Specification and realization of muscle identity, Model studies in Drosophila. <br />The morphological diversity of skeletal muscles underlies animal locomotion. Gene Regulatory Networks directing generic myogenesis have been decrypted in mammals, The control of each muscle morphological identity remains a fundamental and bio-medical issue, with human myopathies affecting distinct sets of muscles. <br />Muscle-Identity aimed at establishing a global view of this control, using an insect model.
The morphological diversity of muscles underlies the precision and strength of movements. Muscle development (myogenesis) involves the fusion of individual precursor cells, the myoblasts, into multinucleate syncitial fibers. Myogenic Regulatory Factors and different other Transcription Factors (TFs) play essential roles. Different combinations may act in myoblasts of different embryonic origin (Sambavisan et al., 2009; Buckhingham,M., 2017). Yet, what controls each muscle identity -orientation, shape, size, innervation- remains unknown. This is a pregnant question, for fundamental and bio-medical reasons, many human myopathies affecting specifically distinct sets of muscles (Emery, A.E, 2002).<br />The stereotypy of the musculature of a Drosophila insect larva is a classical model to study muscle identity. Each muscle is built by fusion of Founder Cells (FCs) and fusion competent myoblasts. Muscle identity reflects the expression by each FC of specific TFs (Tixier et al., 2010). For example, our laboratory showed early that one TF conserved between Drosophila and humans, Collier/Early B-cell Factor, is required in one specific muscle (Crozatier and Vincent, 1999; Dubois et al., 2007). Our project aimed to obtain a more global picture of the specification and realization of muscle identity.
In addition to using Drosophila molecular and reverse genetics, confocal and scanning electron microsocopy, our laboratory has pioneered double fluorescent in situ hybridization (FISH) experiments with intronic probes to determine with high precision time windows of gene expression. We also made a series of original reporter constructs where different exogenous introns have been introduced within the reporter coding region. FISH experiments are performed in embryos expressing fluorescent proteins under the control of dedicated cis-regulatory modules which recapitulate identity Transcription Factor expression throughout muscle development, allowing both visualizing specific muscle progenitor cells and the final shape of wild type or mutant muscles with high resolution. ImageJ software is used to measure the intensity of each nuclear hybridization dot. FISH dot numbers are taken as a proxy for transcription period and dot intensity as a proxy for transcription initiation rate. The whole strategy can be adapted to any gene in any tissue. We use Cas9/CRISPR strategies for genome editing, and developed live-imaging of embryonic muscle development and larval locomotion.
Muscle-Identity included 4 tasks
1.Specification of muscle progenitors : GRNs.
Our screens identified new roles of EGF signaling in controlling the sequential birth of muscle progenitors, endowing them with distinct identities and roles of Eya and Six and MyoD in specifying PC identity. The windows of transcription and cross-regulations between these and other TFs provide a dynamic view of the transcriptional control of muscle identity and an extended framework for studying general myogenic factors in diversification of muscle shapes (de Taffin, 2015; Dubois, 2016).
2. Propagation and memory of muscle identity; the CRM handover mechanism.
Dissection of the cis-regulatory landscape of identity TFs identified cis-elements (CRMs) acting sequentially. Targeted deletion of CRMs showed that propagation of the transcription program from the muscle founder to of fusing myoblasts nuclei is required for establishing muscle identity. Identity loss leads to formation of branched muscles, recalling the Duchenne Muscular Dystrophies phenotype, and locomotion defects. (A. Carayon, PhD, 2018; ms to be submitted).
3. Realisation of muscle identity. (Re)-Programming of fibre nuclei.
By tracking nuclei, we showed that all myoblasts apart founder cells are naive and reprogrammed to a given muscle identity post-fusion. Differential transcription of identity realization genes is regulated at both levels of transcription initiation rate and number of active nuclei. The generic differentiation and identity programs progress independently. Together, these data provide a new framework for studying the differentiation of multinucleate cells (Bataillé, 2017).
4. Evolution of muscle patterns and Gene Regulatory Networks.
Discovery of new thoracic muscles connecting the skeleton and internal organs raises the question of the evolutionary origin and functions of these atypical muscles. (Boukhatmi., 2014 ; Bataillé et al., 2015 ; ms to be submitted).
Our screens in flies revealed an unanticipated complexity in the control of muscle morphological diversity. Among found actors, some act as general myogenic factors in mammals. We suggest that they could additionally specify muscle morphological identity.
Our screen also identified loci involved in various aspects of myogenesis, as many new avenues to explore human muscular dystrophies.One next step is to decrypt the consequences of muscle identity shifts on locomotion
The discovery of a novel type of asymmetric muscles connecting the skeleton to the gut foresees a completely new field of myology research. Finally, deciphering the dynamics of reprogramming of myofiber nuclei provides a first insight into integration of muscle identity with general myogenesis. .
$ co-first authors; * co-corresponding authors
Boukhatmi H. $, Schaub, C. $, Bataillé, L. $, Reim, I., Frendo,J.L., Frasch, M.* and Vincent, A.* (2014). An Org-1–Tup transcriptional cascade reveals different types of alary muscles connecting internal organs in Drosophila. Development, 141, 3761-3771.
De Taffin, M.$, Carrier Y.$, Dubois, L., Bataillé, L., Painset, A., Le Gras, S., Jost, B., Crozatier, M. and Vincent, A.* (2015) Genome-wide mapping of Collier in vivo binding sites highlights its hierarchical position in different transcription regulatory networks. PLoS ONE journal.pone.0133387
Bataille, L.*, Frendo, J.L., and Vincent, A.* (2015) Hox control of Drosophila larval anatomy; The Alary and Thoracic Alary-Related Muscles. Mechanisms of Development, 138, 170-176. (revue invitée)
Dubois, L.*, Frendo J.L., Chanut, H., B., Crozatier, M. and Vincent, A.* (2016) Genetic dissection of the Transcription Factor code controlling serial specification of muscle identities in Drosophila. eLIFE 2016;5:e14979
Bataille, L.*, Boukhatmi, H., Frendo, J.L., and Vincent, A.* (2017) Dynamics of transcriptional (re)-programming of syncytial nuclei in developing muscles. BMC Biology 15, 48-67.
Carayon, A. Thèse de doctorat- PhD thesis, Université de Toulouse (2018). Mise en place de l’identité musculaire durant la myogenèse embryonnaire chez la Drosophile.
Carayon, A., Bataille, L., Carrier, Y., Dubois, L., Lebreton, G., Wystrach, A., Vincent, A. and Frendo, J.L.* (2018) Loss of identity Transcription Factor activation in syncytial nuclei leads to branched muscles and locomotion defects. To be submitted, October 2018
Bataille, L.*, Colombié, N., Paululat, A., Frendo, J.L., and Vincent, A.* (2018) The Drosophila Alary muscles; a cornerstone of the heart. To be submitted, October 2018
The musculature of each animal is composed of a stereotypical array of muscles, which share general properties but are morphologically distinct from each other. Elucidating the mechanisms involved in conferring a specific identity to each muscle remains a major challenge in myogenesis research. Our project aims at deciphering the genetic and molecular programmes underlying the morphological diversity of skeletal muscles by focusing on two phases of muscle development: i) specification of muscle progenitor cell (PC) identity, ii) realisation of muscle identity, that is, translation of PC identity into a specific morphology.
We focus most of our studies on the dorso-lateral DA3 and dorsal DA2 Drosophila muscles, which express the Collier (Col) and Islet1/Tail-up (Tup) identity transcriptions factors (iTFs), respectively.
Our first objective is to identify all the TFs that are required for specification of DA3 identity, using a systematic genetic screen that covers all three chromosomes. A pilot study revealed several classes of DA3 transformation resulting from mutations in TFs. We will study in detail the function of these TFs in order to establish the Gene Regulatory Network controlling the DA3 lineage, before extending this analysis to three other muscle lineages. This screen represents the first genome-wide screen for deciphering combinatorial codes of muscle identity TFs (iTFs).
Activation of specific iTFs, in specific PCs reflects their position along body axes. Based on col expression, we have proposed that positional memory is transmitted to all nuclei of a syncytial fibre, via a CRM (cis-regulatory module) handover mechanism: PC accumulation of a given iTF under the control of a “positional” CRM, is responsible for activation of a secondary “memory” CRM, maintaining this expression during muscle growth. Our second objective is to assess the CRM handover mechanism in a genomic context, and to broaden the conclusions to include tup regulation. Lastly, we will undertake a systematic identification of TFs binding col and tup “memory” CRMs by yeast one-hybrid screens. Together, these studies should decrypt in unprecedented detail, how positional information integrated by muscle PCs is propagated to all nuclei of the muscle fibre to implement muscle identity.
Our major, third objective is to explore the process of transcriptional reprogramming of “naïve” myoblast nuclei incorporated into a growing muscle fibre. We will develop new strategies for RNA-profiling and ChIP-SEQ analyses of specific muscles in order to i) identify Col and/or Tup target genes involved in realisation of muscle identity and ii) establish the dynamics of transcription of a selection of these target genes in myofibre nuclei. One important aim is to discover new genes required for proper muscle selection of epidermal attachment sites. Correlating transcriptional reprogramming dynamics of myofibre nuclei with cellular aspects of muscle differentiation should bring a new integrated view of muscle identity realisation. Identification of Col and Tup direct targets will establish a direct link between the iTF code and muscle morphology.
The stereotypy of the Drosophila muscle pattern raises the question of the evolution of this pattern. Our fourth, longer-term objective is to compare muscle morphology and iTF expression between two Drosophila species, D. melanogaster and D. virilis (wandering larva), one mosquito, Anopheles gambiae (aquatic larva), and the honeybee Apis mellifera (sessile larva). Finally, our recent work on Tup/Islet1 brought to light intriguing parallels based on TF co-option between pharyngeal muscles in chordates and dorsal muscles in Drosophila. We will further explore these parallels, through collaborations with external groups.
Altogether, our proposal will determine how the steps of specification and realisation are linked, and establish a global picture of how muscle identity is integrated into the general myogenic program.
Monsieur Alain VINCENT (Centre de Biologie du Développement)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
CBD Centre de Biologie du Développement
Help of the ANR 405,949 euros
Beginning and duration of the scientific project: October 2013 - 48 Months