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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Acceptance summary:

The authors study the gene encoding a muscle identity transcription factor, Collier, through examination of its cis-regulatory modules. They demonstrate how selection of attachment sites in muscle and locomotion are related to muscle identity and through transcriptional reprogramming of syncytial nuclei.

Decision letter after peer review:

[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]

Thank you for submitting your work entitled "Intrinsic control of muscle attachment sites matching" for consideration by eLife. Your article has been reviewed by a Senior Editor, and three reviewers. The reviewers have opted to remain anonymous.

Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.

We do appreciate that some of the results are interesting and novel. However, we are not persuaded that they are a significant advance and of sufficient interest for eLife readers at this stage. Also, the manuscript should be re-written so that the conclusions are not over-interpreted. In addition, quantification of the FISH analysis must be provided and compared to the protein distribution and intensity. These are amongst the important experiments suggested below that would make the study more widely relevant. We feel that these could take well over two months to do well. We will be pleased to consider a fresh submission, in due course, that addresses all reviewers' concerns, should the authors wish to consider eLife.

Reviewer #1:

The manuscript by Carayon et al. addresses an intriguing question of whether the acquisition of muscle identity could impact muscle function and mobility of an organism. To tackle this they generate a series of regulatory region deficiencies that affect transcription of Drosophila muscle iTF col required for the formation and properties of DA3 muscle. In particular, they generate two viable col regulatory regions mutant lines: one in which a complete DA3 to DA2 transformation takes place and another line with deletion of col autoregulatory region in which DA3 is not fully transformed to DA2 and has branched morphology.

Interestingly, mutant larvae with branched DA3 muscles display several mobility defects demonstrating that inappropriate acquisition of identity of one muscle could have a deleterious systemic effect. From this perspective, it is a valuable and novel work that merits to be published in eLife.

There are a few aspects that require some clarifications:

1) Is the problem of identity propagation observed at stage 14 in col^ΔL0.5 context also seen at later stages in DA3 muscle? In other words, is it only a delay in switching on col or permanent incapacity to make fused nuclei transcriptionally active for col? FISH experiment with intronic col probe on stage 16 embryos would help to clarify this point.

2) Some fraction of DA3 muscles in col^ΔL0.5 embryos does not display branching phenotype. Could this normal DA3 morphology be associated with a partial or total identity propagation? Could authors document such cases? This would further strengthen the link between identity propagation and branching.

3) Why one nucleus is still able to transcribe col and produce Col protein in the context autoregulation is missing. Also, is Col protein expression level in the col-positive nucleus of col^ΔL0.5 DA3 similar to that in wt DA3?

4) Authors show that larvae with branched DA3 move slower than larvae with DA3 fully transformed to DA2 indicating that muscle branching has more impact on muscle function than muscle transformation. This could be the case, however other muscle properties and in particular muscle innervation could contribute to mobility phenotype. Some views showing how branched versus fully transformed DA3 muscles are innervated would be of help.

Reviewer #2:

The manuscript of Carayon et al. investigates how the identity of a muscle cell shapes the choice and structure of muscle attachment to its tendon. Previous work from the group has identified specific muscle enhancers in the identity gene collier that controls its expression during the process of founder specification and muscle morphogenesis. This new manuscript adds to this body of work by making specific genomic deletions of these enhancers via Crispr technology. The group subsequently analyzes the phenotypes that result from the selective removal of these enhancers, through confocal imaging of both fixed and live Drosophila samples, by SEM and by locomotion assays. These new reagents allow the investigators to map the changes in the patterns of collier expression, assess the contribution of each enhancer to collier expression, and assess one important aspect of muscle identity – the attachment to the tendon cells. Of the key conclusions, they find that (1) as expected from previously published work in the field, FC identity controls selection and development of muscle attachment to the tendon; (2) the robustness of the muscle pattern depends on the reprogramming of myonuclei after fusion (as suggested by earlier published work); (3) the morphogenesis of the tendon-muscle interaction requires several steps in the process, that when altered, leads to the development of branched muscles; and (4) these alterations in muscle attachment (branched muscles) can lead to defects in muscle activity as measured by locomotion of the organism.

Overall, this work is solid. However, the issue for me becomes whether this work provides significant enough advancement for the muscle biology field to merit publication in eLife. Unfortunately, I believe that this manuscript would be better suited for a more specialized journal. To merit consideration in eLife, one would want to see, for example, how collier regulates the dynamic attachment process that is described.

Reviewer #3:

In this manuscript, Alexander Carayon et al. analyze the consequences of deletion of transcription cis-regulatory modules of a single muscle identity Transcription Factor (iTF), Collier (Col), which in Drosophila embryo regulates the identity of muscle DA3. Previously the authors characterized 3 cis-regulatory regions of the Col gene, responsible for early, late, as well as autoregulatory transcriptional control of Col gene in DA3 muscle. They now have deleted each of these sites and ask how it affects Col transcription in the DA3 muscle, and what would be the outcome in terms of muscle morphology, attachment, and larval movement. They show that the CRM for late expression of Col (and to some extent the autoregulatory CRM) are essential for providing the DA3 identity since in its absence, this muscle is either abnormal or partially transformed into DA2 muscle. Although the contribution of Col to the identity of the DA3 had been reported previously, the present study adds important information regarding the functional contribution of the different Col regulatory regions to the muscle identity, attachment sites, and overall larval movement. These results are clean and convincing.

Essential revisions:

1) Overinterpretation of the data: in the abstract, the authors write that " We show that both selection of muscle attachment sites and muscle/muscle matching is intrinsic to muscle identity and requires transcriptional reprogramming of syncytial nuclei". Whereas they indeed show that selection of muscle attachment sites is intrinsic to muscle identity, there is no direct evidence that it requires transcriptional reprogramming of syncytial nuclei.

2) The Col protein, as well as FISH analysis in the col^ΔL1.3 or col^ΔL0.5 are both intriguing because they indicate that Col transcription is retained in a single nucleus (presumably the founder nucleus) within the entire syncytium of DA3. Do the authors have an explanation as to why the Col protein, being produced in the syncytial cytoplasm, does not translocate to the neighboring nuclei?

3) It will be also informative to demonstrate where this nucleus resides relative to the other nuclei, and whether this impacts on the outcome in terms of the observed attachment sites. In Figure 3, the authors should show the other nuclei of the DA3 muscle in each of the mutants. This can be easily achieved by co-staining the muscles with GFP-NLS driven by col-Gal4 driver.

4) Why the transformation of DA3 to DA2 is relatively rare? Could it be that additional iTFs exist in the DA3 muscle?

5) Do the authors have any idea as to why the col^ΔECRM is homozygous female sterile. Is it due to an off-target effect?

6) Quantification of the FISH: it would be important to add quantification data as to the reduction in the col transcripts in the mutants shown in Figure 3.

7) The authors should explain better what they mean by homotypic repulsion.

8) Did the authors check that muscle genes, such as MHC, TpnC, actin etc, are expressed normally and in the mutant DA3 muscle, and that its innervation occurs ordinarily? Can they rule out that the muscle does not function due to physiological reasons, rather than the aberrant attachments?

[Editors’ note: further revisions were suggested prior to acceptance, as described below.]

Thank you for re-submitting your article "Intrinsic control of muscle attachment sites matching" for consideration by eLife. Your article has been reviewed K VijayRaghavan as the Senior Editor, and two reviewers. The reviewers have opted to remain anonymous.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, when editors judge that a submitted work as a whole belongs in eLife but that some conclusions require a modest amount of additional new data, as they do with your paper, we are asking that the manuscript be revised to either limit claims to those supported by data in hand, or to explicitly state that the relevant conclusions require additional supporting data.

Our expectation is that the authors will eventually carry out the additional experiments and report on how they affect the relevant conclusions either in a preprint on bioRxiv or medRxiv, or if appropriate, as a Research Advance in eLife, either of which would be linked to the original paper.

Summary:

Carayon et al. describe an interesting functional analysis of distinct cis-regulatory modules (CRMs) controlling the expression of the collier (col) gene in a single DA3 muscle. Previous analysis from the Vincent lab identified a critical role for col in providing the identity of the DA3 muscle. In the present study, the authors analyzed the functional contribution of specific CRMs for the temporal col expression and function, in the DA3 muscle, following their specific deletion. The analysis revealed that deleting the Late CRM (L-CRM, col^ΔL), which contains both Mef2 and Twi binding sites, as well as an autoregulatory Col binding sequences, led to the transformation of the DA3 into the DA2 muscle. Deleting a smaller region within this sequence responsible only for the autoregulatory Col activity led to abnormal split muscle DA3. The authors show that whereas in col^ΔL DA3 muscle the entire transcription of col is undetectable at stage 14, in fused multinucleated DA3 muscle, the deletion of only the auto-regulatory Col sequences led to partial col transcription. Further image analysis of live embryos indicated the sequence of events leading to the split DA3 phenotype. The authors speculate that Col late expression in all the nuclei of DA3 is required for proper detachment of DA3 from the DA2 muscle at its dorsal site and further attraction to the LL1 attachment site. Additional analysis was performed to extract the crawling behavior of the mutant larvae. This analysis implied that the transformation of DA3 to DA2 did not affect significantly the larval step length, however, larvae with split DA3 did show a change in stride length, which was also correlated with aberrant innervation of the split muscle.

Essential revisions:

Overall, the results regarding the functional contribution of the distinct CRMs are convincing and nicely presented. However, the conclusions regarding muscle-muscle repulsion and the model explaining the sequence of events leading to DA3 transformation are less convincing. The link to larvae crawling behaviour is also puzzling due to the stronger phenotype obtained by partial deletion of Col, relative to its complete deletion, and also due to the effect on muscle innervation. These could be discussed in the 'Discussion' section.

1) Figure 2B – the numbers of embryos analyzed is not indicated.

2) Figure 3B – in col^ΔL0.5 st 14 there are extra nuclei labeled below the DA3 muscle. These are not stained in the control. What are these?

3) Figure 3C – why does Col protein not label all nuclei in the control DA3? Why is the green label stronger in col^ΔL0.5 relative to col^ΔL1.3.

4) Figure 3D – The quantification of transcription at stage 14 is missing. We are puzzled by the question of why late CRM is not active when the autoregulatory is missing. Mef2 should be still active in all nuclei, should it not?

5) Figure 3D – there is no information of how the fluorescence quantification was done. Did the authors perform background subtraction? Did the authors check whether nau transcription is affected or not by col depletion? They should provide images and quantification of nau FISH as well or give clear reasons of why these could be future work if they are unable to do this now.

6) Figure 5 – The authors describe numerous membrane protrusions, which we do not see in the image provided, and no quantification of these is described. No information regarding homotypic DA3/DA3 and DA2/DA2 is provided. Are these real homotypic adhesions? Again, please give clear reasons for why these could be future work if they are unable to do further experiments now.

7) Figure 5 – The images are very nice, however, we do not know about repulsion or attraction. It is speculation. There could be other explanations. Please tone down and discuss various possibilities

The suggested experiments can be done speedily, assuming that the FISH data are already available. In addition, they should tone down the sentences regarding attraction/repulsion because they do not bring strong evidence for that, other than the live imaging, which is nice but descriptive.

[Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]

Reviewer #1:

[…]

There are a few aspects that require some clarifications:

1) Is the problem of identity propagation observed at stage 14 in col^ΔL0.5 context also seen at later stages in DA3 muscle? In other words, is it only a delay in switching on col or permanent incapacity to make fused nuclei transcriptionally active for col? FISH experiment with intronic col probe on stage 16 embryos would help to clarify this point.

The suggested experiment has now been done and the results are presented in a supplementary figure (new Figure 3—figure supplement 1) and recorded in Results section subsection “(Re)programming of syncytial nuclei is required for muscle morphological identity”.

Briefly, in wt embryos, col transcription in all nuclei of the DA3 muscle is switched off at stage 16. The same complete switch off is observed in col^ΔL0.5 embryos, showing that there is no delay in activation of col transcription in low Col level conditions, but rather a low number of fused nuclei which is transcriptionally reprogrammed to DA3 identity.

2) Some fraction of DA3 muscles in col^ΔL0.5 embryos does not display branching phenotype. Could this normal DA3 morphology be associated with a partial or total identity propagation? Could authors document such cases? This would further strengthen the link between identity propagation and branching.

We agree with the reviewer that normal DA3 morphology observed in col^ΔL0.5 is likely associated with at least partial identity propagation. A proxy is L-CRM-moeGFP expression (Figure 2A), which depends upon Col levels through its autoregulation site. To more directly link DA3 nuclei reprogramming to normal versus branched DA3 formation would require visualization of col transcription when selected muscle attachment sites have been stabilized (see Figure 4B). As discussed above, col transcription has already ceased at that stage, precluding this experiment.

3) Why one nucleus is still able to transcribe col and produce Col protein in the context autoregulation is missing. Also, is Col protein expression level in the col-positive nucleus of col^ΔL0.5 DA3 similar to that in wt DA3?

The question of the transcriptional outcome of the FC relative to final muscle identity is indeed a key question. To address this question, we have chosen to quantify the col transcription rather than Col protein levels, using stellaris single RNA probes, because of higher accuracy. Results, now presented in new Figure 3D and in subsection “(Re)programming of syncytial nuclei is required for muscle morphological identity” show that the level of col transcription in the DA3 FC is nodal for transcriptional reprogramming of syncytial nuclei and proper muscle identity. This is an important addition to the previous version; thanks to the reviewer.

4) Authors show that larvae with branched DA3 move slower than larvae with DA3 fully transformed to DA2 indicating that muscle branching has more impact on muscle function than muscle transformation. This could be the case, however other muscle properties and in particular muscle innervation could contribute to mobility phenotype. Some views showing how branched versus fully transformed DA3 muscles are innervated would be of help.

We thank the reviewer again for their suggestion. Labelling motoneuron axons using anti-HRP staining, shows that fully transformed DA3^>DA2 muscles are innervated. However, in most cases, only one branch of branched muscles is (new Figure 7—figure supplement 1). This innervation defect could, in part, explain the impaired locomotion specifically observed in larvae with numerous branched muscles, as now stated in subsection “Branched muscles result in subtle locomotion defects”.

Reviewer #2:

[…]

Overall, this work is solid. However, the issue for me becomes whether this work provides significant enough advancement for the muscle biology field to merit publication in eLife. Unfortunately, I believe that this manuscript would be better suited for a more specialized journal. To merit consideration in eLife, one would want to see, for example, how collier regulates the dynamic attachment process that is described.

How Collier regulates DA3 muscle attachment and identifying Col targets in this process is certainly a priority next step. We already tested several candidate genes, either via phenotypic analyses (e.g., slit), or expression analyses (e.g., members of the Dip and Dpr families; Orban et al., 2013; Tan et al., 2015), focusing on Col direct targets (de Taffin et al., 2015) and their proposed partners, but without success. There are hundreds of extracellular protein variants and this is a long-standing investment. The syncytial nature of muscles when the attachment process takes place and dynamic reprogramming of syncytial nuclei has been, so far, an obstacle to transcriptome and ChIP-seq analyses of single muscle, in any organism. New toolsare being developed in our lab to profile in parallel several dorsal muscles in wt and mutant embryos, but we feel that this goal is beyond the scope of our present analysis which connects iTF CRM regulation and locomotion.

Our detailed analysis of DA3 muscle morphological development, both by live-imaging with a dedicated reporter and on fixed embryos and larvae (modified Figure 4, Figure 5 and Figure 6), provides a novel, unprecedented view of the muscle attachment site selection and attachment process (modified scheme Figure 5B). Data in the previous version suggested, for the first time, that homotypic repulsion and heterotypic attraction are involved in precise muscle-muscle attachments leading to the exquisite staggered muscles pattern. In the present manuscript, we visualized both the DA2 and DA3 muscles in wt and mutant embryos using a newly identified driver (new Figure 5A) and the data fully support our previous conclusions.

Reviewer #3:

[…]

Essential revisions:

1) Overinterpretation of the data: in the abstract, the authors write that " We show that both selection of muscle attachment sites and muscle/muscle matching is intrinsic to muscle identity and requires transcriptional reprogramming of syncytial nuclei". Whereas they indeed show that selection of muscle attachment sites is intrinsic to muscle identity, there is no direct evidence that it requires transcriptional reprogramming of syncytial nuclei.

We agree with the reviewer that there is only indirect evidence that muscle identity requires transcriptional reprogramming of syncytial nuclei. We toned down the sentence in the Abstract and modified highlight 2, accordingly.

2) The Col protein, as well as FISH analysis in the col^ΔL1.3 or col^ΔL0.5 are both intriguing because they indicate that Col transcription is retained in a single nucleus (presumably the founder nucleus) within the entire syncytium of DA3. Do the authors have an explanation as to why the Col protein, being produced in the syncytial cytoplasm, does not translocate to the neighboring nuclei?

Immunostainings show thepresence of more Col protein in syncytial nuclei in col^ΔL0.5 than col^ΔL1.3 mutants (Figure 3B), and this correlates with the severity of the phenotype, branched muscle versus DA3^>DA2. We also found this result puzzling. We have now measured the level of col transcription in the DA3 FC in wt and each CRM mutant (new Figure 3D). It shows that this level is significantly lower than wt in both col^ΔL0.5 and col^ΔL1.3 mutants, at stage 12, when there is a single nucleus. Beyond the lower level in col^ΔL1.3 than col^ΔL0.5 at that stage, what differs between these two mutant strains is the maintenance of col transcription in the syncytial muscle precursor, which is only observed in col^ΔL0.5 mutants (Figure 3C). We infer from these different results that the uptake of Col protein by newly fused nuclei reflects the level of newly synthesized Col protein from the FC. One hypothesis is that, in conditions of low transcription/low translation, uptake is mainly limited to the FC nucleus, because of diffusion rate of the protein. In both col^ΔL0.5 and col^ΔL1.3 mutants, and unlike in wt, there is no other source of Col protein than the FC because of the deletion of the Col autoregulation site. We have now revised the text (subsection “(Re)programming of syncytial nuclei is required for muscle morphological identity”) to make this point clearer.

3) It will be also informative to demonstrate where this nucleus resides relative to the other nuclei, and whether this impacts on the outcome in terms of the observed attachment sites. In Figure 3, the authors should show the other nuclei of the DA3 muscle in each of the mutants. This can be easily achieved by co-staining the muscles with GFP-NLS driven by col-Gal4 driver.

This is an interesting question, which we are presently unable to answer. Previous studies of col and other genes transcription in the DA3 muscle have led to the conclusion that the FC nucleus occupies a specific, central position at stages 14 and 15, when the muscle is angled shape (Bataillé et al., 2017). The cease of transcription did, however, not allow determining its position at stage 16. The absence of col transcription, after stage 12 makes impossible to determine whether the FC nucleus adopts a different position in col^ΔL1.3 mutants and no reporter we have tested so far allows to specifically track the DA3 FC nucleus in the DA3 syncytium.

4) Why the transformation of DA3 to DA2 is relatively rare? Could it be that additional iTFs exist in the DA3 muscle?

We do not feel that the transformation of DA3 in DA2 muscle in col^ΔL1.3 mutants is rare (more than 85% in the embryos and 60% + plus 27% branched muscles) in larvae. Previous experiments indicated a similar penetrance for col null mutant embryos (Enriquez et al., 2012), suggesting that other iTFs may contribute DA3 identity. Other iTFs are indeed known to be expressed or/and required in the DA3 muscle (Tixier et al., 2010; Dubois et al., 2016) including Vestigial see Figure 5—figure supplement 1). We added a sentence in subsection “iTF transcription in muscle PCs; redundant CRMs”.

5) Do the authors have any idea as to why the col^ΔE is homozygous female sterile. Is it due to an off-target effect?

We verified that col^ΔL1.3 female sterility (abortive oocyte development, data not shown) is not due to an additional mutation at the col locus, suggesting indeed that it is due to an off-target effect.

6) Quantification of the FISH: it would be important to add quantification data as to the reduction in the col transcripts in the mutants shown in Figure 3.

We thank the reviewer for their important remark. We have now quantified the col transcription level in nuclei of PC and FC cells. Results are presented in new Figure 3D and described (subsection “(Re)programming of syncytial nuclei is required for muscle morphological identity”), and show that the level of col transcription in the DA3 FC is nodal for transcriptional reprogramming of syncytial nuclei and proper muscle identity.

7) The authors should explain better what do they mean by homotypic repulsion.

We name homotypic repulsion the fact that two muscles of the same identity cannot adhere to each other and form indirect musclemuscle attachments. Internal muscle-muscle attachments are only heterotypic, i.e., form between muscle of distinct identities, e.g., DA3/DA2 and DA2/DA1. Repulsion between two DA3 muscles in wt embryos is both supported by movies (Video 1) and stainings of fixed embryos, which show that exploring filopodia issued from one DA3 muscle are repelled by the DA3 muscle of the next anterior segment. Repulsion does not operate upon (partial) loss of DA3 identity (Video 2). We extensively rephrased the text to better explain this point in the revised version (subsection “Muscle attachment: tendon attraction and homotypic repulsion”).

8) Did the authors check that muscle genes, such as MHC, TpnC, actin etc, are expressed normally and in the mutant DA3 muscle, and that its innervation occurs ordinarily? Can they rule out that the muscle does not function due to physiological reasons, rather than the aberrant attachments?

As far as we can tell, the sarcomeric structure of transformed or branched DA3 muscles is normal (see the new supplementary Figure 7—figure supplement 1). We have previously established that activation of “generic” muscle differentiation and attachment genes, such as Mhc and Kon are synchronously activated in all muscle nuclei, making unlikely that is controlled by iTFs (Bataille et al., 2017). Following the suggestion of reviewers 3 and 1, we looked at “DA3” innervation in col^ΔL0.5 and col^ΔL1.3 mutants. Labelling motoneuron axons using anti-HRP staining shows that fully transformed DA3^>DA2 muscles are innervated. However, in most cases, only one branch of branched muscles is (new Figure 7—figure supplement 1). This innervation defect could, in part, explain the impaired locomotion specifically observed in larvae with numerous branched muscles, as now stated in subsection “Branched muscles result in subtle locomotion defects”.

[Editors’ note: what follows is the authors’ response to the second round of review.]

Essential revisions:

Overall, the results regarding the functional contribution of the distinct CRMs are convincing and nicely presented. However, the conclusions regarding muscle-muscle repulsion and the model explaining the sequence of events leading to DA3 transformation are less convincing. The link to larvae crawling behaviour is also puzzling due to the stronger phenotype obtained by partial deletion of Col, relative to its complete deletion, and also due to the effect on muscle innervation. These could be discussed in the 'Discussion' section.

Proper crawling of the larva requires integration of information from neuronal networks, relayed by stereotypic synaptic connections between motorneurons and muscles (Landgraf and Thor, 2006). One possible interpretation of the col^ΔL0.5 mutant locomotion phenotype is that muscle innervation of branched muscles – one connection for two branches- may interfere with its timing of contraction. In case of complete DA3 ^>DA2 muscle transformation in col^ΔL1.3 mutant larvae, the transformed muscle is innervated by the ISN and could contract normally. It will be interesting in the next future to profile the transformed and branched muscle activity in crawling (following the study of Zarin et al., eLife, 2019), or other movements such as larval rolling.

Possible interpretations are discussed in subsection “Branched muscles impact on crawling speed”.

1) Figure 2B – the numbers of embryos analyzed is not indicated.

The number of embryos analyzed in Figure 2B was indicated in subsection “Phenotype quantification at embryonic and larval stages”. It is now also given in the legend of the Figure 2.

2) Figure 3B – in col^ΔL0.5 st 14 there are extra nuclei labeled below the DA3 muscle. These are not stained in the control. What are these?

We thank the reviewer for pointing out this “ectopic” expression. It corresponds to nuclei of other muscles which, in addition to DA3/DO5, originate from progenitors transcribing col (DO4/LL1 and DT1/DO3). At stage 14, under normal conditions, Col expression is only maintained in the DA3 muscle and barely visible in these other muscles. In col^ΔL0.5 mutant embryos where the col binding site is removed, low level of Col expression are still detected in the DT1 and LL1, and possibly D05 nuclei. We previously noticed a similar “ectopic” expression of a 2.6-0.9 late col CRM reporter where the Col binding site was mutated (unpublished data; Author response image 1), while col expression in the DA3 is strongly reduced. Our interpretation is that either mutation or removal of the Col binding site removes a binding site for a repressing factor (perhaps competing with Col for binding) contributing to Col repression in lineages other than DA3. We have previously reported that several TFs contribute to the lineage-specific transcription/repression of col transcription (Figure 7 in Dubois et al., 2016), and this putative repressor remains to be identified (this has been added in subsection “iTF transcription in muscle PCs; redundant CRMs”). Of note Col expression in stage 14 nuclei is dependent in all muscles upon the presence of Twi and Mef2 binding sites in the late CRM (compare col^ΔL1.3 and col^ΔL0.5). This is mentioned in subsection “(Re)programming of syncytial nuclei is required for muscle morphological identity”.

Author response image 1

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3) Figure 3C – why does Col protein not label all nuclei in the control DA3? Why is the green label stronger in col^ΔL0.5 relative to col^ΔL1.3.

We agree with the reviewer that the green channel may obscure the blue channel in some panels of Figure 3C, for example wt/df stage 14. Author response image 2 is the original image showing only the blue and red channels, which shows that Col protein (blue) is present in all nuclei transcribing col (red channel). We chose not to show the blue channel alone in submitted Figure 3C not to overload it.

Author response image 2

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Of note, we have previously shown that Col protein is detected in all DA3 nuclei in stage 14 wt embryos. What we observed, however, was a mixture of ON/OFF nuclei for transcription of identity transcription factors and realization genes, suggesting that col auto-regulation and activation of downstream genes is Col threshold-dependent (Dubois et al., 2007; Bataillé et al., 2017).In the Figure 3C, we use L-CRM-moeGFP expression to visualize the Col-expressing progenitors and DA3 muscle contours. L-CRM-moeGFP activity is depending on the Twi and Mef2 binding sites, from the progenitor stage (see Figure 1—figure supplement 3 and Dubois et al., 2007), explaining why more GFP accumulates in col^ΔL0.5 than col^ΔL1.3 mutants.

4) Figure 3D – The quantification of transcription at stage 14 is missing. We are puzzled by the question of why late CRM is not active when the autoregulatory is missing. Mef2 should be still active in all nuclei, should it not?

As shown in Figure 3C and summary (Figure 3E), col transcription is no more detected in col^ΔL1.3 mutants at stage 14, preventing comparative measurements.

Previous analysis of col mutants has shown that Col is required for maintenance of its own transcription in a few tissues where it is expressed, among which the DA3 muscle lineage beyond the FC stage (Crozatier et al., 1999; Dubois et al., 2007). Mef2 and Twist are also required for L-CRM activity (this manuscript, see reply to point 2). Our working model is that Mef2 and Twist early binding to the L-CRM (Sandmann et al., 2007) primes the late col CRM for Col autoregulation (subsection “(Re)programming of syncytial nuclei is required for muscle morphological identity”).

5) Figure 3D – there is no information of how the fluorescence quantification was done. Did the authors perform background subtraction? Did the authors check whether nau transcription is affected or not by col depletion? They should provide images and quantification of nau FISH as well or give clear reasons of why these could be future work if they are unable to do this now.

We have now modified subsection “Quantification of col transcription “ and provide a new supplementary figure (Figure 3—figure supplement 2) showing that nau transcription is not particularly modified in ΔCRM col mutants.

6) Figure 5 – The authors describe numerous membrane protrusions, which we do not see in the image provided, and no quantification of these is described. No information regarding homotypic DA3/DA3 and DA2/DA2 is provided. Are these real homotypic adhesions? Again, please give clear reasons for why these could be future work if they are unable to do further experiments now.

7) Figure 5 – The images are very nice, however, we do not know about repulsion or attraction. It is speculation. There could be other explanations. Please tone down and discuss various possibilities

Following the reviewer’s suggestions, we now added a supplementary figure (Figure 5—figure supplement 2), stage 15 embryo, to show the membrane protrusions issued from DA muscles and contacting each other.

We think that the accumulation of MoeGFP at the matching surface between the DA2 and DA3 muscles supports our conclusion of heterotypic adhesion. Triple staining for the tendon cell and muscle contours and α-spectrin supports the model of “indirect” muscle-muscle attachment underneath tendon cells schematized in Maartens and Brown, 2014 (Author response image 3). We postulate that stabilization of the posterior, before anterior attachment, a developmental sequence also observed during development of abdominal adult muscles (Currie and Bate, 1991) may be instrumental in the precise matching of muscle-muscle attachments.

Conversion of the heterotypic DA3/DA2 matching into homotypic DA3^>DA2/DA3^>DA2 adhesion in col mutants suggests that DA3>DA3 homotypic repulsion is alleviated. The alternative possibility that heterotypic adhesion is not privileged over homotypic adhesion is also plausible. Future work aims at identifying surface molecules which could mediate heterotypic adhesion and/or homotypic repulsion, using DA3, DA2 or DA3^>DA2-specific RNA-seq experiments. From our previous studies one the dynamics or reprograming of naïve myoblast nuclei during syncytial elongation (Bataille et al., 2017) the time window may be relatively short, making the experiments challenging.

The text has been modified to tone down conclusions on these aspects and discuss various possibilities; subsection “Muscle staggered ends; heterotypic versus homotypic interactions?”.

Author response image 3

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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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