Multiple Cis-Regulatory Modules ensure robust tup/islet1 function in dorsal muscle identity specification

Multiple Cis-Regulatory Modules ensure robust tup/islet1 function in dorsal muscle identity specification | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Multiple Cis-Regulatory Modules ensure robust tup/islet1 function in dorsal muscle identity specification Aurore Pelletier, Alexandre Carayon, Yannick Carrier, Coralie Sengenès, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6063601/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Aug, 2025 Read the published version in Skeletal Muscle → Version 1 posted 9 You are reading this latest preprint version Abstract Background: The development of functional muscles in Drosophila melanogaster relies on precise spatial and temporal transcriptional control, orchestrated by complex gene regulatory networks. Central to this regulation are cis-regulatory modules (CRMs), which integrate inputs from transcription factors to fine-tune gene expression during myogenesis. In this study, we investigate the transcriptional regulation of the LIM-homeodomain transcription factor Tup (Tailup/Islet-1), a key regulator of dorsal muscle development. Methods: Using a combination of CRISPR-Cas9-mediated deletion and transcriptional analyses, we examined the role of multiple CRMs in regulating tup expression. Results: We demonstrate that tup expression is controlled by multiple CRMs that function redundantly to maintain robust tup transcription in dorsal muscles. These mesodermal tup CRMs act sequentially and differentially during the development of dorsal muscles and other tissues, including heart cells and alary muscles. We show that activity of the two late-acting CRMs govern late-phase tup expression through positive autoregulation, whereas an early enhancer initiates transcription independently. Deletion of both late-acting CRMs results in muscle identity shifts and defective muscle patterning. Detailed morphological analyses reveal muscle misalignments at intersegmental borders. Conclusions: Our findings underscore the importance of CRM-mediated autoregulation and redundancy in ensuring robust and precise tup expression during muscle development. These results provide insights into how multiple CRMs coordinate gene regulation to ensure proper muscle identity and function. Myogenesis Enhancers Transcriptional regulation Multiple CRMs Muscle identity Muscle patterning Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Animals display an assembly of skeletal muscles of various sizes and shapes that underlies/allows specific movements. In most cases, multiple muscles are cooperatively involved. The diversity of muscles raises the question of how individual muscles acquire the necessary shapes and specific attachment sites during development. In Drosophila larvae, muscles are single multinucleated fibers which develop by fusion of one founder cell with fusion-competent myoblasts. The identity of each founder cell (FC) is determined by its unique combination of identity transcription factors (iTFs) and by the precise timing and levels at which those factors are expressed (Baylies et al., 1998 ; de Joussineau et al., 2012 ; Dubois et al., 2016 ). FC identity specification is a multi-step process, starting with the delineation of promuscular clusters (PMCs), groups of equipotent cells from which progenitor cells (PCs) are selected. PCs then divide asymmetrically, giving rise to two different FCs, or, in a few cases, one FC and another lineage (Bate, 1990 ; Deng et al., 2017 ). Expression of different iTFs in different FCs starts with iTF activation in PMCs in response to positional information and progressive refinement in PCs and FCs through cross-regulations between different iTFs and response to Notch signaling and Hox information (Carmena et al., 1998 ; Carmena et al., 2002 ; Enriquez et al., 2010; Dubois et al., 2016 ). During the fusion process, the specific iTF code expressed by the founder cell (FC) is transferred to the nuclei of fusion-competent myoblasts (FCMs) in the syncytium, effectively reprogramming FCMs to adopt FC identity (Knirr et al., 1999; Crozatier and Vincent, 1999 ; Bataillé et al., 2017 ). This reprogramming is thought to activate downstream genes that control the final muscle morphology (Bataillé et al., 2010 ; Junion and Jagla, 2022) (Fig. 1 A). The development of functional muscles in Drosophila melanogaster therefore relies on precise spatial and temporal transcriptional control of each iTF expression (Dubois et al., 2016 ). Central to this regulatory architecture are cis-regulatory modules (CRMs); short DNA sequences that integrate inputs from multiple transcription factors/signaling pathways to finely regulate gene expression. CRMs essentially determine when, where, and how much a gene is expressed, providing both flexibility and robustness to developmental gene expression (Zinzen et al., 2009 ; Bonn et al. 2012 ; Kvon et al., 2021). Developmental genes typically display multiple CRMs, often spread across large genomic regions (Visel et al., 2009 ; Schoenfelder and Fraser, 2019 ). Originally, it was thought that CRMs each drive distinct spatiotemporal aspects of gene expression, following a model of modular regulation (Andersson and Sandelin, 2020 ; Field and Adelman, 2020 ; Furlong and Levine, 2018 ; Long et al. 2016; Shlyueva et al., 2014 ). This was an oversimplified model, however, since, in many cases CRMs regulating the same gene exhibit overlapping spatiotemporal activity, leading to the notion of cumulative regulation including by primary and shadow enhancers (Hoch et al., 1990; Jeong et al., 2006; Zeitlinger et al., 2007 ; Bothma et al., 2015 ; Cannavo et al., 2016; Whitney et al., 2022 ). The LIM-homeodomain transcription factor Tailup (Tup) is the Drosophila ortholog of vertebrate Islet1. We have shown that embryos carrying null tup mutations (tup ex4 ) display a disorganized dorsal musculature, indicating that tup plays a key role in establishing the stereotypical pattern of the dorsal muscles of Drosophila larvae (Boukhatmi et al., 2012 ). However, the pleiotropy of tup ex4 mutant phenotypes, including germ band retraction failure and defects in cardiogenesis and hematopoietic organ (lymph gland, LG) formation (Tao et al., 2007 ; Mann et al., 2009) precluded precise analysis of muscle patterning defects. To overcome this difficulty, we undertook to further characterize the tup regulatory region, in order to identify CRMs which could be used to generate and characterize muscle-specific tup mutants. Here, we first report that transcription of tup in 4 dorsal larval muscles and the alary muscles (AM) relies upon three distinct CRMs spread over 26 kb of regulatory region. Two CRMs, namely tupADME and tupHDME , function in a partially redundant manner to maintain robust tup expression in the dorsal FCs and their growing muscle syncytia, and this involves Tup direct autoregulation. The third CRM, tupF4 (Tao et al., 2007 ), initiates tup transcription in the two dorsal progenitors that give rise to these four founder cells, as well as in the alary muscle lineage, but appears not strictly required to trigger tup autoregulation in FCs. CRISPR-Cas9–mediated deletions of late-acting tup muscle CRMs show that tup function in specifying dorsal muscle identity relies on the combined activity of these two CRMs. Loss of tup transcription in growing muscle syncitia, leads to mis-matching of dorsal muscles attachment sites at segmental borders. In summary, this study provides new insights into the transcriptional regulation of tup/Islet1 during Drosophila muscle development. By dissecting the contributions of individual CRMs, we propose a model in which sequential and lineage-specific activity of different CRMs allows for the progressive acquisition of muscle identity, ensuring robust muscle patterning. These findings contribute to a broader understanding of how multiple CRM-mediated gene regulation orchestrates complex developmental processes and provides resilience against genetic or environmental perturbations. Methods Fly strains All Drosophila melanogaster stocks and genetic crosses were grown using standard medium at 25°C. All the lines were provided by the Bloomington Drosophila Stock Center except tup ex4 (de Navascues and Modolell, 2007). Lines used are white [1118] (BDSC_3605), vasa-cas9 VK00027 (BDSC_51324 ) and the 21 tup Janelia-Gal4 lines (GMR) (Pfeiffer and al., 2008) listed in the Table 1 . We also used 5 Vienna tiles enhancer-Gal4 lines (VT) from the Vienna Drosophila Resource Center (Kvon et al., 2014) listed in the Table 1 . CRM deletions generated by Crispr/Cas9 Genomic tup target sites were identified using http://tools.flycrispr.molbio.wisc.edu/targetFinder/ (Gratz et al., 2014 ). Prior to final selection of RNA guides (gRNA) for deletions of tup CRMs, genomic PCR and sequencing of DNA from vasa-cas9 VK00027 flies was performed to check for polymorphisms in the targeted regions. Guides targeting tup F4, tup ADME and tup HDME were inserted in the pCFD4: U6:3-gRNA vector (Addgene n o : 49411) as described (Port et al., 2014 ), (see http://www.crisprflydesign.org/wpcontent/uploads/2014/06/Cloning-with-pCFD4.pdf ). All guides were verified by sequencing. The sequences of the oligonucleotides used to construct each gRNA expression plasmid are: tup F4: gRNA1: 5’-TTGTTGGCACTCCGATCTGAAGG-3’ and gRNA2: 5’-TTGTCTGC GGCAAGCGTCGAAGG-3’; tup ADME: gRNA1: 5’-GCAGCCCTGATCCTGACCGTTGG-3' and gRNA2: 5’-GGCAG ATTTAGTCCGTCAGTCGG-3'; tup HDME: gRNA1: 5’-CTCTTTAAAGGGAAGCTCAACC-3' and gRNA2: 5'-CACCAAC TGGAGTGCCAGTGCC-3' To delete the core region of tupF4, tupADME and tupHDME, vasa-cas9 embryos were microinjected with gRNAs in pCFD4 (200 ng/µl). Each adult hatched from an injected embryo was crossed to the balancer stock sna Sco / CyO, { wg en11 -LacZ } and 100–200 F1 flies were individually tested for either tup CRM deletion by PCR on genomic DNA. Reporter constructs, immunohistochemistry, in situ hybridization The yellow intron ( yi ), FlyBase ID #FBgn0004034 (position: 356918–359616) was inserted in the lacZ coding region between aa (Tyr 952) and aa (Ser 953) by standard PCR-based cloning position (Carayon et al, 2020 ). The resulting fragment was cloned downstream of tup CRM ( tup F4, tup HDME and tup ADME) inserted in a pAttB vector, and micro-injected in embryos for chromosomal insertion at position 68A4. Antibody staining and in situ hybridization with intronic probes were as described previously (Dubois et al, 2016 ). Primary antibodies were: rabbit anti-Kr (1/300), mouse anti-LacZ (1/1000; Promega), rabbit anti-GFP (1/1000; Torrey Pines Biolabs), chicken anti-GFP (1/500; Abcam), Phalloidine-Texas RedX (1/500; Thermofisher Scientific). Secondary antibodies were: Alexa Fluor 488-, 555- and 647- conjugated antibodies (1/300; Molecular Probes) and biotinylated goat anti-mouse (1/2000; Vector Laboratories). Digoxygenin-labelled antisense RNA probes were transcribed in vitro from PCR-amplified DNA sequences, using T7 polymerase (Roche Digoxigenin labelling Kit). In situ Hybridization were done as described (Dubois et al, 2016 ). When antibody staining and FISH were combined, the standard immuno-histochemistry protocol was performed first, with 1U/µl of RNase inhibitor from Promega included in all solutions, followed by the FISH protocol. Confocal sections were acquired on Leica SP8 or SPE microscopes at 40x magnifications, 1024x1024 pixels resolution. Images were assembled using ImageJ and Photoshop softwares. Phenotype quantification at embryonic and larval stages To quantify embryonic phenotypes, wt; VgM1-moeGFP and Δ tup ADME + HDME; VgM1-moeGFP embryos were immunostained with a primary mouse anti-GFP (1/500) (Roche) and secondary biotinylated goat anti-mouse (1/2000) (VECTASTAIN® ABC Kit). Stained embryos were imaged using a Nikon eclipse 80i microscope and a Nikon digital camera DXM 1200C. A minimum 50 abdominal segments (n) of stage 16 embryos were analyzed for each genotype. (wt: 10 embryos; Δ tup ADME + HDME: 10 embryos). To quantify larval phenotypes, wandering L3 larvae were analyzed. 6 WT and 11 Δ tup ADME + HDME larvae were used. Morphologies of DA2 and DA1 muscles were examined in 31 segments (WT) and in 55 segments (Δ tup ADME + HDME) by scanning electron microscopy. Scanning Electron Microscopy (SEM) To prepare fillets, third instar wild type and homozygous Δ tup ADME + HDME larvae raised at 25°C were dissected in myorelaxant buffer, according to (Yalgin et al., 2011 ). Larvae were cut longitudinally on the ventral side to preserve and expose the dorsal and dorso-lateral musculature. Fillets were then fixed 1 hour in a 4% formaldehyde/ 2.5% glutaraldehyde mixture in 1X PBS, washed in water and dehydrated gradually in ethanol. Fillets were dried at the critical point (Leica EM CPD 300 critical point apparatus), covered with a platinum layer (Leica EM MED 020 metalliser) and imaged with a Quanta 250 FEG FEI scanning microscope. Results Three separate tup CRMs regulate tup expression in dorsal skeletal muscles . Tup is expressed in the muscle progenitor cells (PCs) at the origin of the four dorsal-most somatic muscles of the Drosophila embryo and larva: DA1, DA2, DO1, and DO2 (Boukhatmi et al., 2012 ; Fig. 1 A). Two cis-regulatory modules (CRMs) have previously been associated to this expression: tup F4, a 1.5-kb DNA fragment located between − 13.5 and − 12 kb upstream of the tup transcription start site drives tup expression in various cell types issued from the dorsal mesoderm: lymph gland cells, alary muscles (AMs), pericardial cells, and cardioblasts, in addition to skeletal muscles (Tao et al., 2007 ); tup ADME, a 1,3-kb fragment located between − 19.8 and − 18.5 kb upstream of the transcription start site which drives tup expression only in the alary muscles and dorsal somatic muscles (Boukhatmi et al., 2014 ; Fig. 1 B). These two CRMs were identified through in silico search of evolutionarily conserved sequence blocks and ChiP-seq-experiments targeting mesodermal transcription factors (MesoTFs), including Tinman (Tin) (Philippakis et al., 2006; Zinzen et al., 2009 ; Jin et al., 2013; Fig. 1 B and Fig.S1-S2 ). Both tup F4 and tup ADME also contain conserved Org-1 binding sites critical for regulating tup transcription in the alary muscles (Boukhatmi et al., 2014 ). ChIP-seq analyses identified a third cluster of MesoTFs binding sites upstream of tup ADME, suggesting the existence of an additional mesodermal tup CRM. (Fig. 1 B). To sustain in silico analyses, we screened the full set of publicly available (GMR and VT) expression reporter lines (Pfeiffer et al., 2008; Kvon et al., 2014) covering 55-kb of tup genomic region (Fig. 1 B). Five GMR lines, GMR76B01, GMR76B03, GMR76B07, GMR76E06 and GMR76E11 showed expression in heart cells, alary muscles and dorsal muscles ( Table 1) . GMR76B03 overlaps with tup F4, while GMR76B01 and GMR76B07 overlap with tup ADME (Fig. 1 B and Table 1) . The other lines, GMR76E06 and GMR76E11 displayed expression in heart cells and dorsal muscles, identifying and additional tup mesodermal CRM located between − 26.7 and − 25 kb upstream of the tup transcription start site, i.e , around 5 kbp upstream of tup ADME (Fig. 1 B and Table 1 ). Interestingly, GMR76E11 showed delayed and weaker expression in DA2 and DO2 muscles than GMR76E06 ( Fig.S3 ), suggesting that GMR76E06 contains regulatory information absent in GMR76E11. In support of this, DNA sequence analysis indicated that GMR76E06 contains evolutionarily conserved Tup binding sites not present in GMR76E11, while both reporters contain Tin binding sites ( Fig.S4 ). Based on sequence and expression data, a new reporter construct was designed to both encompass conserved sequence blocks shared by GMR76E06 and GMR76E11 and the Tup binding sites ( Fig.S4 ). Expression analysis of this construct confirmed activity both in heart cells and dorsal muscles, leading us to name it tup HDME (Heart and Dorsal Muscles Enhancer) (Fig. 1 B, 1 C). We then compared the activity of tup F4, tup ADME, and tup HDME at different embryonic stages, using moe-GFP as reporter (Fig. 1 C). The expression of tup F4-moeGFP in promuscular clusters (st10-11), suggested that it plays an early role in early tup mesodermal expression. From embryonic stages 13 to 15, the three CRMs showed overlapping expression in dorsal muscles precursors, suggesting a relay of the proximal tup activating CRM by the distal CRMs. By stage 15, tup F4-moeGFP expression began to decrease in DA1 and DO1, to become undetectable in all dorsal muscles by stage 16. Dorsal views of stage 16 embryos showed both tup F4-moeGFP and tup HDME-moeGFP expression in the lymph gland, pericardial cells, and cardiomyocytes, and tup ADME-moeGFP expression only in Svp-positive cardioblasts which give rise to ostiae (Molina and Cripps, 2001; Tao et al., 2007 ) ( Fig.S5A ). On top of that, tup F4-moeGFP and tup ADME-moeGFP expression is detected in the alary muscles, whereas tup HDME-moeGFP expression is detected in anterior pharyngeal muscles ( Fig.S5A, B ). In summary, characterisation of the tup cis-regulatory landscape shows three distinct CRMs, tup F4, tup ADME, and tup HDME, scattered within 26kb of tup upstream DNA, contribute to control tup transcription in different mesodermal tissues in Drosophila embryos (Fig. 1 and S5B). Their partly overlapping patterns of activity, both spatially and temporally, suggests that these three CRMs combinatorically ensure precise control of tup expression in somatic muscles and AM, as well as heart and lymph gland cells during embryonic development. Direct autoregulation of tupHDME in dorsal muscles. We previously showed that tup transcription in dorsal muscles is lost in Tup protein null (tup ex4 ) embryos, indicating a positive autoregulation mechanism (Boukhatmi et al., 2012 ). To investigate whether Tup protein directly regulates its own expression through the three CRMs, we performed in situ hybridization using a LacZ probe in tup ex4 mutant embryos carrying LacZ reporter gene driven by each individual CRM ( Fig.S6 ). We observed that tup F4 activity in dorsal muscles is unaffected by the loss of Tup protein ( Fig.S6A ), consistent with the absence of Tup binding sites in this CRM ( Fig.S1 ). In contrast, tup ADME ( Fig.S6B ) and tup HDME ( Fig.S6C ) showed a complete loss of LacZ transcription in the dorsal muscles, indicating that Tup protein is required for the activation of these CRMs. Both tup ADME and tup HDME contain predicted Tup binding sites ( Fig.S2 and S4 ), suggesting that Tup directly autoregulates its expression through one or both these CRMs in the embryos. In support of this, previous work showed that mutation of the single tup ADME Tup binding site reduced tup ADME activity in dorsal muscles, while not in alary muscles (Boukhatmi et al., 2014 ). tup HDME contains three clustered conserved Tup binding sites (Fig. 2 A and Fig. S4 ). To determine whether tup HDME is also subject to autoregulation, we generated a modified tup HDME reporter construct lacking all three predicted Tup binding sites, tup HDME ΔTup (Fig. 2 A). We found that tup HDME ΔTup activity was completely lost in dorsal muscles while maintained in heart cells and the lymph gland (Fig. 2 B), showing that Tup autoregulation is required for tup HDME activity in developing dorsal muscles. In conclusion, these results demonstrate that Tup autoregulation is essential for maintaining tup expression specifically in dorsal muscles by directly binding to the tup HDME in addition of tup ADME CRM. However, tup expression in other mesodermal tissues, such as heart cells and the lymph gland, occurs independently of Tup protein, suggesting that other combinations of transcription factors, which include Org-1, tunes the level of tup expression in different mesodermal tissues. Combinatorial regulation of tup transcription in dorsal muscles. To further investigate temporal and lineage-specific aspects of tup regulation by different CRMs and overcome limitations associated with reporter protein stability, we compared the patterns of endogenous and reporter nascent transcripts using in situ hybridization with intronic probes (Fig. 3 ). We first examined endogenous tup transcription throughout embryonic development, using a probe against the first tup intron. To help identifying individual dorsal muscles, we immunostained embryos for Krüppel (Kr) which marks the DA1, DO1 (and LL1) progenitor cells, founder cells and muscle precursors, in addition to amnioserosa cells (Beckett and Baylies, 2007 ; Dobi et al. 2015 ) ( Fig.S7 ). In situ data confirmed that tup transcription starts at the promuscular stage, embryonic stage 10 and is maintained in dorsal muscles until stages 14–15. Overlap between tup nascent transcripts and Kr immunostaining from stages 12 to 14 ( Fig.S7 - yellow frames A, B, C) confirmed that tup is transcribed in the nuclei of DA1 and DO1 founder cells and later in the nuclei of developing DA1 and DO1 fibers. By stage 16, tup transcription was no longer observed in dorsal muscles, while persisting in cardiac and pericardial cells and the alary muscles. At stage 16, Kr expression is also lost in the muscle fibers and solely detected in the trachea, which runs internal to the somatic musculature. To precisely determine which aspects of tup transcription correlated with each tup CRM, we introduced a yellow intron into the LacZ reporter coding region (Fig. 3 A). Using dual in situ hybridization for intronic probes enabled us to precisely compare endogenous tup transcription ( tup int ) and each tup CRM-driven transcription ( tup F4 int , tup ADME int and tup HDME int ) at each step of muscle development. To facilitate their identification, DA1/DO1 nuclei were labeled by Kr immunostaining. The results showed that only tup F4 is active in promuscular clusters, stage 10, confirming its role in initiating tup transcription. By stage 14, tup F4-driven transcription was no longer detected in dorsal muscles, while it persisted in cardiac and pericardial cells, and alary muscles. tup HDME activity was first detected at stage 11, in dorsal muscle progenitor cells, whereas tup ADME activity was detected later, in dorsal muscles founder cells (FCs), stage 12 (Fig. 3 A). Unlike tup F4, tup HDME and tup ADME kept being active during myofiber elongation, stage 14. tup ADME and tup HDME-driven transcription was no more detected in dorsal muscles by stage 16 (Fig. 3 A). Together, these findings show that tup F4 is active in dorsal promuscular clusters and that tup ADME and tup HDME regulate tup transcription during muscle fiber development, with partial temporal overlap between the three CRMS (Fig. 3 B). Previous analysis of dorso-lateral muscle lineages has shown that the birth time of different muscle FCs follows a precise sequence and that temporal windows of a given iTF expression may differ in different muscle lineages (Dubois et al., 2016 ). To determine the lineage-specificity of individual tup CRMs, we focused our analysis on two key stages of muscle development: stage 12, when FC identity is specified (Frasch, 1999; De Joussineau et al., 2012 ; Dubois et al., 2016 ), and stage 15, when FC transcriptional identity has been propagated to nuclei of fused myoblasts, a process known as identity reprogramming of syncytial nuclei (Crozatier and Vincent, 1999 ; Dubois et al., 2007 ; Bataillé et al., 2017 ). Kr immunostaining and positional information were used to identify each dorsal muscle lineage at stage 12 (Fig. 3 C). tup in situ confirmed tup transcription in the four dorsal muscle FCs, and initiation of tup transcription in the nuclei of “naïve” myoblasts (FCMs) incorporated into developing dorsal muscles. It also showed that tup F4 remains active in the DA2 and DO2 (and AM) FCs and not the dorsal-most DA1 and DO1 FCs. At that stage, both tup ADME and tup HDME become active in the four dorsal FCs. By stage 15, the dynamics have shifted, with tup F4 remaining active only in DO2, tup ADME being active in all four muscle lineages, and tup HDME active only in the DO1 and DO2 lineages (Fig. 3 C). As documented above, (Fig. 2 ), the temporal relay between early and late CRMs in dorsal muscles involves direct Tup autoregulation, a mechanism which operates neither in heart nor lymph gland cells, nor in the alary muscles. Taken together, analysis of nascent endogenous and reporter transcripts revealed a temporal, lineage-specific sequence of tup regulation during Drosophila muscle development. tup F4-driven early tup transcription in promuscular clusters is relayed by tup ADME and tup HDME activities which ensure propagation of tup transcription to fused nuclei during muscle fiber formation in a lineage-specific manner. Deletion of tupADME and tupHDME impairs tup transcription in dorsal muscles. To investigate the individual roles of each tup cis-regulatory module (CRM) and their combined effect on somatic muscle identity, we generated dorsal muscle-specific tup mutants by using CRISPR-Cas9 to delete the muscle-specific tup CRMs (Fig. 4 A). Neither individual CRM deletion resulted in germ band retraction failure, allowing to analyse in detail muscle development. As a first step, we analysed tup transcription in embryos homozygous for each CRM deletion (Fig. 4 B). Global analysis at stages 12 and 14 showed that deletion of neither individual CRMs was sufficient to eliminate tup transcription in developing muscles. The detection of tup transcription in Δ tup F4 embryos, indicated that, while subject to direct autoregulation, tup ADME or/and tup HDME activity does not depend upon earlier tupF4 activity. tup transcription in at least a fraction of dorsal muscle nuclei, was detected as well at stage 14 upon deletion of either tup ADME or tup HDME, suggesting their redundancy. In contrast, tup transcription was lost stage 14 in the double tup ADME and tup HDME (Δ tup ADME + HDME) deletion mutant (clear arrowheads), indicating that these two CRMs are together required for propagating transcriptional identity of FCs during reprogramming of fused FCMs into growing syncitia (Fig. 4 B). Deletion of tupADME and tupHDME disrupts the dorsal muscle pattern. Since tup transcription in developing muscles was only abolished in the double Δ tupADME + Δ tupHDME CRM deletion mutants, we focused our analysis on muscle morphology in these mutants in late embryos and third instar larvae, as the new tup alleles did not display the embryonic lethality and pleiotropic effects typically associated with complete tup loss-of-function. We first analysed the muscle patterns in stage 16 embryos, using Tropomyosin 2 immuno-staining of the somatic musculature together with the VgM1-moeGFP reporter line (Carayon et al., 2020 ) which allows morphological inspection of the DA2 and DA3 muscle lineages. In wt embryos, VgM1-moeGFP expression illustrates the staggered rows pattern of DA muscles, with the posterior attachment sites of the DA2 and DA3 muscles of one segment facing the anterior attachments of DA1 and DA2, respectively, in the next segment (Fig. 5 A), a pattern repeated between successive adjacent segments reflecting muscle attachment sites matching (Carayon et al., 2020 ). Δ tup ADME + HDME mutant embryos displayed a disorganised dorsal muscle pattern (Fig. 5 A), showing that tup ADME/HDME combinatorial activity is required for tup role in dorsal muscle identity. Detailed analysis or VgM1-moeGFP expression shows that the DA1 and the DA2 muscles are misshaped or absent in 59.6% (n = 31) and 34.3% (n = 23) of segments, respectively, compared to 5.2% and 5.3%, respectively in wt-type embryos (Fig. 5 A-B). Δ tupADME + Δ tupHDME mutant embryos exhibit a partial transformation of the DA2 muscle into a DA3 identity, rather than the complete transformation observed in tup null embryos (Boukhatmi et al., 2014 ). Misalignment of DA1/DA2 and DA2/DA3 are illustrated by schematic drawings across three consecutive segments S n−1 , S n , and S n+1 (Fig. 5 A). Dorsal muscle attachment matching is disrupted in ΔtupADME + HDME 3rd instar larvae. To further investigate muscle morphological defects due to Δ tup ADME + HDME CRMs deletion, we analysed the somatic musculature of third instar larvae using scanning electron microscopy (SEM) of dissected fillets (Fig. 6 ). This allows precise investigation of the DA muscles attachments (Carayon et al., 2020 ). As previously described, the staggered ends pattern of dorsal muscles established in wt late embryos (Fig. 5 A) is maintained in wt larvae (Fig. 6 A). In Δ tup ADME + HDME larvae, the muscle attachment matching is severely disrupted, with defects in DA1 and DA2 muscle morphology and attachment sites observed in 52.7% (n = 29) and 43.6% of segments (n = 24), respectively, compared to 6.5% and 3.2%, respectively, in wt embryos (Fig. 6 B). Beside the loss of dorsal attachment of the DA1 muscle, one striking phenotype is the homotypic (DA2/DA2) attachment of DA2 muscles in consecutive segments, while only heterotypic DA3/DA2 and DA2/DA1 matchings are observed in wt embryos. This observation is consistent with an identity shift of the DA2 muscle towards a DA3 muscle identity. The similarity between the late embryonic and 3rd instar larval phenotypes confirm the key role of Tup is establishing the correct morphological identity of dorsal muscles during embryogenesis. Together with previous analysis of mutants for another DA identity gene, collier (Carayon et al., 2020 ), analysis of muscle-specific tup mutations shows that the precise heterotypic matching of muscles attachment sites at segmental borders is a sensitive read-out of muscle transcriptional identity. Discussion A stereotyped set of 30 somatic muscles in each abdominal segment underlies Drosophila larval crawling (Bate, 1990 ). Each muscle morphology reflects expression of a specific combination of "identity transcription factors" (iTFs) by its founder cell (Frasch, 1999; De Joussineau et al., 2012 ; Dubois et al., 2016 ). A subset of iTFs is already activated in PMCs from which PCs and FCs are selected. For example, the PCs which give rise to the DA2 and DA3 FCs are both selected from a PMC expressing Tinman (NKx2.5), Collier (Col/Kn) and Tup. It is the sequential birth of the DA2 and DA3 PCs which determines that Tup remains expressed in the DA2 PC and FC and Col in the DA3 FC (Enriquez et al., 2010; Boukhatmi et al., 2012 ). Here, we further investigated the regulation and role of Tup/Islet1 in PCs/FCs seeding the formation of dorsal muscles. CRM redundancy and robustness of tup expression in the dorsal mesoderm. We identified a third, distal, tup CRM, which we named Heart and Dorsal Muscles ( tup HDME) CRM, which regulates tup transcription in the dorsal mesoderm. Comparison with the previously known CRMs, tup F4 and tup ADME (Tao et al., 2007 ; Boukhatmi et al., 2012 ), using detailed transcriptional analyses with endogenous and reporter intronic probes shows that each CRM displays a specific timing of activity during dorsal muscle development. Both tup HDME and tup ADME control tup transcription in dorsal muscles beyond the FC step. CRISPR-Cas9-mediated deletion of either CRM individually did not result in significant disruption of muscle tup expression, whereas deletion of both supressed tup transcription at late stages of muscle development (stages 14–16). This, at least partial, CRM redundancy suggests that robust tup function is key in specifying skeletal muscle identities. The redundancy and robustness of tup regulation via multiple CRMs could be part of a more general mechanism of transcriptional control during muscle development. Other transcription factors, either "generic", such as Mef2 (Nguyen and Xu, 1998; Sandmann et al., 2007 ), or specific to a subset of skeletal muscles (iTFs) like Collier (Enriquez et al., 2012 ;) are regulated by multiple CRMs working sequentially or/and synergistically during muscle development, an evolutionary strategy for ensuring a precise level of expression across different stages of development (Reddington et al., 2020 ; Kvon et al., 2021). This could be part of a general mechanism for robustness of integrating generic and identity aspects of muscle development (Bataille et al., 2017). In case of tup , each of skeletal muscle CRM is also active in other mesodermal derivatives which differ between CRMs. An evolutionarily selection of this combinatorial set up could be essential to coordinate development of a stereotypical muscle pattern and development of the heart and associated tissues, the cardiac outflow, valves cells, AMs and lymph gland (Tao et al., 2007 ; Zmojdzian and Jagla, 2013 ; Boukhatmi et al., 2014 ; Meyer et al., 2023). Autoregulation of tup expression in dorsal muscles. Interestingly, the deletion of the early-active tup F4 CRM did not abolish tup transcription or perturb dorsal muscle patterning, suggesting that tup transcription at the PMC stage is not critical for dorsal muscle identity. Moreover, since tup HDME activity, which overlaps with tup F4 at the PC stage, remains unaffected by tup F4 deletion, this rules out a simple handover mechanism initiating tup HDME autoregulation. One possible explanation is that the same upstream transcription factors (TFs) or chromatin-opening factors bind to both tupF4 and tupHDME , but at different times or concentrations, with tupHDME maintaining tup transcription via its direct autoregulatory sites (Fig. 2 ). On support of this, Chip-SEQ analyses have shown Tin binding to both tup F4 and tup HDME in 4–6 hours embryos, Twi sequentially binds to tup F4 and tup ADME in 2–4 and 6–8 hour embryos, respectively and Mef2 sequentially binds to tup F4 and tup ADME in 4–6 and 6–12 hour embryos, respectively (Zinzen et al., 2009 ; Philippakis et al., 2013; http://furlonglab.embl.de/ tissue_specific_DHS). We attempted to confirm these observations by generating a double CRISPR-Cas9-mediated deletion of tup F4 and tup HDME, but for technical reasons, we were unable to collect double mutant individuals. This is not the first occurence where deletion of a CRM driving iTF expression in PMCs does not lead to muscle patterning defects. Similar results were indeed observed in studying the respective roles of early (PMC) and late (FC/muscle) col CRMs. In this case as well, the early CRM was not required to prime autoregulation (Carayon et al. 2020 ). This raises important questions about the specific role of early-active CRMs in muscle identity specification within specific subsets of muscle, while highlighting the pivotal role of late-active CRMs via autoregulation in maintaining and propagating muscle identity. Whether there is a specific role of iTFs expression in PMC prior to the process of PC/FC identity specification remains to be fully elucidated. Our results support a model of muscle identity specification that progressively refined at each stage of muscle development - PMC to PC, PC to FC, and FC to syncytial fiber - through distinct CRM activities. This temporal refinement through separate regulatory elements may ensure that muscle identity is established and maintained through each critical step of muscle development. Sustained tup transcription in dorsal muscles is dependent on a positive autoregulatory mechanism exerted on both tup ADME (Boukhatmi et al., 2012 ) and tup HDME (Fig. 2 ). Yet, while tup HDME deleted of its Tup binding sites is inactive in dorsal muscles, it remains active in heart cells or the lymph gland, indicating a tissue specific autoregulation. tup autoregulation mediated by tup ADME is also skeletal muscle-specific and is not exerted in AMs. In turn, tupHDME , is not active in AMs, consistent with the absence of Org-1/Tbx1 binding sites which directly control tup ADME activity in these peculiar heart-associated muscles (Boukhatmi et al., 2012 ). Other transcription factors may regulate tup expression in cardiac cells or the lymph gland, including Tinman, Twist, and/or Bagpipe (Zinzen et al., 2009 ; Sandmann et al., 2006 ; Azpiazu and Frasch, 1993 ). Future understanding of the regulatory roles of other TF binding motifs present within combinations of tup enhancers and enhancer-promoter interactions will certainly benefit from newly developed technologies such as Quantitative enhancer-FACS-Seq analysis (Waters et al., 2021 ) and Capture-C in purified myogenic cells (Pollex et al., 2024 ). Impact of tup transcription loss on muscle patterning and alignment. Previous analysis of tup null ( tup ex4 ) embryos, using a DA3 muscle marker Collier, revealed a DA2-to-DA3 (DA2 > DA3) identity shift. tup ex4 embryonic lethality, including germ band retraction defects, precluded, however detailing this identity shift at the morphological level (Boukhatmi et al., 2012 ). Expression of the VgM1-moeGFP reporter in double tup CRM mutants allowed to visualise the DA2 > DA3 transformation in fully developed embryos (Fig. 5 A). In wild-type embryos and larvae, the DA2 muscle of segment S n aligns with the DA3 muscle of S n−1 and the DA1 muscle of segment S n+1 , at intersegmental borders (Fig. 5 A; Carayon et al., 2020 ). However, in the double tup ADME and tup HDME deletion mutants, this DA3-DA2, DA2-DA1 intersegmental matching is often replaced by homotypic alignment between DA2 muscles in consecutive segments (Fig. 5 A, B). Residual heterotypic matching could possibly reflect either the late timing of loss of tup transcription, in other terms, residual expression driven by tupF4 or/and the involvement of other iTFs expressed in DA muscles FCs (Dubois et al., 2017). DA muscle mismatching at intersegmental borders persists until the end of larval development (Fig. 6 A, B), confirming that the specific morphology of somatic muscles at work in larval crawling, which includes muscle growth, elongation and choice of tendon and/or muscle attachment sites, reflects the combination of iTFs expressed by muscle FCs in early embryos. The DA3-DA2-DA1 misalignments mirror those observed in collier ( col ) muscle CRMs mutants, which show a DA3-to-DA2 identity shift with DA3-DA3 homotypic matching replacing DA3-DA2 matching at intersegmental segmental borders (Carayon et al., 2020 ). The similarity of the symmetrically opposite tup and col dorsal muscle phenotypes shows that the col > tup transcriptional regulation which ensures that Tup is expressed in the DA2 FC and Col in the DA3 FC (Boukhatmi et al., 2012 ), is critical for the proper alignment and orientation of dorsal muscles and larval locomotion. The defects observed in Δ tup ADME + HDME mutants further underscore the importance of coordinated CRM activity in establishing a stereotypical muscle pattern, with in the case of tup , tup ADME and tup HDME redundant function ensuring robustness to this pattern. Conclusions The involvement of multiple CRMs for precise tup regulation in skeletal muscles, similar to previously reported for col , another TF critical for muscle identity and optimal larval locomotion (Enriquez et al., 2012 ; Dubois et al. 2007 ; Carayon et al., 2020 ) supports that temporal cascade strategies provide robustness to transcription control of muscle identity, and resilience of species-specific muscle patterns against genetic or environmental perturbations. Future work should investigate the physiological consequences of muscle mismatching observed in tup mutants. Additionally, exploring the interaction of tup with other muscle-specific transcription factors and their associated CRMs may uncover further complexities in the regulatory networks that govern muscle identity and development. Abbreviations CRM - Cis-Regulatory Module iTFs - Identity Transcription Factors FC - Founder Cell FCM - Fusion-Competent Myoblast PMCs - Promuscular Clusters PCs - Progenitor Cells tup - Tailup (also known as Islet1 in vertebrates) tup ex4 - tup mutant allele Mef2 - Myocyte Enhancer Factor 2 Col - Collier Tin - Tinman Twi - Twist DA - Dorsal Acute Muscles DO - Dorsal Oblique Muscle 1 HDME - Heart and Dorsal Muscles Enhancer ADME – Alary and Dorsal Muscles Enhancer AMs - Alary Muscles CRISPR-Cas9 - Clustered Regularly Interspaced Short Palindromic Repeats and Caspase 9-associated protein gRNA - Guide RNA (used for CRISPR/Cas9 gene editing) ChIP-Seq - Chromatin Immunoprecipitation Sequencing Declarations Availability of data and materials: The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests: The authors declare that they have no competing interests. Funding : This work was supported by CNRS, Association Française contre les Myopathies (AFM) Research Grant 21887, ANR grant 13-BSVE2-0010-01. Authors' contributions: J-LF managed the project. LD and J-LF conceptualized and designed the experiments. AP, AC, YC, LD and J-LF performed the experiments. AP, YC, LD, CS and J-LF analysed the data and prepared the figures for the manuscript. J-LF wrote the manuscript with input from co-authors. Acknowledgements: The authors sincerely thank Alain Vincent, whose initiative led to this project. They are grateful for his unwavering commitment, valuable assistance in writing and proofreading, and insightful advice and discussions. We thank the Bloomington Stock Center and the Vienna Drosophila Resource Center for Drosophila strains, and Julien Favier, Drosophila embryos microinjection platform from the Center of Integrative Biology in Toulouse – France. References Andersson R, Sandelin A. 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The tup F4 sequence was annotated using EvoPrinter, a comparative genomics tool designed to identify conserved DNA sequences shared among several orthologous DNAs. Black capital letters represent bases in the Drosophila melanogaster reference sequence that are conserved in the orthologous sequences of other Drosophila species: D. simulans , D. sechellia , D. yakuba , D. erecta , D. pseudoobscura , and D. persimilis . Transcription factor binding sites were predicted using the JASPAR core database, an open-access resource of curated, non-redundant transcription factor binding profiles. Binding sites for Org-1 (blue) and Tin (grey) are highlighted. The blue box indicates a cluster of mesodermal transcription factor binding sites identified in previous ChIP-seq experiments (ChIP Tinman and ChIP Meso TFs). The coordinates of the tup F4 cis-regulatory module (CRM) are chr2L:18892809-18894301 (FlyBase release: r6.53). The nucleotide sequences of PCR primers used to amplify the tup F4 CRM are underlined in red. figsup2.jpg Figure sup2: Conserved sequences and transcription factor binding sites in the tupADME Cis-Regulatory Module. The tup ADME sequence was annotated as indicated in Fig.S1. Binding sites for Org-1 (blue), Tup/Islet-1 (yellow), Mef2 (purple), and Twist (green) are highlighted. The coordinates of the tup ADME cis-regulatory module (CRM) are chr2L:18899345-18900688 (FlyBase release: r6.53). The nucleotide sequences of PCR primers used to amplify the tup ADME CRM are underlined in red. figsup3.jpg Figure sup3: Expression of GMR 76E06 and GMR 76E11 delineate a new tup cis-regulatory module. (A) GMR 76E06 and 76E11 expression during embryonic stages 11 to 16 (nuclear LacZ immunostaining). Both GMRs are expressed in the heart and dorsal muscles. At stage 16, GMR 76E06 is expressed in the DA1/DO1 and DA2/DO2 muscles, while GMR 76E11 expression is restricted to DA1/DO1 (white box). (B) GMR 76E06 and 76E11 sequences partly overlap. The newly identified CRM Tup HDME (in red) spans this overlap and the predicted binding sites for the transcription factors Tup/Islet-1 (Tup) and Tinman (Tin). figsup4.jpg Figure sup4: Conserved sequences and transcription factor binding sites in the tupHDME Cis-Regulatory Module. The tup HDME sequence was annotated as indicated in Fig.S1. Binding sites for Tup/Islet-1 (yellow) and Tin (grey) binding sites are highligthed. A cluster of binding sites for mesodermal transcription factors, determined by previous ChIP-seq experiments (ChIP Tinman and ChIP Meso TFs), is indicated by the blue box. The coordinates of the tup HDME-CRM are chr2L:18905826-18907523 (FlyBase release: r6.53). Nucleotide sequences of the PCR primers used to amplify the tup HDME CRM are underlined in red. The primer used to delete the three Tup binding sites and generate the tup HDMEDTup construct for autoregulation experiments (see Fig.2A) is underlined in grey. figsup5.jpg Figure sup5: Combinatorial activity of the three tup CRMs in different mesodermal cell derivatives. (A) Dorsal view of the expression patterns of tup F4-moeGFP, tup ADME-moeGFP and Tup HDME-moeGFP CRMs in late embryos (st 16) immunostained with an anti-GFP antibody. Depending on the CRM observed, expression is detected in the pharyngeal muscles (pm), alary muscles (AM), and the lymph gland (LG) (top pannels), in cardiac cells (CC) or pericardiac cells (pc) (bottom pannels). (B)The table summarizes the expression of tup F4, tup ADME and tup HDME in mesodermal tissues during the embryonic development (from stage 13 to stage 16). figsup6.jpg Figure sup6: Autoregulation by Tup maintains tupADME and tupHDME activity in dorsal muscles. tup F4 (A), tup ADME (B) and tup HDME (C) LacZ reporter expression in wild-type ( + ) and tup ex4 mutant embryos lacking Tup protein visualized by in situ hybridization using a LacZ probe. (A) tup F4-LacZ activity in promuscular clusters and progenitor cells is independent of Tup. (B-C) Both activity of tup ADME (B) and tup HDME (C) is lost in dorsal muscles in tup ex4 mutant embryos, showing that Tup is required. figsup7.jpg Figure sup7: tup transcription during embryonic development. tup transcription revealed by in situ hybridization (green signal) in stage 10 to 16 embryos immuno-stained for Krüppel (Kr), blue, which labels amniosera cells (amn), and the dorsal muscles DA1 and DO1 in addition to lateral (LM) muscle lineages. tup is transcribed in amn, cardiac cells (cc), pericardial cells (pc), dorsal muscles (DM), and alary muscles (AM) up to stage 14, before being restricted to cc and pc cells and AMs, stage 16. Kr is initially expressed in the amniosera at stage 10, followed by expression in DA1 and DO1 progenitor cells at stages 11–12. Later in development, Kr expression is restricted to the trachea (tr). The bottom panels are enlarged views of the yellow framed regions, designated A, B and C . (A, B) At stages 12 (A) and 13 (B), tup is transcribed (green dots) in the nuclei of the DA1 and DO1 founder cells identified by Kr expression and the AM founder cell. (C) At stage 14, multiple nuclei of the DA1 and DO1 (and AM) developing muscles actively transcribe tup . table1.jpg Table1: Expression patterns of Janelia (GMR) and Vienna Tiles (VT) lines. Summary of Janelia (GMR) and Vienna Tiles (VT) lines used to characterize the cis-regulatory sequences of Tup/Islet-1, with expression in different mesodermal tissues indicated by (x). A total of 26 lines were tested (21 Janelia and 5 VT) covering 50 kb of tup genomic region (see also Fig.1B). Five lines displayed expression in somatic muscles (x in red): lines 76B03, 76B07, 76B01, 76E11, and 76E06, two of which, 76B01 and 76B07 showed also expression in alary muscles (AMs). Cite Share Download PDF Status: Published Journal Publication published 25 Aug, 2025 Read the published version in Skeletal Muscle → Version 1 posted Editorial decision: Revision requested 22 Apr, 2025 Reviews received at journal 14 Apr, 2025 Reviewers agreed at journal 24 Mar, 2025 Reviews received at journal 21 Mar, 2025 Reviewers agreed at journal 25 Feb, 2025 Reviewers invited by journal 25 Feb, 2025 Editor assigned by journal 25 Feb, 2025 Submission checks completed at journal 20 Feb, 2025 First submitted to journal 19 Feb, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6063601","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":429172095,"identity":"e09653e9-83d4-43ab-befc-4fff7952fb1d","order_by":0,"name":"Aurore Pelletier","email":"","orcid":"","institution":"Centre de Biologie Intégrative, Université de Toulouse, CNRS, France","correspondingAuthor":false,"prefix":"","firstName":"Aurore","middleName":"","lastName":"Pelletier","suffix":""},{"id":429172097,"identity":"f2772dfa-6700-41d2-8145-2ac23aa410b9","order_by":1,"name":"Alexandre Carayon","email":"","orcid":"","institution":"Centre de Biologie Intégrative, Université de Toulouse, CNRS, France","correspondingAuthor":false,"prefix":"","firstName":"Alexandre","middleName":"","lastName":"Carayon","suffix":""},{"id":429172101,"identity":"37ec15fa-1193-4f6a-a22e-cfc1ddb769a7","order_by":2,"name":"Yannick Carrier","email":"","orcid":"","institution":"Centre de Biologie Intégrative, Université de Toulouse, CNRS, France","correspondingAuthor":false,"prefix":"","firstName":"Yannick","middleName":"","lastName":"Carrier","suffix":""},{"id":429172106,"identity":"e08f03ed-c8a7-47de-adc6-cd7120d4f70c","order_by":3,"name":"Coralie Sengenès","email":"","orcid":"","institution":"RESTORE: A geroscience and rejuvenation research center, Université de Toulouse, CNRS","correspondingAuthor":false,"prefix":"","firstName":"Coralie","middleName":"","lastName":"Sengenès","suffix":""},{"id":429172108,"identity":"6eb0bd2c-c659-405e-9b11-3e8b36a946af","order_by":4,"name":"Laurence Dubois","email":"","orcid":"","institution":"RESTORE: A geroscience and rejuvenation research center, Université de Toulouse, CNRS","correspondingAuthor":false,"prefix":"","firstName":"Laurence","middleName":"","lastName":"Dubois","suffix":""},{"id":429172109,"identity":"798cd1ce-e2d9-43a8-867b-c13d061af1f0","order_by":5,"name":"Jean-Louis Frendo","email":"data:image/png;base64,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","orcid":"","institution":"RESTORE: A geroscience and rejuvenation research center, Université de Toulouse, CNRS","correspondingAuthor":true,"prefix":"","firstName":"Jean-Louis","middleName":"","lastName":"Frendo","suffix":""}],"badges":[],"createdAt":"2025-02-19 11:23:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6063601/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6063601/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13395-025-00392-4","type":"published","date":"2025-08-25T15:57:07+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":78947676,"identity":"6143e472-0088-4084-85a5-31cade1dc6d0","added_by":"auto","created_at":"2025-03-21 08:09:07","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":147548,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eThree distinct Cis-Regulatory Modules regulate tup expression in dorsal muscle tissue.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Diagrammatic representation of the external (left) and internal (middle) somatic muscles in a late embryo (st17). The shape and position of the four dorsal muscles (DA1/DO1 and DA2/DO2) within an abdominal segment are highlighted in red. The dorso-lateral (DA3) and lateral (LL1) muscles are also indicated. Scanning electron microscopy of an abdominal segment (right) from a third instar larva (L3) shows the shape and position of the four dorsal muscles and the alary muscle (AM). \u003cstrong\u003e(B)\u003c/strong\u003eSchematic representation of the \u003cem\u003etup\u003c/em\u003etranscribed region. The positions of tested GMR and VT fragments are shown as grey horizontal bars, with numbers indicating those active in dorsal muscles. Clusters of \u003cem\u003ein vivo\u003c/em\u003e mesodermal transcription factor binding sites (chip meso TFs) and Tinman (chip Tinman) binding sites are marked by purple bars. Two previously identified cis-regulatory modules (\u003cem\u003etup\u003c/em\u003eF4 and \u003cem\u003etup\u003c/em\u003eADME) and a newly identified module (\u003cem\u003etup\u003c/em\u003eHDME, red bar) are shown. \u003cstrong\u003e(C) \u003c/strong\u003eEach CRM was fused to moe-GFP reporter gene, and GFP expression detected using an anti-GFP antibody. The three CRMs together recapitulate \u003cem\u003etup\u003c/em\u003eexpression in promuscular clusters (PMC), dorsal muscles (DA1/DO1 and DA2/DO2), alary muscles (AM), thoracic alary-related muscles (TARM), and heart cells during embryonic development (stages 10 to 17).\u003c/p\u003e","description":"","filename":"fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6063601/v1/8c3cee0f59e46b1aecf5124a.jpg"},{"id":78947671,"identity":"a9b8dcab-8490-4325-a6ad-ea816284c6c5","added_by":"auto","created_at":"2025-03-21 08:09:07","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":61723,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eTup binding sites are required for specific tupHDME autoregulation in dorsal muscles.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e A deletion removing all the three Tup binding sites gave the \u003cem\u003etup\u003c/em\u003eHDME\u003csup\u003eDTup\u003c/sup\u003e-moeGFP reporter. \u003cstrong\u003e(B)\u003c/strong\u003e While \u003cem\u003etup\u003c/em\u003eHDME-moeGFP is expressed both in the heart, lymph gland (LG) and dorsal muscles (DM), \u003cem\u003etup\u003c/em\u003eHDME\u003csup\u003eDTup\u003c/sup\u003e-moeGFP is only expressed in the heart and LG, highlighting \u003cem\u003etup\u003c/em\u003e direct autoregulation in muscles.\u003c/p\u003e","description":"","filename":"fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6063601/v1/c03572615c01958bb016906c.jpg"},{"id":78947669,"identity":"fa377ad2-9f69-45be-a76f-9d448d4a85a0","added_by":"auto","created_at":"2025-03-21 08:09:07","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":200829,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003etupCRMs sequential activities during dorsal muscles development.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Activity of \u003cem\u003etup\u003c/em\u003eF4, \u003cem\u003etup\u003c/em\u003eADME and \u003cem\u003etup\u003c/em\u003eHDME compared to endogenous \u003cem\u003etup\u003c/em\u003eexpression between embryonic stages 10 and 16. Nascent transcripts were visualized using intronic probes for \u003cem\u003etup\u003c/em\u003e(green dots) and the reporter gene (yellow intron into \u003cem\u003elacZ\u003c/em\u003e) red dots. Krüppel (Kr) antibody staining (blue) identifies the amnioserosa (amn), cardiac and pericardial cells (cc and pc), the DA1/DO1 progenitors and muscles, and the trachea (tr) (see also \u003cstrong\u003eFig.S8\u003c/strong\u003e). \u003cstrong\u003e(B)\u003c/strong\u003e Diagram summarizing the sequential activity of \u003cem\u003etup\u003c/em\u003e CRMs at the different stages of muscle development: promuscular cluster (PMC), progenitor cell (PC), founder cell (FC) and myofiber. \u003cstrong\u003e(C) \u003c/strong\u003eLeft panels: detailed view of \u003cem\u003etup\u003c/em\u003eF4, \u003cem\u003etup\u003c/em\u003eADME and \u003cem\u003etup\u003c/em\u003eHDME activity at the FC (stage 12) and syncytial fiber (stage 15) stages, using \u003cem\u003ein situ\u003c/em\u003e hybridization as in \u003cstrong\u003eFig.3A\u003c/strong\u003e. Right panels: schematized activity of each CRM in the DA1, DO1, DA2, DO2 and AM FCs (stage 12, red dots) and growing muscles (stage 15, red colored).\u003c/p\u003e","description":"","filename":"fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6063601/v1/f0d51f57a1106f48ab003fd7.jpg"},{"id":78947690,"identity":"2260aecf-04d6-40c8-a9e0-57b929bfcfc7","added_by":"auto","created_at":"2025-03-21 08:09:08","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":119176,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eDeletion of ADME+HDME CRMs abolishes tup transcription in dorsal muscles.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Schematic representation of the different deletions of \u003cem\u003etup\u003c/em\u003e CRMs. Homozygous flies lines were generated for each CRM (Δ\u003cem\u003etup\u003c/em\u003eF4, Δ\u003cem\u003etup\u003c/em\u003eADME, Δ\u003cem\u003etup\u003c/em\u003eHDME) and a double \u003cem\u003etup\u003c/em\u003eADME and \u003cem\u003etup\u003c/em\u003eHDME deletion (Δ\u003cem\u003etup\u003c/em\u003eADME+HDME). \u003cstrong\u003e(B)\u003c/strong\u003e \u003cem\u003etup \u003c/em\u003etranscription in these homozygous lines compared to wt, using \u003cem\u003ein situ\u003c/em\u003ehybridization to nascent transcripts (small black dots). \u003cem\u003etup\u003c/em\u003e transcription in the dorsal muscles is lost at stage 14 upon removing both ADME+HDME CRMs (clear arrowheads).\u003c/p\u003e","description":"","filename":"fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6063601/v1/84a2233fcb548d9e86000e8e.jpg"},{"id":78948247,"identity":"6fff4369-398d-47d4-9837-c0dd6c2780f3","added_by":"auto","created_at":"2025-03-21 08:17:07","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":171482,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eLoss of tup transcription disrupts the pattern of embryonic dorsal muscle.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Dorsolateral views of stage 16 wt (top panels) and homozygous Δ\u003cem\u003etup\u003c/em\u003eADME+HDME embryos (bottom panels) carrying the Vg1-4-moeGFP reporter, stained for tropomyosin 2 (red) and GFP (green). Four consecutive segments are shown. Vg1-4-moeGFP is strongly expressed in the DA2 and weakly the DA3 muscles. Right panels are schematic representations of the position, shape, and segmental attachments of the DA1 (red), DA2 (green), and DA3 (light green) muscles in three consecutive segments (S\u003csub\u003en-1\u003c/sub\u003e, S\u003csub\u003en\u003c/sub\u003e, and S\u003csub\u003en+1\u003c/sub\u003e). The intersegmental boundaries are represented by dashed black lines. In absence of \u003cem\u003etup\u003c/em\u003e transcription (Δ\u003cem\u003etup\u003c/em\u003eADME+HDME; Vg1-4-moeGFP embryos), the DA1 and DA2 shapes and attachment sites are strongly abnormal and the matching of DA3-DA2, DA2-DA1 intersegmental attachment sites vastly disturbed \u003cstrong\u003e(B)\u003c/strong\u003e Quantification of the relative proportions of normal and altered DA1 and DA2 muscles in wt (red) and Δ\u003cem\u003etup\u003c/em\u003eADME+HDME (blue). 10 embryos of each genotype were analyzed and the morphology of 76 DA2 and 77 DA1 muscles was recorded in wt segments, and 67 DA1 and 52 DA1 muscles in Δ\u003cem\u003etup\u003c/em\u003eADME+HDME segments.\u003c/p\u003e","description":"","filename":"fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6063601/v1/9955805d8b225ff4a14f3cc1.jpg"},{"id":78948256,"identity":"8a5711da-77cf-4ca0-bf6f-cc76d85ee96a","added_by":"auto","created_at":"2025-03-21 08:17:08","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":151879,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eMuscle mismatching in ΔtupADME+HDME L3 larvae.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Left, scanning electron microscopy of filleted wt and Δ\u003cem\u003etup\u003c/em\u003eADME+HDME larvae showing dorso-lateral muscles in two consecutive segments. Right, schematic representations of the position, shape and attachment sites of DA1 (red), DA2 (green), and DA3 (light green) muscles. In the absence of \u003cem\u003etup\u003c/em\u003e, there is muscle attachment sites mismatching at the intersegmental boundary (dashed black line) \u003cstrong\u003e(B)\u003c/strong\u003e Quantification of the relative proportions of normal and altered DA1 and DA2 muscles in wt (red) and Δ\u003cem\u003etup\u003c/em\u003eADME+HDME (blue) larvae. 6 wt and 11 Δ\u003cem\u003etup\u003c/em\u003eADME+HDME were analyzed, and the morphology of DA2 and DA1 muscles recorded in 31 wt and 55 Δ\u003cem\u003etup\u003c/em\u003eADME+HDME segments, respectively.\u003c/p\u003e","description":"","filename":"fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6063601/v1/027709f7cf69be82243a5d76.jpg"},{"id":90344844,"identity":"f90e560c-13bb-4077-a5ba-8bd39d711d1c","added_by":"auto","created_at":"2025-09-01 16:05:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2234971,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6063601/v1/da30bda7-2135-4186-bd6c-1cb75e411e14.pdf"},{"id":78947678,"identity":"711a53ea-4023-4f04-b6ca-512906163fb6","added_by":"auto","created_at":"2025-03-21 08:09:07","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":136592,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eFigure sup1: Conserved sequences and transcription factor binding sites in the tupF4 Cis-Regulatory Module.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003etup\u003c/em\u003eF4 sequence was annotated using EvoPrinter, a comparative genomics tool designed to identify conserved DNA sequences shared among several orthologous DNAs. Black capital letters represent bases in the \u003cem\u003eDrosophila melanogaster\u003c/em\u003ereference sequence that are conserved in the orthologous sequences of other \u003cem\u003eDrosophila\u003c/em\u003especies: \u003cem\u003eD. simulans\u003c/em\u003e, \u003cem\u003eD. sechellia\u003c/em\u003e, \u003cem\u003eD. yakuba\u003c/em\u003e, \u003cem\u003eD. erecta\u003c/em\u003e, \u003cem\u003eD. pseudoobscura\u003c/em\u003e, and \u003cem\u003eD. persimilis\u003c/em\u003e. Transcription factor binding sites were predicted using the JASPAR core database, an open-access resource of curated, non-redundant transcription factor binding profiles. Binding sites for Org-1 (blue) and Tin (grey) are highlighted. The blue box indicates a cluster of mesodermal transcription factor binding sites identified in previous ChIP-seq experiments (ChIP Tinman and ChIP Meso TFs). The coordinates of the \u003cem\u003etup\u003c/em\u003eF4 cis-regulatory module (CRM) are chr2L:18892809-18894301 (FlyBase release: r6.53). The nucleotide sequences of PCR primers used to amplify the \u003cem\u003etup\u003c/em\u003eF4 CRM are underlined in red.\u003c/p\u003e","description":"","filename":"figsup1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6063601/v1/c97c0b44b6858ee1bee11baf.jpg"},{"id":78948935,"identity":"ac11071e-dd81-40d5-aa90-cc4e9d413462","added_by":"auto","created_at":"2025-03-21 08:25:07","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":131727,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eFigure sup2: Conserved sequences and transcription factor binding sites in the tupADME Cis-Regulatory Module.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003etup\u003c/em\u003eADME sequence was annotated as indicated in \u003cstrong\u003eFig.S1\u003c/strong\u003e. Binding sites for Org-1 (blue), Tup/Islet-1 (yellow), Mef2 (purple), and Twist (green) are highlighted. The coordinates of the \u003cem\u003etup\u003c/em\u003eADME cis-regulatory module (CRM) are chr2L:18899345-18900688 (FlyBase release: r6.53). The nucleotide sequences of PCR primers used to amplify the \u003cem\u003etup\u003c/em\u003eADME CRM are underlined in red.\u003c/p\u003e","description":"","filename":"figsup2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6063601/v1/aebdaafd0dde1c93360093d9.jpg"},{"id":78947677,"identity":"eea5ba58-512a-4e4d-a8d6-e9b6aae3f636","added_by":"auto","created_at":"2025-03-21 08:09:07","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":57466,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eFigure sup3: Expression of GMR 76E06 and GMR 76E11 delineate a new tup cis-regulatory module.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e GMR 76E06 and 76E11 expression during embryonic stages 11 to 16 (nuclear LacZ immunostaining). Both GMRs are expressed in the heart and dorsal muscles. At stage 16, GMR 76E06 is expressed in the DA1/DO1 and DA2/DO2 muscles, while GMR 76E11 expression is restricted to DA1/DO1 (white box). \u003cstrong\u003e(B)\u003c/strong\u003e GMR 76E06 and 76E11 sequences partly overlap. The newly identified CRM \u003cem\u003eTup\u003c/em\u003eHDME (in red) spans this overlap and the predicted binding sites for the transcription factors Tup/Islet-1 (Tup) and Tinman (Tin).\u003c/p\u003e","description":"","filename":"figsup3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6063601/v1/6619efdee51636addb709c95.jpg"},{"id":78947693,"identity":"95091f55-4124-410c-9910-753bf0f0a5d1","added_by":"auto","created_at":"2025-03-21 08:09:08","extension":"jpg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":159069,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eFigure sup4: Conserved sequences and transcription factor binding sites in the tupHDME Cis-Regulatory Module.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003etup\u003c/em\u003eHDME sequence was annotated as indicated in \u003cstrong\u003eFig.S1\u003c/strong\u003e. Binding sites for Tup/Islet-1 (yellow) and Tin (grey) binding sites are highligthed. A cluster of binding sites for mesodermal transcription factors, determined by previous ChIP-seq experiments (ChIP Tinman and ChIP Meso TFs), is indicated by the blue box. The coordinates of the \u003cem\u003etup\u003c/em\u003eHDME-CRM are chr2L:18905826-18907523 (FlyBase release: r6.53). Nucleotide sequences of the PCR primers used to amplify the \u003cem\u003etup\u003c/em\u003eHDME CRM are underlined in red. The primer used to delete the three Tup binding sites and generate the \u003cem\u003etup\u003c/em\u003eHDMEDTup construct for autoregulation experiments (see \u003cstrong\u003eFig.2A\u003c/strong\u003e) is underlined in grey.\u003c/p\u003e","description":"","filename":"figsup4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6063601/v1/705528346478385996cc367c.jpg"},{"id":78948937,"identity":"a4fc10f7-9a23-49e3-9583-c471a1149acd","added_by":"auto","created_at":"2025-03-21 08:25:07","extension":"jpg","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":98236,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eFigure sup5: Combinatorial activity of the three tup CRMs in different mesodermal cell derivatives.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Dorsal view of the expression patterns of \u003cem\u003etup\u003c/em\u003eF4-moeGFP, \u003cem\u003etup\u003c/em\u003eADME-moeGFP and \u003cem\u003eTup\u003c/em\u003eHDME-moeGFP CRMs in late embryos (st 16) immunostained with an anti-GFP antibody. Depending on the CRM observed, expression is detected in the pharyngeal muscles (pm), alary muscles (AM), and the lymph gland (LG) (top pannels), in cardiac cells (CC) or pericardiac cells (pc) (bottom pannels). \u003cstrong\u003e(B)\u003c/strong\u003eThe table summarizes the expression of \u003cem\u003etup\u003c/em\u003eF4, \u003cem\u003etup\u003c/em\u003eADME and \u003cem\u003etup\u003c/em\u003eHDME in mesodermal tissues during the embryonic development (from stage 13 to stage 16).\u003c/p\u003e","description":"","filename":"figsup5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6063601/v1/6421baa997d6985a307c902c.jpg"},{"id":78948251,"identity":"433b55bd-5f31-4e7c-aed1-9fab4c177a8b","added_by":"auto","created_at":"2025-03-21 08:17:07","extension":"jpg","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":127015,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eFigure sup6: Autoregulation by Tup maintains tupADME and tupHDME activity in dorsal muscles.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003etup\u003c/em\u003eF4 \u003cstrong\u003e(A)\u003c/strong\u003e, \u003cem\u003etup\u003c/em\u003eADME \u003cstrong\u003e(B)\u003c/strong\u003e and \u003cem\u003etup\u003c/em\u003eHDME \u003cstrong\u003e(C)\u003c/strong\u003e LacZ reporter expression in wild-type (\u003cem\u003e+\u003c/em\u003e) and \u003cem\u003etup\u003c/em\u003e\u003csup\u003e\u003cem\u003eex4\u003c/em\u003e\u003c/sup\u003e mutant embryos lacking Tup protein visualized by \u003cem\u003ein situ\u003c/em\u003e hybridization using a \u003cem\u003eLacZ\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eprobe. \u003cstrong\u003e(A) \u003c/strong\u003e\u003cem\u003etup\u003c/em\u003eF4-LacZ activity in promuscular clusters and progenitor cells is independent of Tup. \u003cstrong\u003e(B-C)\u003c/strong\u003e Both activity of \u003cem\u003etup\u003c/em\u003eADME \u003cstrong\u003e(B)\u003c/strong\u003e and \u003cem\u003etup\u003c/em\u003eHDME \u003cstrong\u003e(C)\u003c/strong\u003e is lost in dorsal muscles in \u003cem\u003etup\u003c/em\u003e\u003csup\u003e\u003cem\u003eex4\u003c/em\u003e\u003c/sup\u003e mutant embryos, showing that Tup is required.\u003c/p\u003e","description":"","filename":"figsup6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6063601/v1/bc746bf93886dbf4a230759c.jpg"},{"id":78948249,"identity":"e4a232d1-5736-482c-83d2-90b88f5d645e","added_by":"auto","created_at":"2025-03-21 08:17:07","extension":"jpg","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":226749,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eFigure sup7: tup transcription during embryonic development.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003etup\u003c/em\u003e transcription revealed by \u003cem\u003ein situ\u003c/em\u003e hybridization (green signal) in stage 10 to 16 embryos immuno-stained for Krüppel (Kr), blue, which labels amniosera cells (amn), and the dorsal muscles DA1 and DO1 in addition to lateral (LM) muscle lineages.\u003cem\u003etup\u003c/em\u003e is transcribed in amn, cardiac cells (cc), pericardial cells (pc), dorsal muscles (DM), and alary muscles (AM) up to stage 14, before being restricted to cc and pc cells and AMs, stage 16. Kr is initially expressed in the amniosera at stage 10, followed by expression in DA1 and DO1 progenitor cells at stages 11–12. Later in development, Kr expression is restricted to the trachea (tr). The bottom panels are enlarged views of the yellow framed regions, designated A, B and C\u003cem\u003e. \u003c/em\u003e\u003cstrong\u003e(A, B)\u003c/strong\u003e At stages 12 \u003cstrong\u003e(A)\u003c/strong\u003e and 13 \u003cstrong\u003e(B)\u003c/strong\u003e, \u003cem\u003etup\u003c/em\u003e is transcribed (green dots) in the nuclei of the DA1 and DO1 founder cells identified by Kr expression and the AM founder cell. \u003cstrong\u003e(C)\u003c/strong\u003e At stage 14, multiple nuclei of the DA1 and DO1 (and AM) developing muscles actively transcribe \u003cem\u003etup\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"figsup7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6063601/v1/573b89da54d4b850df42b9c2.jpg"},{"id":78948936,"identity":"1bed2fd6-9b09-4c20-ace0-532af60bd9f1","added_by":"auto","created_at":"2025-03-21 08:25:07","extension":"jpg","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":156472,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eTable1: Expression patterns of Janelia (GMR) and Vienna Tiles (VT) lines.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eSummary of Janelia (GMR) and Vienna Tiles (VT) lines used to characterize the cis-regulatory sequences of Tup/Islet-1, with expression in different mesodermal tissues indicated by (x). A total of 26 lines were tested (21 Janelia and 5 VT) covering 50 kb of \u003cem\u003etup\u003c/em\u003e genomic region (see also \u003cstrong\u003eFig.1B\u003c/strong\u003e). Five lines displayed expression in somatic muscles (x in red): lines 76B03, 76B07, 76B01, 76E11, and 76E06, two of which, 76B01 and 76B07 showed also expression in alary muscles (AMs).\u003c/p\u003e","description":"","filename":"table1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6063601/v1/e2df5e48bdf1e012c491d07e.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Multiple Cis-Regulatory Modules ensure robust tup/islet1 function in dorsal muscle identity specification","fulltext":[{"header":"Background","content":"\u003cp\u003eAnimals display an assembly of skeletal muscles of various sizes and shapes that underlies/allows specific movements. In most cases, multiple muscles are cooperatively involved. The diversity of muscles raises the question of how individual muscles acquire the necessary shapes and specific attachment sites during development. In \u003cem\u003eDrosophila\u003c/em\u003e larvae, muscles are single multinucleated fibers which develop by fusion of one founder cell with fusion-competent myoblasts. The identity of each founder cell (FC) is determined by its unique combination of identity transcription factors (iTFs) and by the precise timing and levels at which those factors are expressed (Baylies et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; de Joussineau et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Dubois et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). FC identity specification is a multi-step process, starting with the delineation of promuscular clusters (PMCs), groups of equipotent cells from which progenitor cells (PCs) are selected. PCs then divide asymmetrically, giving rise to two different FCs, or, in a few cases, one FC and another lineage (Bate, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Deng et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Expression of different iTFs in different FCs starts with iTF activation in PMCs in response to positional information and progressive refinement in PCs and FCs through cross-regulations between different iTFs and response to Notch signaling and Hox information (Carmena et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Carmena et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Enriquez et al., 2010; Dubois et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). During the fusion process, the specific iTF code expressed by the founder cell (FC) is transferred to the nuclei of fusion-competent myoblasts (FCMs) in the syncytium, effectively reprogramming FCMs to adopt FC identity (Knirr et al., 1999; Crozatier and Vincent, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Bataill\u0026eacute; et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). This reprogramming is thought to activate downstream genes that control the final muscle morphology (Bataill\u0026eacute; et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Junion and Jagla, 2022) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe development of functional muscles in \u003cem\u003eDrosophila melanogaster\u003c/em\u003e therefore relies on precise spatial and temporal transcriptional control of each iTF expression (Dubois et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Central to this regulatory architecture are cis-regulatory modules (CRMs); short DNA sequences that integrate inputs from multiple transcription factors/signaling pathways to finely regulate gene expression. CRMs essentially determine when, where, and how much a gene is expressed, providing both flexibility and robustness to developmental gene expression (Zinzen et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Bonn et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Kvon et al., 2021). Developmental genes typically display multiple CRMs, often spread across large genomic regions (Visel et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Schoenfelder and Fraser, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Originally, it was thought that CRMs each drive distinct spatiotemporal aspects of gene expression, following a model of modular regulation (Andersson and Sandelin, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Field and Adelman, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Furlong and Levine, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Long et al. 2016; Shlyueva et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). This was an oversimplified model, however, since, in many cases CRMs regulating the same gene exhibit overlapping spatiotemporal activity, leading to the notion of cumulative regulation including by primary and shadow enhancers (Hoch et al., 1990; Jeong et al., 2006; Zeitlinger et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Bothma et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Cannavo et al., 2016; Whitney et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe LIM-homeodomain transcription factor Tailup (Tup) is the \u003cem\u003eDrosophila\u003c/em\u003e ortholog of vertebrate Islet1. We have shown that embryos carrying null \u003cem\u003etup\u003c/em\u003e mutations (tup\u003csup\u003eex4\u003c/sup\u003e) display a disorganized dorsal musculature, indicating that \u003cem\u003etup\u003c/em\u003e plays a key role in establishing the stereotypical pattern of the dorsal muscles of \u003cem\u003eDrosophila\u003c/em\u003e larvae (Boukhatmi et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). However, the pleiotropy of tup\u003csup\u003eex4\u003c/sup\u003e mutant phenotypes, including germ band retraction failure and defects in cardiogenesis and hematopoietic organ (lymph gland, LG) formation (Tao et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Mann et al., 2009) precluded precise analysis of muscle patterning defects. To overcome this difficulty, we undertook to further characterize the \u003cem\u003etup\u003c/em\u003e regulatory region, in order to identify CRMs which could be used to generate and characterize muscle-specific \u003cem\u003etup\u003c/em\u003e mutants.\u003c/p\u003e \u003cp\u003eHere, we first report that transcription of \u003cem\u003etup\u003c/em\u003e in 4 dorsal larval muscles and the alary muscles (AM) relies upon three distinct CRMs spread over 26 kb of regulatory region. Two CRMs, namely \u003cem\u003etupADME\u003c/em\u003e and \u003cem\u003etupHDME\u003c/em\u003e, function in a partially redundant manner to maintain robust \u003cem\u003etup\u003c/em\u003e expression in the dorsal FCs and their growing muscle syncytia, and this involves Tup direct autoregulation. The third CRM, \u003cem\u003etupF4\u003c/em\u003e (Tao et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), initiates \u003cem\u003etup\u003c/em\u003e transcription in the two dorsal progenitors that give rise to these four founder cells, as well as in the alary muscle lineage, but appears not strictly required to trigger \u003cem\u003etup\u003c/em\u003e autoregulation in FCs. CRISPR-Cas9\u0026ndash;mediated deletions of late-acting \u003cem\u003etup\u003c/em\u003e muscle CRMs show that \u003cem\u003etup\u003c/em\u003e function in specifying dorsal muscle identity relies on the combined activity of these two CRMs. Loss of \u003cem\u003etup\u003c/em\u003e transcription in growing muscle syncitia, leads to mis-matching of dorsal muscles attachment sites at segmental borders.\u003c/p\u003e \u003cp\u003eIn summary, this study provides new insights into the transcriptional regulation of tup/Islet1 during Drosophila muscle development. By dissecting the contributions of individual CRMs, we propose a model in which sequential and lineage-specific activity of different CRMs allows for the progressive acquisition of muscle identity, ensuring robust muscle patterning. These findings contribute to a broader understanding of how multiple CRM-mediated gene regulation orchestrates complex developmental processes and provides resilience against genetic or environmental perturbations.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eFly strains\u003c/h2\u003e \u003cp\u003eAll \u003cem\u003eDrosophila melanogaster\u003c/em\u003e stocks and genetic crosses were grown using standard medium at 25\u0026deg;C. All the lines were provided by the Bloomington Drosophila Stock Center except \u003cem\u003etup\u003c/em\u003e\u003csup\u003e\u003cem\u003eex4\u003c/em\u003e\u003c/sup\u003e (de Navascues and Modolell, 2007). Lines used are \u003cem\u003ewhite\u003c/em\u003e\u003csup\u003e\u003cem\u003e[1118]\u003c/em\u003e\u003c/sup\u003e (BDSC_3605), \u003cem\u003evasa-cas9\u003c/em\u003e\u003csup\u003e\u003cem\u003eVK00027\u003c/em\u003e\u003c/sup\u003e (BDSC_51324 ) and the 21 \u003cem\u003etup\u003c/em\u003e Janelia-Gal4 lines (GMR) (Pfeiffer and al., 2008) listed in the \u003cb\u003eTable\u0026nbsp;1\u003c/b\u003e. We also used 5 Vienna tiles enhancer-Gal4 lines (VT) from the Vienna Drosophila Resource Center (Kvon et al., 2014) listed in the \u003cb\u003eTable\u0026nbsp;1\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCRM deletions generated by Crispr/Cas9\u003c/h3\u003e\n\u003cp\u003eGenomic \u003cem\u003etup\u003c/em\u003e target sites were identified using \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://tools.flycrispr.molbio.wisc.edu/targetFinder/\u003c/span\u003e\u003cspan address=\"http://tools.flycrispr.molbio.wisc.edu/targetFinder/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (Gratz et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Prior to final selection of RNA guides (gRNA) for deletions of \u003cem\u003etup\u003c/em\u003e CRMs, genomic PCR and sequencing of DNA from \u003cem\u003evasa-cas9\u003c/em\u003e\u003csup\u003e\u003cem\u003eVK00027\u003c/em\u003e\u003c/sup\u003e flies was performed to check for polymorphisms in the targeted regions. Guides targeting \u003cem\u003etup\u003c/em\u003eF4, \u003cem\u003etup\u003c/em\u003eADME and \u003cem\u003etup\u003c/em\u003eHDME were inserted in the pCFD4: U6:3-gRNA vector (Addgene n\u003csup\u003eo\u003c/sup\u003e: 49411) as described (Port et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), (see \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.crisprflydesign.org/wpcontent/uploads/2014/06/Cloning-with-pCFD4.pdf\u003c/span\u003e\u003cspan address=\"http://www.crisprflydesign.org/wpcontent/uploads/2014/06/Cloning-with-pCFD4.pdf\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). All guides were verified by sequencing. The sequences of the oligonucleotides used to construct each gRNA expression plasmid are:\u003c/p\u003e \u003cp\u003e \u003cem\u003etup\u003c/em\u003eF4: gRNA1: 5\u0026rsquo;-TTGTTGGCACTCCGATCTGAAGG-3\u0026rsquo; and gRNA2: 5\u0026rsquo;-TTGTCTGC GGCAAGCGTCGAAGG-3\u0026rsquo;;\u003c/p\u003e \u003cp\u003e \u003cem\u003etup\u003c/em\u003eADME: gRNA1: 5\u0026rsquo;-GCAGCCCTGATCCTGACCGTTGG-3' and gRNA2: 5\u0026rsquo;-GGCAG ATTTAGTCCGTCAGTCGG-3';\u003c/p\u003e \u003cp\u003e \u003cem\u003etup\u003c/em\u003eHDME: gRNA1: 5\u0026rsquo;-CTCTTTAAAGGGAAGCTCAACC-3' and gRNA2: 5'-CACCAAC TGGAGTGCCAGTGCC-3'\u003c/p\u003e \u003cp\u003eTo delete the core region of \u003cem\u003etupF4, tupADME and tupHDME, vasa-cas9\u003c/em\u003e embryos were microinjected with gRNAs in pCFD4 (200 ng/\u0026micro;l). Each adult hatched from an injected embryo was crossed to the balancer stock \u003cem\u003esna\u003c/em\u003e\u003csup\u003e\u003cem\u003eSco\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/\u003c/em\u003eCyO, {\u003cem\u003ewg\u003c/em\u003e\u003csup\u003e\u003cem\u003een11\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-LacZ\u003c/em\u003e} and 100\u0026ndash;200 F1 flies were individually tested for either \u003cem\u003etup\u003c/em\u003e CRM deletion by PCR on genomic DNA.\u003c/p\u003e\n\u003ch3\u003eReporter constructs, immunohistochemistry, in situ hybridization\u003c/h3\u003e\n\u003cp\u003eThe \u003cem\u003eyellow\u003c/em\u003e intron (\u003cem\u003eyi\u003c/em\u003e), FlyBase ID #FBgn0004034 (position: 356918\u0026ndash;359616) was inserted in the \u003cem\u003elacZ\u003c/em\u003e coding region between aa (Tyr 952) and aa (Ser 953) by standard PCR-based cloning position (Carayon et al, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The resulting fragment was cloned downstream of \u003cem\u003etup\u003c/em\u003eCRM (\u003cem\u003etup\u003c/em\u003eF4, \u003cem\u003etup\u003c/em\u003eHDME and \u003cem\u003etup\u003c/em\u003eADME) inserted in a pAttB vector, and micro-injected in embryos for chromosomal insertion at position 68A4. Antibody staining and \u003cem\u003ein situ\u003c/em\u003e hybridization with intronic probes were as described previously (Dubois et al, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Primary antibodies were: rabbit anti-Kr (1/300), mouse anti-LacZ (1/1000; Promega), rabbit anti-GFP (1/1000; Torrey Pines Biolabs), chicken anti-GFP (1/500; Abcam), Phalloidine-Texas RedX (1/500; Thermofisher Scientific). Secondary antibodies were: Alexa Fluor 488-, 555- and 647- conjugated antibodies (1/300; Molecular Probes) and biotinylated goat anti-mouse (1/2000; Vector Laboratories). Digoxygenin-labelled antisense RNA probes were transcribed \u003cem\u003ein vitro\u003c/em\u003e from PCR-amplified DNA sequences, using T7 polymerase (Roche Digoxigenin labelling Kit).\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn situ\u003c/em\u003e Hybridization were done as described (Dubois et al, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). When antibody staining and FISH were combined, the standard immuno-histochemistry protocol was performed first, with 1U/\u0026micro;l of RNase inhibitor from Promega included in all solutions, followed by the FISH protocol. Confocal sections were acquired on Leica SP8 or SPE microscopes at 40x magnifications, 1024x1024 pixels resolution. Images were assembled using ImageJ and Photoshop softwares.\u003c/p\u003e\n\u003ch3\u003ePhenotype quantification at embryonic and larval stages\u003c/h3\u003e\n\u003cp\u003eTo quantify embryonic phenotypes, wt; VgM1-moeGFP and Δ\u003cem\u003etup\u003c/em\u003eADME\u0026thinsp;+\u0026thinsp;HDME; VgM1-moeGFP embryos were immunostained with a primary mouse anti-GFP (1/500) (Roche) and secondary biotinylated goat anti-mouse (1/2000) (VECTASTAIN\u0026reg; ABC Kit). Stained embryos were imaged using a Nikon eclipse 80i microscope and a Nikon digital camera DXM 1200C. A minimum 50 abdominal segments (n) of stage 16 embryos were analyzed for each genotype. (wt: 10 embryos; Δ\u003cem\u003etup\u003c/em\u003eADME\u0026thinsp;+\u0026thinsp;HDME: 10 embryos). To quantify larval phenotypes, wandering L3 larvae were analyzed. 6 WT and 11 Δ\u003cem\u003etup\u003c/em\u003eADME\u0026thinsp;+\u0026thinsp;HDME larvae were used. Morphologies of DA2 and DA1 muscles were examined in 31 segments (WT) and in 55 segments (Δ\u003cem\u003etup\u003c/em\u003eADME\u0026thinsp;+\u0026thinsp;HDME) by scanning electron microscopy.\u003c/p\u003e\n\u003ch3\u003eScanning Electron Microscopy (SEM)\u003c/h3\u003e\n\u003cp\u003eTo prepare fillets, third instar wild type and homozygous Δ\u003cem\u003etup\u003c/em\u003eADME\u0026thinsp;+\u0026thinsp;HDME larvae raised at 25\u0026deg;C were dissected in myorelaxant buffer, according to (Yalgin et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Larvae were cut longitudinally on the ventral side to preserve and expose the dorsal and dorso-lateral musculature. Fillets were then fixed 1 hour in a 4% formaldehyde/ 2.5% glutaraldehyde mixture in 1X PBS, washed in water and dehydrated gradually in ethanol. Fillets were dried at the critical point (Leica EM CPD 300 critical point apparatus), covered with a platinum layer (Leica EM MED 020 metalliser) and imaged with a Quanta 250 FEG FEI scanning microscope.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eThree separate tup CRMs regulate tup expression in dorsal skeletal muscles\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eTup is expressed in the muscle progenitor cells (PCs) at the origin of the four dorsal-most somatic muscles of the \u003cem\u003eDrosophila\u003c/em\u003e embryo and larva: DA1, DA2, DO1, and DO2 (Boukhatmi et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Two cis-regulatory modules (CRMs) have previously been associated to this expression: \u003cem\u003etup\u003c/em\u003eF4, a 1.5-kb DNA fragment located between \u0026minus;\u0026thinsp;13.5 and \u0026minus;\u0026thinsp;12 kb upstream of the \u003cem\u003etup\u003c/em\u003e transcription start site drives \u003cem\u003etup\u003c/em\u003e expression in various cell types issued from the dorsal mesoderm: lymph gland cells, alary muscles (AMs), pericardial cells, and cardioblasts, in addition to skeletal muscles (Tao et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2007\u003c/span\u003e); \u003cem\u003etup\u003c/em\u003eADME, a 1,3-kb fragment located between \u0026minus;\u0026thinsp;19.8 and \u0026minus;\u0026thinsp;18.5 kb upstream of the transcription start site which drives \u003cem\u003etup\u003c/em\u003e expression only in the alary muscles and dorsal somatic muscles (Boukhatmi et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). These two CRMs were identified through \u003cem\u003ein silico\u003c/em\u003e search of evolutionarily conserved sequence blocks and ChiP-seq-experiments targeting mesodermal transcription factors (MesoTFs), including Tinman (Tin) (Philippakis et al., 2006; Zinzen et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Jin et al., 2013; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and \u003cb\u003eFig.S1-S2\u003c/b\u003e). Both \u003cem\u003etup\u003c/em\u003eF4 and \u003cem\u003etup\u003c/em\u003eADME also contain conserved Org-1 binding sites critical for regulating \u003cem\u003etup\u003c/em\u003e transcription in the alary muscles (Boukhatmi et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). ChIP-seq analyses identified a third cluster of MesoTFs binding sites upstream of \u003cem\u003etup\u003c/em\u003eADME, suggesting the existence of an additional mesodermal \u003cem\u003etup\u003c/em\u003eCRM. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). To sustain \u003cem\u003ein silico\u003c/em\u003e analyses, we screened the full set of publicly available (GMR and VT) expression reporter lines (Pfeiffer et al., 2008; Kvon et al., 2014) covering 55-kb of \u003cem\u003etup\u003c/em\u003e genomic region (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Five GMR lines, GMR76B01, GMR76B03, GMR76B07, GMR76E06 and GMR76E11 showed expression in heart cells, alary muscles and dorsal muscles (\u003cb\u003eTable\u0026nbsp;1)\u003c/b\u003e. GMR76B03 overlaps with \u003cem\u003etup\u003c/em\u003eF4, while GMR76B01 and GMR76B07 overlap with \u003cem\u003etup\u003c/em\u003eADME (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and \u003cb\u003eTable\u0026nbsp;1)\u003c/b\u003e. The other lines, GMR76E06 and GMR76E11 displayed expression in heart cells and dorsal muscles, identifying and additional \u003cem\u003etup\u003c/em\u003e mesodermal CRM located between \u0026minus;\u0026thinsp;26.7 and \u0026minus;\u0026thinsp;25 kb upstream of the \u003cem\u003etup\u003c/em\u003e transcription start site, \u003cem\u003ei.e\u003c/em\u003e, around 5 kbp upstream of \u003cem\u003etup\u003c/em\u003eADME (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and \u003cb\u003eTable\u0026nbsp;1\u003c/b\u003e). Interestingly, GMR76E11 showed delayed and weaker expression in DA2 and DO2 muscles than GMR76E06 (\u003cb\u003eFig.S3\u003c/b\u003e), suggesting that GMR76E06 contains regulatory information absent in GMR76E11. In support of this, DNA sequence analysis indicated that GMR76E06 contains evolutionarily conserved Tup binding sites not present in GMR76E11, while both reporters contain Tin binding sites (\u003cb\u003eFig.S4\u003c/b\u003e). Based on sequence and expression data, a new reporter construct was designed to both encompass conserved sequence blocks shared by GMR76E06 and GMR76E11 and the Tup binding sites (\u003cb\u003eFig.S4\u003c/b\u003e). Expression analysis of this construct confirmed activity both in heart cells and dorsal muscles, leading us to name it \u003cem\u003etup\u003c/em\u003eHDME (Heart and Dorsal Muscles Enhancer) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eWe then compared the activity of \u003cem\u003etup\u003c/em\u003eF4, \u003cem\u003etup\u003c/em\u003eADME, and \u003cem\u003etup\u003c/em\u003eHDME at different embryonic stages, using moe-GFP as reporter (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The expression of \u003cem\u003etup\u003c/em\u003eF4-moeGFP in promuscular clusters (st10-11), suggested that it plays an early role in early \u003cem\u003etup\u003c/em\u003e mesodermal expression. From embryonic stages 13 to 15, the three CRMs showed overlapping expression in dorsal muscles precursors, suggesting a relay of the proximal tup activating CRM by the distal CRMs. By stage 15, \u003cem\u003etup\u003c/em\u003eF4-moeGFP expression began to decrease in DA1 and DO1, to become undetectable in all dorsal muscles by stage 16. Dorsal views of stage 16 embryos showed both \u003cem\u003etup\u003c/em\u003eF4-moeGFP and \u003cem\u003etup\u003c/em\u003eHDME-moeGFP expression in the lymph gland, pericardial cells, and cardiomyocytes, and \u003cem\u003etup\u003c/em\u003eADME-moeGFP expression only in Svp-positive cardioblasts which give rise to ostiae (Molina and Cripps, 2001; Tao et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) (\u003cb\u003eFig.S5A\u003c/b\u003e). On top of that, \u003cem\u003etup\u003c/em\u003eF4-moeGFP and \u003cem\u003etup\u003c/em\u003eADME-moeGFP expression is detected in the alary muscles, whereas \u003cem\u003etup\u003c/em\u003eHDME-moeGFP expression is detected in anterior pharyngeal muscles (\u003cb\u003eFig.S5A, B\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eIn summary, characterisation of the \u003cem\u003etup\u003c/em\u003e cis-regulatory landscape shows three distinct CRMs, \u003cem\u003etup\u003c/em\u003eF4, \u003cem\u003etup\u003c/em\u003eADME, and \u003cem\u003etup\u003c/em\u003eHDME, scattered within 26kb of \u003cem\u003etup\u003c/em\u003e upstream DNA, contribute to control \u003cem\u003etup\u003c/em\u003e transcription in different mesodermal tissues in \u003cem\u003eDrosophila\u003c/em\u003e embryos (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and S5B). Their partly overlapping patterns of activity, both spatially and temporally, suggests that these three CRMs combinatorically ensure precise control of \u003cem\u003etup\u003c/em\u003e expression in somatic muscles and AM, as well as heart and lymph gland cells during embryonic development.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDirect autoregulation of tupHDME in dorsal muscles.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe previously showed that \u003cem\u003etup\u003c/em\u003e transcription in dorsal muscles is lost in Tup protein null (tup\u003csup\u003eex4\u003c/sup\u003e) embryos, indicating a positive autoregulation mechanism (Boukhatmi et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). To investigate whether Tup protein directly regulates its own expression through the three CRMs, we performed \u003cem\u003ein situ\u003c/em\u003e hybridization using a LacZ probe in tup\u003csup\u003eex4\u003c/sup\u003e mutant embryos carrying LacZ reporter gene driven by each individual CRM (\u003cb\u003eFig.S6\u003c/b\u003e). We observed that \u003cem\u003etup\u003c/em\u003eF4 activity in dorsal muscles is unaffected by the loss of Tup protein (\u003cb\u003eFig.S6A\u003c/b\u003e), consistent with the absence of Tup binding sites in this CRM (\u003cb\u003eFig.S1\u003c/b\u003e). In contrast, \u003cem\u003etup\u003c/em\u003eADME (\u003cb\u003eFig.S6B\u003c/b\u003e) and \u003cem\u003etup\u003c/em\u003eHDME (\u003cb\u003eFig.S6C\u003c/b\u003e) showed a complete loss of LacZ transcription in the dorsal muscles, indicating that Tup protein is required for the activation of these CRMs. Both \u003cem\u003etup\u003c/em\u003eADME and \u003cem\u003etup\u003c/em\u003eHDME contain predicted Tup binding sites (\u003cb\u003eFig.S2\u003c/b\u003e and \u003cb\u003eS4\u003c/b\u003e), suggesting that Tup directly autoregulates its expression through one or both these CRMs in the embryos. In support of this, previous work showed that mutation of the single \u003cem\u003etup\u003c/em\u003eADME Tup binding site reduced \u003cem\u003etup\u003c/em\u003eADME activity in dorsal muscles, while not in alary muscles (Boukhatmi et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). \u003cem\u003etup\u003c/em\u003eHDME contains three clustered conserved Tup binding sites (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cb\u003eFig. S4\u003c/b\u003e). To determine whether \u003cem\u003etup\u003c/em\u003eHDME is also subject to autoregulation, we generated a modified \u003cem\u003etup\u003c/em\u003eHDME reporter construct lacking all three predicted Tup binding sites, \u003cem\u003etup\u003c/em\u003eHDME\u003csup\u003eΔTup\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). We found that \u003cem\u003etup\u003c/em\u003eHDME\u003csup\u003eΔTup\u003c/sup\u003e activity was completely lost in dorsal muscles while maintained in heart cells and the lymph gland (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), showing that Tup autoregulation is required for \u003cem\u003etup\u003c/em\u003eHDME activity in developing dorsal muscles.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn conclusion, these results demonstrate that Tup autoregulation is essential for maintaining \u003cem\u003etup\u003c/em\u003e expression specifically in dorsal muscles by directly binding to the \u003cem\u003etup\u003c/em\u003eHDME in addition of \u003cem\u003etup\u003c/em\u003eADME CRM. However, \u003cem\u003etup\u003c/em\u003e expression in other mesodermal tissues, such as heart cells and the lymph gland, occurs independently of Tup protein, suggesting that other combinations of transcription factors, which include Org-1, tunes the level of \u003cem\u003etup\u003c/em\u003e expression in different mesodermal tissues.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCombinatorial regulation of tup transcription in dorsal muscles.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo further investigate temporal and lineage-specific aspects of \u003cem\u003etup\u003c/em\u003e regulation by different CRMs and overcome limitations associated with reporter protein stability, we compared the patterns of endogenous and reporter nascent transcripts using \u003cem\u003ein situ\u003c/em\u003e hybridization with intronic probes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). We first examined endogenous \u003cem\u003etup\u003c/em\u003e transcription throughout embryonic development, using a probe against the first \u003cem\u003etup\u003c/em\u003e intron. To help identifying individual dorsal muscles, we immunostained embryos for Kr\u0026uuml;ppel (Kr) which marks the DA1, DO1 (and LL1) progenitor cells, founder cells and muscle precursors, in addition to amnioserosa cells (Beckett and Baylies, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Dobi et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) (\u003cb\u003eFig.S7\u003c/b\u003e). \u003cem\u003eIn situ\u003c/em\u003e data confirmed that \u003cem\u003etup\u003c/em\u003e transcription starts at the promuscular stage, embryonic stage 10 and is maintained in dorsal muscles until stages 14\u0026ndash;15. Overlap between tup nascent transcripts and Kr immunostaining from stages 12 to 14 (\u003cb\u003eFig.S7\u003c/b\u003e - yellow frames A, B, C) confirmed that \u003cem\u003etup\u003c/em\u003e is transcribed in the nuclei of DA1 and DO1 founder cells and later in the nuclei of developing DA1 and DO1 fibers. By stage 16, \u003cem\u003etup\u003c/em\u003e transcription was no longer observed in dorsal muscles, while persisting in cardiac and pericardial cells and the alary muscles. At stage 16, Kr expression is also lost in the muscle fibers and solely detected in the trachea, which runs internal to the somatic musculature.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo precisely determine which aspects of \u003cem\u003etup\u003c/em\u003e transcription correlated with each \u003cem\u003etup\u003c/em\u003e CRM, we introduced a \u003cem\u003eyellow\u003c/em\u003e intron into the LacZ reporter coding region (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Using dual \u003cem\u003ein situ\u003c/em\u003e hybridization for intronic probes enabled us to precisely compare endogenous \u003cem\u003etup\u003c/em\u003e transcription (\u003cem\u003etup\u003c/em\u003e\u003csup\u003e\u003cem\u003eint\u003c/em\u003e\u003c/sup\u003e) and each \u003cem\u003etup\u003c/em\u003eCRM-driven transcription (\u003cem\u003etup\u003c/em\u003eF4\u003csup\u003eint\u003c/sup\u003e, \u003cem\u003etup\u003c/em\u003eADME\u003csup\u003eint\u003c/sup\u003e and \u003cem\u003etup\u003c/em\u003eHDME\u003csup\u003eint\u003c/sup\u003e) at each step of muscle development. To facilitate their identification, DA1/DO1 nuclei were labeled by Kr immunostaining. The results showed that only \u003cem\u003etup\u003c/em\u003eF4 is active in promuscular clusters, stage 10, confirming its role in initiating \u003cem\u003etup\u003c/em\u003e transcription. By stage 14, \u003cem\u003etup\u003c/em\u003eF4-driven transcription was no longer detected in dorsal muscles, while it persisted in cardiac and pericardial cells, and alary muscles. \u003cem\u003etup\u003c/em\u003eHDME activity was first detected at stage 11, in dorsal muscle progenitor cells, whereas \u003cem\u003etup\u003c/em\u003eADME activity was detected later, in dorsal muscles founder cells (FCs), stage 12 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Unlike \u003cem\u003etup\u003c/em\u003eF4, \u003cem\u003etup\u003c/em\u003eHDME and \u003cem\u003etup\u003c/em\u003eADME kept being active during myofiber elongation, stage 14. \u003cem\u003etup\u003c/em\u003eADME and \u003cem\u003etup\u003c/em\u003eHDME-driven transcription was no more detected in dorsal muscles by stage 16 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Together, these findings show that \u003cem\u003etup\u003c/em\u003eF4 is active in dorsal promuscular clusters and that \u003cem\u003etup\u003c/em\u003eADME and \u003cem\u003etup\u003c/em\u003eHDME regulate \u003cem\u003etup\u003c/em\u003e transcription during muscle fiber development, with partial temporal overlap between the three CRMS (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Previous analysis of dorso-lateral muscle lineages has shown that the birth time of different muscle FCs follows a precise sequence and that temporal windows of a given iTF expression may differ in different muscle lineages (Dubois et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). To determine the lineage-specificity of individual \u003cem\u003etup\u003c/em\u003eCRMs, we focused our analysis on two key stages of muscle development: stage 12, when FC identity is specified (Frasch, 1999; De Joussineau et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Dubois et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), and stage 15, when FC transcriptional identity has been propagated to nuclei of fused myoblasts, a process known as identity reprogramming of syncytial nuclei (Crozatier and Vincent, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Dubois et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Bataill\u0026eacute; et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Kr immunostaining and positional information were used to identify each dorsal muscle lineage at stage 12 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). \u003cem\u003etup in situ\u003c/em\u003e confirmed \u003cem\u003etup\u003c/em\u003e transcription in the four dorsal muscle FCs, and initiation of \u003cem\u003etup\u003c/em\u003e transcription in the nuclei of \u0026ldquo;na\u0026iuml;ve\u0026rdquo; myoblasts (FCMs) incorporated into developing dorsal muscles. It also showed that \u003cem\u003etup\u003c/em\u003eF4 remains active in the DA2 and DO2 (and AM) FCs and not the dorsal-most DA1 and DO1 FCs. At that stage, both \u003cem\u003etup\u003c/em\u003eADME and \u003cem\u003etup\u003c/em\u003eHDME become active in the four dorsal FCs. By stage 15, the dynamics have shifted, with \u003cem\u003etup\u003c/em\u003eF4 remaining active only in DO2, \u003cem\u003etup\u003c/em\u003eADME being active in all four muscle lineages, and \u003cem\u003etup\u003c/em\u003eHDME active only in the DO1 and DO2 lineages (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). As documented above, (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), the temporal relay between early and late CRMs in dorsal muscles involves direct Tup autoregulation, a mechanism which operates neither in heart nor lymph gland cells, nor in the alary muscles.\u003c/p\u003e \u003cp\u003eTaken together, analysis of nascent endogenous and reporter transcripts revealed a temporal, lineage-specific sequence of \u003cem\u003etup\u003c/em\u003e regulation during \u003cem\u003eDrosophila\u003c/em\u003e muscle development. \u003cem\u003etup\u003c/em\u003eF4-driven early \u003cem\u003etup\u003c/em\u003e transcription in promuscular clusters is relayed by \u003cem\u003etup\u003c/em\u003eADME and \u003cem\u003etup\u003c/em\u003eHDME activities which ensure propagation of \u003cem\u003etup\u003c/em\u003e transcription to fused nuclei during muscle fiber formation in a lineage-specific manner.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDeletion of tupADME and tupHDME impairs tup transcription in dorsal muscles.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo investigate the individual roles of each \u003cem\u003etup\u003c/em\u003e cis-regulatory module (CRM) and their combined effect on somatic muscle identity, we generated dorsal muscle-specific \u003cem\u003etup\u003c/em\u003e mutants by using CRISPR-Cas9 to delete the muscle-specific \u003cem\u003etup\u003c/em\u003e CRMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Neither individual CRM deletion resulted in germ band retraction failure, allowing to analyse in detail muscle development. As a first step, we analysed \u003cem\u003etup\u003c/em\u003e transcription in embryos homozygous for each CRM deletion (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Global analysis at stages 12 and 14 showed that deletion of neither individual CRMs was sufficient to eliminate \u003cem\u003etup\u003c/em\u003e transcription in developing muscles. The detection of \u003cem\u003etup\u003c/em\u003e transcription in Δ\u003cem\u003etup\u003c/em\u003eF4 embryos, indicated that, while subject to direct autoregulation, \u003cem\u003etup\u003c/em\u003eADME or/and \u003cem\u003etup\u003c/em\u003eHDME activity does not depend upon earlier \u003cem\u003etupF4\u003c/em\u003e activity. \u003cem\u003etup\u003c/em\u003e transcription in at least a fraction of dorsal muscle nuclei, was detected as well at stage 14 upon deletion of either \u003cem\u003etup\u003c/em\u003eADME or \u003cem\u003etup\u003c/em\u003eHDME, suggesting their redundancy. In contrast, \u003cem\u003etup\u003c/em\u003e transcription was lost stage 14 in the double \u003cem\u003etup\u003c/em\u003eADME and \u003cem\u003etup\u003c/em\u003eHDME (Δ\u003cem\u003etup\u003c/em\u003eADME\u0026thinsp;+\u0026thinsp;HDME) deletion mutant (clear arrowheads), indicating that these two CRMs are together required for propagating transcriptional identity of FCs during reprogramming of fused FCMs into growing syncitia (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDeletion of tupADME and tupHDME disrupts the dorsal muscle pattern.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSince \u003cem\u003etup\u003c/em\u003e transcription in developing muscles was only abolished in the double Δ\u003cem\u003etupADME\u003c/em\u003e\u0026thinsp;+\u0026thinsp;Δ\u003cem\u003etupHDME\u003c/em\u003e CRM deletion mutants, we focused our analysis on muscle morphology in these mutants in late embryos and third instar larvae, as the new \u003cem\u003etup\u003c/em\u003e alleles did not display the embryonic lethality and pleiotropic effects typically associated with complete \u003cem\u003etup\u003c/em\u003e loss-of-function. We first analysed the muscle patterns in stage 16 embryos, using Tropomyosin 2 immuno-staining of the somatic musculature together with the VgM1-moeGFP reporter line (Carayon et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) which allows morphological inspection of the DA2 and DA3 muscle lineages. In wt embryos, VgM1-moeGFP expression illustrates the staggered rows pattern of DA muscles, with the posterior attachment sites of the DA2 and DA3 muscles of one segment facing the anterior attachments of DA1 and DA2, respectively, in the next segment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), a pattern repeated between successive adjacent segments reflecting muscle attachment sites matching (Carayon et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Δ\u003cem\u003etup\u003c/em\u003eADME\u0026thinsp;+\u0026thinsp;HDME mutant embryos displayed a disorganised dorsal muscle pattern (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), showing that \u003cem\u003etup\u003c/em\u003eADME/HDME combinatorial activity is required for \u003cem\u003etup\u003c/em\u003e role in dorsal muscle identity. Detailed analysis or VgM1-moeGFP expression shows that the DA1 and the DA2 muscles are misshaped or absent in 59.6% (n\u0026thinsp;=\u0026thinsp;31) and 34.3% (n\u0026thinsp;=\u0026thinsp;23) of segments, respectively, compared to 5.2% and 5.3%, respectively in wt-type embryos (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B). Δ\u003cem\u003etupADME\u003c/em\u003e\u0026thinsp;+\u0026thinsp;Δ\u003cem\u003etupHDME\u003c/em\u003e mutant embryos exhibit a partial transformation of the DA2 muscle into a DA3 identity, rather than the complete transformation observed in \u003cem\u003etup\u003c/em\u003e null embryos (Boukhatmi et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Misalignment of DA1/DA2 and DA2/DA3 are illustrated by schematic drawings across three consecutive segments S\u003csub\u003en\u0026minus;1\u003c/sub\u003e, S\u003csub\u003en\u003c/sub\u003e, and S\u003csub\u003en+1\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDorsal muscle attachment matching is disrupted in ΔtupADME\u0026thinsp;+\u0026thinsp;HDME 3rd instar larvae.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo further investigate muscle morphological defects due to Δ\u003cem\u003etup\u003c/em\u003eADME\u0026thinsp;+\u0026thinsp;HDME CRMs deletion, we analysed the somatic musculature of third instar larvae using scanning electron microscopy (SEM) of dissected fillets (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). This allows precise investigation of the DA muscles attachments (Carayon et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). As previously described, the staggered ends pattern of dorsal muscles established in wt late embryos (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) is maintained in wt larvae (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). In Δ\u003cem\u003etup\u003c/em\u003eADME\u0026thinsp;+\u0026thinsp;HDME larvae, the muscle attachment matching is severely disrupted, with defects in DA1 and DA2 muscle morphology and attachment sites observed in 52.7% (n\u0026thinsp;=\u0026thinsp;29) and 43.6% of segments (n\u0026thinsp;=\u0026thinsp;24), respectively, compared to 6.5% and 3.2%, respectively, in wt embryos (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Beside the loss of dorsal attachment of the DA1 muscle, one striking phenotype is the homotypic (DA2/DA2) attachment of DA2 muscles in consecutive segments, while only heterotypic DA3/DA2 and DA2/DA1 matchings are observed in wt embryos. This observation is consistent with an identity shift of the DA2 muscle towards a DA3 muscle identity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe similarity between the late embryonic and 3rd instar larval phenotypes confirm the key role of Tup is establishing the correct morphological identity of dorsal muscles during embryogenesis. Together with previous analysis of mutants for another DA identity gene, collier (Carayon et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), analysis of muscle-specific \u003cem\u003etup\u003c/em\u003e mutations shows that the precise heterotypic matching of muscles attachment sites at segmental borders is a sensitive read-out of muscle transcriptional identity.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eA stereotyped set of 30 somatic muscles in each abdominal segment underlies \u003cem\u003eDrosophila\u003c/em\u003e larval crawling (Bate, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). Each muscle morphology reflects expression of a specific combination of \"identity transcription factors\" (iTFs) by its founder cell (Frasch, 1999; De Joussineau et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Dubois et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). A subset of iTFs is already activated in PMCs from which PCs and FCs are selected. For example, the PCs which give rise to the DA2 and DA3 FCs are both selected from a PMC expressing Tinman (NKx2.5), Collier (Col/Kn) and Tup. It is the sequential birth of the DA2 and DA3 PCs which determines that Tup remains expressed in the DA2 PC and FC and Col in the DA3 FC (Enriquez et al., 2010; Boukhatmi et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Here, we further investigated the regulation and role of Tup/Islet1 in PCs/FCs seeding the formation of dorsal muscles.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCRM redundancy and robustness of tup expression in the dorsal mesoderm.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe identified a third, distal, \u003cem\u003etup\u003c/em\u003e CRM, which we named Heart and Dorsal Muscles (\u003cem\u003etup\u003c/em\u003eHDME) CRM, which regulates \u003cem\u003etup\u003c/em\u003e transcription in the dorsal mesoderm. Comparison with the previously known CRMs, \u003cem\u003etup\u003c/em\u003eF4 and \u003cem\u003etup\u003c/em\u003eADME (Tao et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Boukhatmi et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), using detailed transcriptional analyses with endogenous and reporter intronic probes shows that each CRM displays a specific timing of activity during dorsal muscle development. Both \u003cem\u003etup\u003c/em\u003eHDME and \u003cem\u003etup\u003c/em\u003eADME control \u003cem\u003etup\u003c/em\u003e transcription in dorsal muscles beyond the FC step. CRISPR-Cas9-mediated deletion of either CRM individually did not result in significant disruption of muscle \u003cem\u003etup\u003c/em\u003e expression, whereas deletion of both supressed \u003cem\u003etup\u003c/em\u003e transcription at late stages of muscle development (stages 14\u0026ndash;16). This, at least partial, CRM redundancy suggests that robust \u003cem\u003etup\u003c/em\u003e function is key in specifying skeletal muscle identities.\u003c/p\u003e \u003cp\u003eThe redundancy and robustness of \u003cem\u003etup\u003c/em\u003e regulation \u003cem\u003evia\u003c/em\u003e multiple CRMs could be part of a more general mechanism of transcriptional control during muscle development. Other transcription factors, either \"generic\", such as Mef2 (Nguyen and Xu, 1998; Sandmann et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), or specific to a subset of skeletal muscles (iTFs) like \u003cem\u003eCollier\u003c/em\u003e (Enriquez et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2012\u003c/span\u003e;) are regulated by multiple CRMs working sequentially or/and synergistically during muscle development, an evolutionary strategy for ensuring a precise level of expression across different stages of development (Reddington et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Kvon et al., 2021). This could be part of a general mechanism for robustness of integrating generic and identity aspects of muscle development (Bataille et al., 2017). In case of \u003cem\u003etup\u003c/em\u003e, each of skeletal muscle CRM is also active in other mesodermal derivatives which differ between CRMs. An evolutionarily selection of this combinatorial set up could be essential to coordinate development of a stereotypical muscle pattern and development of the heart and associated tissues, the cardiac outflow, valves cells, AMs and lymph gland (Tao et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Zmojdzian and Jagla, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Boukhatmi et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Meyer et al., 2023).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAutoregulation of tup expression in dorsal muscles.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eInterestingly, the deletion of the early-active \u003cem\u003etup\u003c/em\u003eF4 CRM did not abolish \u003cem\u003etup\u003c/em\u003e transcription or perturb dorsal muscle patterning, suggesting that \u003cem\u003etup\u003c/em\u003e transcription at the PMC stage is not critical for dorsal muscle identity. Moreover, since \u003cem\u003etup\u003c/em\u003eHDME activity, which overlaps with \u003cem\u003etup\u003c/em\u003eF4 at the PC stage, remains unaffected by \u003cem\u003etup\u003c/em\u003eF4 deletion, this rules out a simple handover mechanism initiating \u003cem\u003etup\u003c/em\u003eHDME autoregulation. One possible explanation is that the same upstream transcription factors (TFs) or chromatin-opening factors bind to both \u003cem\u003etupF4\u003c/em\u003e and \u003cem\u003etupHDME\u003c/em\u003e, but at different times or concentrations, with \u003cem\u003etupHDME\u003c/em\u003e maintaining \u003cem\u003etup\u003c/em\u003e transcription via its direct autoregulatory sites (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). On support of this, Chip-SEQ analyses have shown Tin binding to both \u003cem\u003etup\u003c/em\u003eF4 and \u003cem\u003etup\u003c/em\u003eHDME in 4\u0026ndash;6 hours embryos, Twi sequentially binds to \u003cem\u003etup\u003c/em\u003eF4 and \u003cem\u003etup\u003c/em\u003eADME in 2\u0026ndash;4 and 6\u0026ndash;8 hour embryos, respectively and Mef2 sequentially binds to \u003cem\u003etup\u003c/em\u003eF4 and \u003cem\u003etup\u003c/em\u003eADME in 4\u0026ndash;6 and 6\u0026ndash;12 hour embryos, respectively (Zinzen et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Philippakis et al., 2013; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://furlonglab.embl.de/\u003c/span\u003e\u003cspan address=\"http://furlonglab.embl.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e tissue_specific_DHS). We attempted to confirm these observations by generating a double CRISPR-Cas9-mediated deletion of \u003cem\u003etup\u003c/em\u003eF4 and \u003cem\u003etup\u003c/em\u003eHDME, but for technical reasons, we were unable to collect double mutant individuals.\u003c/p\u003e \u003cp\u003eThis is not the first occurence where deletion of a CRM driving iTF expression in PMCs does not lead to muscle patterning defects. Similar results were indeed observed in studying the respective roles of early (PMC) and late (FC/muscle) \u003cem\u003ecol\u003c/em\u003e CRMs. In this case as well, the early CRM was not required to prime autoregulation (Carayon et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This raises important questions about the specific role of early-active CRMs in muscle identity specification within specific subsets of muscle, while highlighting the pivotal role of late-active CRMs \u003cem\u003evia\u003c/em\u003e autoregulation in maintaining and propagating muscle identity. Whether there is a specific role of iTFs expression in PMC prior to the process of PC/FC identity specification remains to be fully elucidated. Our results support a model of muscle identity specification that progressively refined at each stage of muscle development - PMC to PC, PC to FC, and FC to syncytial fiber - through distinct CRM activities. This temporal refinement through separate regulatory elements may ensure that muscle identity is established and maintained through each critical step of muscle development.\u003c/p\u003e \u003cp\u003eSustained \u003cem\u003etup\u003c/em\u003e transcription in dorsal muscles is dependent on a positive autoregulatory mechanism exerted on both \u003cem\u003etup\u003c/em\u003eADME (Boukhatmi et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) and \u003cem\u003etup\u003c/em\u003eHDME (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Yet, while \u003cem\u003etup\u003c/em\u003eHDME deleted of its Tup binding sites is inactive in dorsal muscles, it remains active in heart cells or the lymph gland, indicating a tissue specific autoregulation. \u003cem\u003etup\u003c/em\u003e autoregulation mediated by \u003cem\u003etup\u003c/em\u003eADME is also skeletal muscle-specific and is not exerted in AMs. In turn, \u003cem\u003etupHDME\u003c/em\u003e, is not active in AMs, consistent with the absence of Org-1/Tbx1 binding sites which directly control \u003cem\u003etup\u003c/em\u003eADME activity in these peculiar heart-associated muscles (Boukhatmi et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Other transcription factors may regulate \u003cem\u003etup\u003c/em\u003e expression in cardiac cells or the lymph gland, including Tinman, Twist, and/or Bagpipe (Zinzen et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Sandmann et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Azpiazu and Frasch, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). Future understanding of the regulatory roles of other TF binding motifs present within combinations of \u003cem\u003etup\u003c/em\u003e enhancers and enhancer-promoter interactions will certainly benefit from newly developed technologies such as Quantitative enhancer-FACS-Seq analysis (Waters et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and Capture-C in purified myogenic cells (Pollex et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eImpact of tup transcription loss on muscle patterning and alignment.\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePrevious analysis of tup null (\u003cem\u003etup\u003c/em\u003e\u003csup\u003e\u003cem\u003eex4\u003c/em\u003e\u003c/sup\u003e) embryos, using a DA3 muscle marker Collier, revealed a DA2-to-DA3 (DA2\u0026thinsp;\u0026gt;\u0026thinsp;DA3) identity shift. \u003cem\u003etup\u003c/em\u003e\u003csup\u003e\u003cem\u003eex4\u003c/em\u003e\u003c/sup\u003e embryonic lethality, including germ band retraction defects, precluded, however detailing this identity shift at the morphological level (Boukhatmi et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Expression of the VgM1-moeGFP reporter in double \u003cem\u003etup\u003c/em\u003eCRM mutants allowed to visualise the DA2\u0026thinsp;\u0026gt;\u0026thinsp;DA3 transformation in fully developed embryos (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eIn wild-type embryos and larvae, the DA2 muscle of segment S\u003csub\u003en\u003c/sub\u003e aligns with the DA3 muscle of S\u003csub\u003en\u0026minus;1\u003c/sub\u003e and the DA1 muscle of segment S\u003csub\u003en+1\u003c/sub\u003e, at intersegmental borders (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA; Carayon et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, in the double \u003cem\u003etup\u003c/em\u003eADME and \u003cem\u003etup\u003c/em\u003eHDME deletion mutants, this DA3-DA2, DA2-DA1 intersegmental matching is often replaced by homotypic alignment between DA2 muscles in consecutive segments (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B). Residual heterotypic matching could possibly reflect either the late timing of loss of \u003cem\u003etup\u003c/em\u003e transcription, in other terms, residual expression driven by \u003cem\u003etupF4\u003c/em\u003e or/and the involvement of other iTFs expressed in DA muscles FCs (Dubois et al., 2017). DA muscle mismatching at intersegmental borders persists until the end of larval development (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B), confirming that the specific morphology of somatic muscles at work in larval crawling, which includes muscle growth, elongation and choice of tendon and/or muscle attachment sites, reflects the combination of iTFs expressed by muscle FCs in early embryos.\u003c/p\u003e \u003cp\u003eThe DA3-DA2-DA1 misalignments mirror those observed in \u003cem\u003ecollier\u003c/em\u003e (\u003cem\u003ecol\u003c/em\u003e) muscle CRMs mutants, which show a DA3-to-DA2 identity shift with DA3-DA3 homotypic matching replacing DA3-DA2 matching at intersegmental segmental borders (Carayon et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The similarity of the symmetrically opposite \u003cem\u003etup\u003c/em\u003e and \u003cem\u003ecol\u003c/em\u003e dorsal muscle phenotypes shows that the col\u0026thinsp;\u0026gt;\u0026thinsp;tup transcriptional regulation which ensures that Tup is expressed in the DA2 FC and Col in the DA3 FC (Boukhatmi et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), is critical for the proper alignment and orientation of dorsal muscles and larval locomotion. The defects observed in Δ\u003cem\u003etup\u003c/em\u003eADME\u0026thinsp;+\u0026thinsp;HDME mutants further underscore the importance of coordinated CRM activity in establishing a stereotypical muscle pattern, with in the case of \u003cem\u003etup\u003c/em\u003e, \u003cem\u003etup\u003c/em\u003eADME and \u003cem\u003etup\u003c/em\u003eHDME redundant function ensuring robustness to this pattern.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe involvement of multiple CRMs for precise \u003cem\u003etup\u003c/em\u003e regulation in skeletal muscles, similar to previously reported for \u003cem\u003ecol\u003c/em\u003e, another TF critical for muscle identity and optimal larval locomotion (Enriquez et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Dubois et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Carayon et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) supports that temporal cascade strategies provide robustness to transcription control of muscle identity, and resilience of species-specific muscle patterns against genetic or environmental perturbations. Future work should investigate the physiological consequences of muscle mismatching observed in \u003cem\u003etup\u003c/em\u003e mutants. Additionally, exploring the interaction of \u003cem\u003etup\u003c/em\u003e with other muscle-specific transcription factors and their associated CRMs may uncover further complexities in the regulatory networks that govern muscle identity and development.\u003c/p\u003e "},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003eCRM\u003c/strong\u003e - Cis-Regulatory Module\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eiTFs\u003c/strong\u003e - Identity Transcription Factors\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFC\u003c/strong\u003e - Founder Cell\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFCM\u003c/strong\u003e - Fusion-Competent Myoblast\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePMCs\u003c/strong\u003e - Promuscular Clusters\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePCs\u003c/strong\u003e - Progenitor Cells\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003etup\u003c/strong\u003e - \u003cem\u003eTailup\u003c/em\u003e (also known as \u003cem\u003eIslet1\u003c/em\u003e in vertebrates)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003etup\u003csup\u003eex4\u003c/sup\u003e\u003c/strong\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003e- \u003cem\u003etup\u003c/em\u003e mutant allele\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMef2\u003c/strong\u003e - Myocyte Enhancer Factor 2\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCol\u003c/strong\u003e - Collier\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTin\u003c/strong\u003e - Tinman\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTwi\u003c/strong\u003e - Twist\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDA\u003c/strong\u003e - Dorsal Acute Muscles\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDO\u003c/strong\u003e - Dorsal Oblique Muscle 1\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHDME\u003c/strong\u003e - Heart and Dorsal Muscles Enhancer\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eADME\u003c/strong\u003e \u0026ndash; Alary and Dorsal Muscles Enhancer\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAMs\u003c/strong\u003e - Alary Muscles\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRISPR-Cas9\u003c/strong\u003e - Clustered Regularly Interspaced Short Palindromic Repeats and Caspase 9-associated protein\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003egRNA\u003c/strong\u003e - Guide RNA (used for CRISPR/Cas9 gene editing)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChIP-Seq\u003c/strong\u003e - Chromatin Immunoprecipitation Sequencing\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAvailability of data and materials:\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCompeting interests:\u003c/em\u003e\u003c/strong\u003e The authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/strong\u003e:\u0026nbsp;This work was supported by CNRS, Association Fran\u0026ccedil;aise contre les Myopathies (AFM) Research Grant 21887, ANR grant 13-BSVE2-0010-01.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthors\u0026apos; contributions:\u0026nbsp;\u003c/em\u003e\u003c/strong\u003eJ-LF managed the project. LD and J-LF conceptualized and designed the experiments. AP, AC, YC, LD and J-LF performed the experiments. AP, YC, LD, CS and J-LF analysed the data and prepared the figures for the manuscript. J-LF wrote the manuscript with input from co-authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAcknowledgements:\u0026nbsp;\u003c/em\u003e\u003c/strong\u003eThe authors sincerely thank Alain Vincent, whose initiative led to this project. They are grateful for his unwavering commitment, valuable assistance in writing and proofreading, and insightful advice and discussions.\u003c/p\u003e\n\u003cp\u003eWe thank the Bloomington Stock Center and the Vienna Drosophila Resource Center for \u003cem\u003eDrosophila\u003c/em\u003e strains, and Julien Favier, \u003cem\u003eDrosophila\u003c/em\u003e embryos microinjection platform from the Center of Integrative Biology in Toulouse \u0026ndash; France.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAndersson R, Sandelin A. Determinants of enhancer and promoter activities of regulatory elements. Nat Rev Genet. 2020;21:71-87. doi:10.1038/s41576-019-0173-8.\u003c/li\u003e\n\u003cli\u003eAzpiazu N, Frasch M. tinman and bagpipe: two homeobox genes that determine cell fates in the dorsal mesoderm of Drosophila. 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Quantitative-enhancer-FACS-seq (QeFS) reveals epistatic interactions among motifs within transcriptional enhancers in developing \u003cem\u003eDrosophila\u003c/em\u003e tissue. \u003cem\u003eGenome Biol\u003c/em\u003e. 2021;22:348. doi:10.1186/s13059-021-02574-x.\u003c/li\u003e\n\u003cli\u003eWhitney PH, Shrestha B, Xiong J, Zhang T, Rushlow CA. Shadow enhancers modulate distinct transcriptional parameters that differentially affect downstream patterning events. \u003cem\u003eDevelopment\u003c/em\u003e. 2022;149:dev200940. doi:10.1242/dev.200940.\u003c/li\u003e\n\u003cli\u003eYalgin C, Karim MR, Moore AW. Immunohistological labeling of microtubules in sensory neuron dendrites, tracheae, and muscles in the \u003cem\u003eDrosophila\u003c/em\u003e larva body wall. \u003cem\u003eJ Vis Exp\u003c/em\u003e. 2011;10:3662. doi:10.3791/3662.\u003c/li\u003e\n\u003cli\u003eZeitlinger J, Zinzen RP, Stark A, Kellis M, Zhang H, Young RA, Levine M. Whole-genome ChIP-chip analysis of Dorsal, Twist, and Snail suggests integration of diverse patterning processes in the \u003cem\u003eDrosophila\u003c/em\u003e embryo. \u003cem\u003eGenes Dev\u003c/em\u003e. 2007;21:385-390. doi:10.1101/gad.1509607.\u003c/li\u003e\n\u003cli\u003eZinzen RP, Girardot C, Gagneur J, Braun M, Furlong EE. Combinatorial binding predicts spatio-temporal cis-regulatory activity. \u003cem\u003eNature\u003c/em\u003e. 2009;462:65\u0026ndash;70. doi:10.1038/nature08531.\u003c/li\u003e\n\u003cli\u003eZmojdzian M, Jagla K. Tailup plays multiple roles during cardiac outflow assembly in \u003cem\u003eDrosophila\u003c/em\u003e. \u003cem\u003eCell Tissue Res\u003c/em\u003e. 2013;354:639-645. doi:10.1007/s00441-013-1644-4.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e\n"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"skeletal-muscle","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"skem","sideBox":"Learn more about [Skeletal Muscle](http://skeletalmusclejournal.biomedcentral.com/)","snPcode":"13395","submissionUrl":"https://submission.nature.com/new-submission/13395/3","title":"Skeletal Muscle","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Myogenesis, Enhancers, Transcriptional regulation, Multiple CRMs, Muscle identity, Muscle patterning","lastPublishedDoi":"10.21203/rs.3.rs-6063601/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6063601/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e The development of functional muscles in \u003cem\u003eDrosophila melanogaster\u003c/em\u003e relies on precise spatial and temporal transcriptional control, orchestrated by complex gene regulatory networks. Central to this regulation are cis-regulatory modules (CRMs), which integrate inputs from transcription factors to fine-tune gene expression during myogenesis. In this study, we investigate the transcriptional regulation of the LIM-homeodomain transcription factor Tup (Tailup/Islet-1), a key regulator of dorsal muscle development.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e Using a combination of CRISPR-Cas9-mediated deletion and transcriptional analyses, we examined the role of multiple CRMs in regulating \u003cem\u003etup\u003c/em\u003eexpression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e We demonstrate that \u003cem\u003etup\u003c/em\u003e expression is controlled by multiple CRMs that function redundantly to maintain robust \u003cem\u003etup\u003c/em\u003e transcription in dorsal muscles. These mesodermal \u003cem\u003etup\u003c/em\u003e CRMs act sequentially and differentially during the development of dorsal muscles and other tissues, including heart cells and alary muscles. We show that activity of the two late-acting CRMs govern late-phase \u003cem\u003etup\u003c/em\u003e expression through positive autoregulation, whereas an early enhancer initiates transcription independently. Deletion of both late-acting CRMs results in muscle identity shifts and defective muscle patterning. Detailed morphological analyses reveal muscle misalignments at intersegmental borders.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e Our findings underscore the importance of CRM-mediated autoregulation and redundancy in ensuring robust and precise \u003cem\u003etup\u003c/em\u003eexpression during muscle development. These results provide insights into how multiple CRMs coordinate gene regulation to ensure proper muscle identity and function.\u003c/p\u003e","manuscriptTitle":"Multiple Cis-Regulatory Modules ensure robust tup/islet1 function in dorsal muscle identity specification","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-21 08:09:02","doi":"10.21203/rs.3.rs-6063601/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-22T10:36:42+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-14T14:19:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"306572859109569517772878293598065148451","date":"2025-03-24T10:44:22+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-21T16:26:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"138897329260471595863580804193386586224","date":"2025-02-25T11:45:31+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-02-25T11:32:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-02-25T11:30:26+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-02-20T11:42:42+00:00","index":"","fulltext":""},{"type":"submitted","content":"Skeletal Muscle","date":"2025-02-19T11:16:39+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"skeletal-muscle","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"skem","sideBox":"Learn more about [Skeletal Muscle](http://skeletalmusclejournal.biomedcentral.com/)","snPcode":"13395","submissionUrl":"https://submission.nature.com/new-submission/13395/3","title":"Skeletal Muscle","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2a8b008b-b27e-4a7e-9979-f8c92e5afe72","owner":[],"postedDate":"March 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-09-01T15:59:31+00:00","versionOfRecord":{"articleIdentity":"rs-6063601","link":"https://doi.org/10.1186/s13395-025-00392-4","journal":{"identity":"skeletal-muscle","isVorOnly":false,"title":"Skeletal Muscle"},"publishedOn":"2025-08-25 15:57:07","publishedOnDateReadable":"August 25th, 2025"},"versionCreatedAt":"2025-03-21 08:09:02","video":"","vorDoi":"10.1186/s13395-025-00392-4","vorDoiUrl":"https://doi.org/10.1186/s13395-025-00392-4","workflowStages":[]},"version":"v1","identity":"rs-6063601","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6063601","identity":"rs-6063601","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}