larvae raised the question of whether it impacted on locomotion. To address this question, we compared the locomotion of +/Df, col^ΔL1.3/Df and col^ΔL0.5/Df larvae using FIM (FTIR-based Imaging Method) and the FIMTrack software, which allows tracking simultaneously many larvae and quantitatively describing a variety of stereotypic movements (Risse et al., 2013; Risse et al., 2014; Risse et al., 2017). Here, we focused on three parameters: crawling speed, stride length, stride duration. We first recorded the ‘walking rate’, also called crawling speed, during the first 20 s, after larvae have been dropped on the agarose gel. At that time, larvae engage an ‘escape response’ corresponding to an active crawling phase (Figure 7A). Box-plot graphs (left) show intra-variability for each of the three genotypes. Beyond this variability, we observe, however, that col^ΔL0.5/Df larvae display a significantly reduced crawling speed on average 1.05 ± 0.034 mm/sec (n = 108), compared to 1.15 ± 0.031 mm/sec for +/Df controls (n = 118), (p=0.03) (Figure 7A). To further investigate the origin of this speed reduction, we measured two crawling speed parameters: stride length and stride duration (Figure 7B–C). A significantly shorter stride was measured for col^ΔL0.5/Df larvae (1.08 ± 0.025 mm) compared to control +/Df larvae (1.17 ± 0.024 mm), (p=0.008). Furthermore, stride duration was extended, from (1.09 ± 0.014 s) for +/Df larvae to 1.15 ± 0.017 s for col^ΔL0.5/Df larvae. For both crawling speed, stride length and stride duration, col^ΔL1.3/Df larvae (n = 112) display intermediate values. Yet, differences with either control or col^ΔL1.3/Df larvae fall below the significance threshold level, suggesting that mis-orientation of the DA3^>DA2 muscle does not, by itself, significantly impair the efficiency of segment contraction. Since larval crawling integrates information provided by neuronal networks, relayed by synaptic connections between motoneurons (MNs) and muscles, the mobility phenotype of col^ΔL0.5/Df larvae could indicate defects motor innervation of DA3^>DA2 muscles. The DA3 and DA2 muscles are innervated by the intersegmental nerve (ISN) which fasciculates motor axons reaching dorsal muscles (Hoang and Chiba, 2001; Landgraf and Thor, 2006). To examine the DA3^>DA2 and branched DA3 innervation, we used anti-HRP and phalloidin staining to view ISN motoneuron projections and muscles, respectively (Figure 7—figure supplement 1). In wt embryos, the MN projection which innervates DA3 leaves the ISN ventral to the DA3 position and orients left such that the neuromuscular junction locates to the first anterior third of the muscle (100%; n = 28 segments). In case of a complete DA3^>DA2 transformation, a MN projection is still observed (100%; n = 15 segments), which leaves the ISN at a more dorsal position than wt, reflecting the dorsal shift of the DA3^>DA2, relative to DA3 muscle. In case of branched muscles, the lower branch is always innervated (100%, n = 30 segments). Only in 20% of the cases, the second, upper branch is also innervated.

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These innervation data indicate that fully transformed DA3 muscles are innervated, but only one branch of branched muscles is, in most cases. It remains to be established whether this asymmetric innervation contributes to the reduced crawling speed of col^ΔL0.5 larvae.

The stereotyped set of 30 somatic muscles in each abdominal segment which underlies Drosophila larval crawling has been thoroughly described many years ago (Bate, 1990; Bate and Rushton, 1993). One essential aspect laid out at that time was the concept of founder cell (FC), i.e., the assignment to form a distinctive muscle to a single founder myoblast able to recruit other myoblasts by fusion. The morphological identity of each muscle is foreseen by a specific pattern of iTF expression in its FC (Tixier et al., 2010; Frasch, 1999). Here, we engineered a CRM deletion strategy connecting iTF expression to the larval musculature architecture and locomotion.

All data generated or analysed during this study are included in the manuscript and supporting files.

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Author details

1. Alexandre Carayon

   1. Centre de Biologie du Développement (CBD), Toulouse, France
   2. Centre de Recherche sur la Cognition Animale (CRCA), Toulouse, France
   3. Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, UPS, Toulouse, France

   Contribution

   Data curation, Formal analysis, Methodology

   Competing interests

   No competing interests declared

2. Laetitia Bataillé

   1. Centre de Biologie du Développement (CBD), Toulouse, France
   2. Centre de Recherche sur la Cognition Animale (CRCA), Toulouse, France
   3. Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, UPS, Toulouse, France

   Contribution

   Resources, Data curation, Formal analysis, Methodology

   Competing interests

   No competing interests declared

3. Gaëlle Lebreton

   1. Centre de Biologie du Développement (CBD), Toulouse, France
   2. Centre de Recherche sur la Cognition Animale (CRCA), Toulouse, France
   3. Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, UPS, Toulouse, France

   Contribution

   Resources, Data curation, Formal analysis, Methodology

   Competing interests

   No competing interests declared

4. Laurence Dubois

   1. Centre de Biologie du Développement (CBD), Toulouse, France
   2. Centre de Recherche sur la Cognition Animale (CRCA), Toulouse, France
   3. Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, UPS, Toulouse, France

   Contribution

   Data curation, Formal analysis, Methodology

   Competing interests

   No competing interests declared

5. Aurore Pelletier

   1. Centre de Biologie du Développement (CBD), Toulouse, France
   2. Centre de Recherche sur la Cognition Animale (CRCA), Toulouse, France
   3. Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, UPS, Toulouse, France

   Contribution

   Data curation, Formal analysis, Methodology

   Competing interests

   No competing interests declared

6. Yannick Carrier

   1. Centre de Biologie du Développement (CBD), Toulouse, France
   2. Centre de Recherche sur la Cognition Animale (CRCA), Toulouse, France
   3. Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, UPS, Toulouse, France

   Contribution

   Resources, Data curation, Methodology

   Competing interests

   No competing interests declared

7. Antoine Wystrach

   1. Centre de Recherche sur la Cognition Animale (CRCA), Toulouse, France
   2. Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, UPS, Toulouse, France

   Contribution

   Resources, Software, Formal analysis

   Competing interests

   No competing interests declared

8. Alain Vincent

   1. Centre de Biologie du Développement (CBD), Toulouse, France
   2. Centre de Recherche sur la Cognition Animale (CRCA), Toulouse, France
   3. Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, UPS, Toulouse, France

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Validation, Investigation, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2769-7501

9. Jean-Louis Frendo

   1. Centre de Biologie du Développement (CBD), Toulouse, France
   2. Centre de Recherche sur la Cognition Animale (CRCA), Toulouse, France
   3. Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, UPS, Toulouse, France

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Methodology, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   jean-louis.frendo@univ-tlse3.fr

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0118-5556

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