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

Aurore Pelletier, Alexandre Carayon, Yannick Carrier, Coralie Sengenès, Laurence Dubois, Jean-Louis Frendo

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Aurore Pelletier, Alexandre Carayon, Yannick Carrier, Coralie Sengenès, Laurence Dubois, et al. Multiple Cis-Regulatory Modules ensure robust tup/islet1 function in dorsal muscle identity specification. 2025. [hal-05384131](https://hal.science/hal-05384131v1)

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

**Aurore Pelletier**

Centre de Biologie Intégrative, Université de Toulouse, CNRS, France

**Alexandre Carayon**

Centre de Biologie Intégrative, Université de Toulouse, CNRS, France

**Yannick Carrier**

Centre de Biologie Intégrative, Université de Toulouse, CNRS, France

**Coralie Sengenès**

RESTORE: A geroscience and rejuvenation research center, Université de Toulouse, CNRS

**Laurence Dubois**

RESTORE: A geroscience and rejuvenation research center, Université de Toulouse, CNRS

**Jean-Louis Frendo**

jean-louis.frendo@univ-tlse3.fr

RESTORE: A geroscience and rejuvenation research center, Université de Toulouse, CNRS

## Research Article

**Keywords:** Myogenesis, Enhancers, Transcriptional regulation, Multiple CRMs, Muscle identity, Muscle patterning

**Posted Date:** March 21st, 2025

**DOI:** https://doi.org/10.21203/rs.3.rs-6063601/v1

**License:** © Ⓟ This work is licensed under a Creative Commons Attribution 4.0 International License.

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**Additional Declarations:** No competing interests reported.
**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.

**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 (PMC), 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 PMC 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; Union and Jagla, 2022) (Fig. 1A).

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 syncytia, 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*<sup>ex4</sup> (de Navascues and Modolell, 2007). Lines used are *white*<sup>[1118]</sup> (BDSC_3605), *vasa-cas9<sup>VK00027</sup>* (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<sup>VK00027</sup>* flies was performed to check for polymorphisms in the targeted regions. Guides targeting *tupF4*, *tupADME* and *tupHDME* were inserted in the pCFD4: U6:3-gRNA vector (Addgene n°: 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:

*tupF4*: gRNA1: 5'-TTGTTGGCACTCCGATCTGAAGG-3' and gRNA2: 5'-TTGTCTGC GGCAAGCGTCGAAGG-3';

*tupADME*: gRNA1: 5'-GCAGCCCTGATCCTGACCGTTGG-3' and gRNA2: 5'-GGCAG ATTTAGTCCGTCAGTCGG-3';

*tupHDME*: 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<sup>Sco</sup>/CyO, {wg<sup>en11</sup>-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 (*y*I), 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 *tupCRM* (*tupF4*, *tupHDME* and *tupADME*) 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). Digoxigenin-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. 1A). Two cis-regulatory modules (CRMs) have previously been associated to this expression: *tupF4*, 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); *tupADME*, 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. 1B). 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. 1B and Fig.S1-S2). Both *tupF4* and *tupADME* 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 *tupADME*, suggesting the existence of an additional mesodermal *tupCRM*. (Fig. 1B). 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. 1B). Five GMR lines, GMR76B01, GMR76B03, GMR76B07, GMR76E06 and GMR76E11 showed expression in heart cells, alary muscles and dorsal muscles (**Table 1**). GMR76B03 overlaps with *tupF4*, while GMR76B01 and GMR76B07 overlap with *tupADME* (Fig. 1B 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 *tupADME* (Fig. 1B 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 *tupHDME* (Heart and Dorsal Muscles Enhancer) (Fig. 1B, 1C).

We then compared the activity of *tupF4*, *tupADME*, and *tupHDME* at different embryonic stages, using moe-GFP as reporter (Fig. 1C). The expression of *tupF4-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, *tupF4-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 *tupF4-moeGFP* and *tupHDME-moeGFP* expression in the lymph gland, pericardial cells, and cardiomyocytes, and *tupADME-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, *tupF4-moeGFP* and *tupADME-moeGFP* expression is detected in the alary muscles, whereas *tupHDME-moeGFP* expression is detected in anterior pharyngeal muscles (Fig.S5A, B).

In summary, characterisation of the *tup* cis-regulatory landscape shows three distinct CRMs, *tupF4*, *tupADME*, and *tupHDME*, 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*<sup>ex4</sup>) 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*<sup>ex4</sup> mutant embryos carrying LacZ reporter gene driven by each individual CRM (Fig.S6). We observed that *tupF4* 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, *tupADME* (Fig.S6B) and *tupHDME* (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 *tupADME* and *tupHDME* 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 *tupADME* Tup binding site reduced *tupADME* activity in dorsal muscles, while not in alary muscles (Boukhatmi et al., 2014). *tupHDME* contains three clustered conserved Tup binding sites (Fig. 2A and Fig. S4). To determine whether *tupHDME* is also subject to autoregulation, we generated a modified *tupHDME* reporter construct lacking all three predicted Tup binding sites, *tupHDME*<sup>ΔTup</sup> (Fig. 2A). We found that *tupHDME*<sup>ΔTup</sup> activity was completely lost in dorsal muscles while maintained in heart cells and the lymph gland (Fig. 2B), showing that Tup autoregulation is required for *tupHDME* 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 *tupHDME* in addition of *tupADME* 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. 3A). 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*F4int, *tup*ADMEint and *tup*HDMEint) 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. 3A). 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. 3A). 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. 3B). 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. 3C). *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 *tupF4* remains active in the DA2 and DO2 (and AM) FCs and not the dorsal-most DA1 and DO1 FCs. At that stage, both *tupADME* and *tupHDME* become active in the four dorsal FCs. By stage 15, the dynamics have shifted, with *tupF4* remaining active only in DO2, *tupADME* being active in all four muscle lineages, and *tupHDME* active only in the DO1 and DO2 lineages (Fig. 3C). 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. *tupF4*-driven early *tup*
transcription in promuscular clusters is relayed by *tupADME* and *tupHDME* 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. 4A). 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. 4B). 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*tupF4* embryos, indicated that, while subject to direct autoregulation, *tupADME* or/and *tupHDME* 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 *tupADME* or *tupHDME*, suggesting their redundancy. In contrast, *tup* transcription was lost stage 14 in the double *tupADME* and *tupHDME* (Δ*tupADME* + 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. 4B).

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. 5A), a pattern repeated between successive adjacent segments reflecting muscle attachment sites matching (Carayon et al., 2020). Δ*tupADME* + HDME mutant embryos displayed a disorganised dorsal muscle pattern (Fig. 5A), showing that *tupADME/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. 5A-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<sub>n-1</sub>, S<sub>n</sub>, and S<sub>n+1</sub> (Fig. 5A).

**Dorsal muscle attachment matching is disrupted in ΔtupADME + HDME 3rd instar larvae.**

To further investigate muscle morphological defects due to Δ*tupADME* + 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. 5A) is maintained in wt larvae (Fig. 6A). In Δ*tupADME* + 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. 6B). 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 *tup*F4 and *tup*HDME, but at different times or concentrations, with *tup*HDME 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 *tupF4* and *tupHDME*, but
for technical reasons, we were unable to collect double mutant individuals.

This is not the first occurrence 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 *tupADME* (Boukhatmi et al., 2012) and *tupHDME* (Fig. 2). Yet, while *tupHDME* 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 *tupADME* 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 *tupADME* 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*<sup>ex4</sup>) embryos, using a DA3 muscle marker Collier, revealed a DA2-to-DA3 (DA2 > DA3) identity shift. *tup*<sup>ex4</sup> 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. 5A).

In wild-type embryos and larvae, the DA2 muscle of segment S<sub>n</sub> aligns with the DA3 muscle of S<sub>n-1</sub> and
the DA1 muscle of segment S<sub>n+1</sub>, at intersegmental borders (Fig. 5A; Carayon et al., 2020). However, in
the double *tupADME* and *tupHDME* deletion mutants, this DA3-DA2, DA2-DA1 intersegmental matching
is often replaced by homotypic alignment between DA2 muscles in consecutive segments (Fig. 5A, 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. 6A, 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**<sup>ex4</sup> - *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.
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**Table 1**

Table 1 is available in the Supplementary Files section.

**Figures**
<figure><img src="image_2.png" /><figcaption>Figure 1</figcaption></figure>

Figure 1

*Three distinct Cis-Regulatory Modules regulate tup expression in dorsal muscle tissue.*

(A) 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). (B) Schematic representation of the *tup* transcribed 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 *in vivo* 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 (*tup*F4 and *tup*ADME) and a newly identified module (*tup*HDME, red bar) are shown. (C) Each CRM was fused to moe-GFP reporter gene, and GFP expression detected using an anti-GFP antibody. The three CRMs together recapitulate *tup* expression 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).

A

<figure><img src="image_3.png" /></figure>

B

<figure><img src="image_4.png" /><figcaption>Figure 2</figcaption></figure>

Figure 2
Tup binding sites are required for specific tupHDME autoregulation in dorsal muscles.

(A) A deletion removing all the three Tup binding sites gave the *tupHDME*<sup>DTup</sup>-moeGFP reporter. (B) While *tupHDME*-moeGFP is expressed both in the heart, lymph gland (LG) and dorsal muscles (DM), *tupHDME*<sup>DTup</sup>-moeGFP is only expressed in the heart and LG, highlighting *tup* direct autoregulation in muscles.

<figure><img src="image_5.png" /><figcaption>Figure 3</figcaption></figure>
(A) Activity of *tupF4*, *tupADME* and *tupHDME* compared to endogenous *tup* expression between embryonic stages 10 and 16. Nascent transcripts were visualized using intronic probes for *tup*(green dots) and the reporter gene (yellow intron into *lacZ*) 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 **Fig.S8**). (B) Diagram summarizing the sequential activity of *tup* CRMs at the different stages of muscle development: promuscular cluster (PMC), progenitor cell (PC), founder cell (FC) and myofiber. (C) Left panels: detailed view of *tupF4*, *tupADME* and *tupHDME* activity at the FC (stage 12) and syncytial fiber (stage 15) stages, using *in situ* hybridization as in **Fig.3A**. 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).
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /><figcaption>Figure 4</figcaption></figure>

Figure 4

*Deletion of ADME+HDME CRMs abolishes tup transcription in dorsal muscles.*

(A) Schematic representation of the different deletions of *tup* CRMs. Homozygous flies lines were generated for each CRM ($\Delta$*tupF4*, $\Delta$*tupADME*, $\Delta$*tupHDME*) and a double *tupADME* and *tupHDME* deletion ($\Delta$*tupADME+HDME*). (B) *tup* transcription in these homozygous lines compared to *wt*, using *in*
situ hybridization to nascent transcripts (small black dots). *tup* transcription in the dorsal muscles is lost at stage 14 upon removing both ADME+HDME CRMs (clear arrowheads).

<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /><figcaption>Figure 5</figcaption></figure>

Figure 5

*Loss of tup transcription disrupts the pattern of embryonic dorsal muscle.*
(A) Dorsolateral views of stage 16 wt (top panels) and homozygous Δ*tupADME*+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<sub>n-1</sub>, S<sub>n</sub>, and S<sub>n+1</sub>). The intersegmental boundaries are represented by dashed black lines. In absence of *tup* transcription (Δ*tupADME*+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 (B) Quantification of the relative proportions of normal and altered DA1 and DA2 muscles in wt (red) and Δ*tupADME*+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*tupADME*+HDME segments.
A

<figure><img src="image_25.png" /></figure>

B

<figure><img src="image_26.png" /><figcaption>Figure 6</figcaption></figure>

Figure 6

*Muscle mismatching in ΔtupADME+HDME L3 larvae.*

(A) Left, scanning electron microscopy of filleted wt and ΔtupADME+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 tup, there is
muscle attachment sites mismatching at the intersegmental boundary (dashed black line) (B)

Quantification of the relative proportions of normal and altered DA1 and DA2 muscles in wt (red) and
Δ*tupADME*+HDME (blue) larvae. 6 wt and 11 Δ*tupADME*+HDME were analyzed, and the morphology of
DA2 and DA1 muscles recorded in 31 wt and 55 Δ*tupADME*+HDME segments, respectively.

**Supplementary Files**

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* figsup1.jpg

• figsup2.jpg

* figsup3.jpg

* figsup4.jpg

* figsup5.jpg

* figsup6.jpg

* figsup7.jpg

* table1.jpg