CE13 - Biologie cellulaire, biologie du développement et de l’évolution

Regulatory networks governing craniofacial muscle diversity – MyoHead

Regulatory networks governing craniofacial muscle diversity

Craniofacial malformations are among the most common human birth defects and have multiple etiologies that affect diverse tissues. The underlying genetic, developmental and clinical causes of these malformations are largely unknown. Therefore we aim to identify all of the cell types that give rise to craniofacial structures and the molecular regulators that lead to the establishment and morphogenesis of the various cell lineages in the head.

The upstream myogenic program in head and neck muscles is distinct from that operating in the trunk and limbs and converges on activation of myogenic determination factors of the MyoD family.

Branchiomeric muscles of the head and neck share a common origin in cardiopharyngeal mesoderm (CPM) with parts of the heart derived from second heart field cardiac progenitor cells. Clonal analysis has identified three common skeletal and cardiac muscle lineages along the anterior-posterior axis of the developing pharynx, giving rise to jaw opening muscles and the right ventricle, derived from the first branchial arch, facial expression muscles and the cardiac outflow tract, derived from the second arch, and neck muscles, including the trapezius, and venous pole myocardium from caudal arches. Investigation of the upstream CPM myogenic program operating in CPM reveals that while some CPM regulators, such as the transcription factor and 22q11.2 deletion syndrome gene TBX1, are broadly required, different regulatory subprograms operate in posterior and anterior arches.

Partners 1 and 2 contributed to the study of cranial mesoderm-derived striated muscles in the embryo by examining progenitor cell contribution to several structures including the esophagus, and extraocular muscles and trapezius muscle. Partner 1 also progressed towards establishing a 4D map of craniofacial muscle development by performing temporal scRNAseq analysis in the early mouse embryo using different genetic driver mice and different developmental stages. The 10X genomics platform was used with pipelines that were procured from the emerging literature or adapted in the lab (SCENIC, SEURAT, scVelo, etc.). Validations were done on cryostat sections at the appropriate stage using antibody staining and RNAscope.

Partner 1 identified bipotent progenitors expressing Myf5 that give rise to both muscle and juxtaposed connective tissue. Following this bifurcation, muscle and connective tissue cells retained complementary signalling features and maintained spatial proximity. Disrupting myogenic identity shifted muscle progenitors to a connective tissue fate.
Partner 1 showed that the transcription factors Tbx1 and Isl1 are required cell-autonomously for myogenic specification of ESM progenitors. Further, genetic loss-of-function and pharmacological studies point to MET/HGF signaling for antero-posterior migration of esophagus muscle progenitors, where Hgf ligand is expressed in adjacent smooth muscle cells (Comai et al. 2019).
Partner 1 also examined EOM formation in mouse. Partner 1 investigated the morphogenesis of EOMs, an evolutionary conserved cranial muscle group that is crucial for the coordinated movement of the eyeballs and for visual acuity. We redefined the cellular origins of periocular connective tissues interacting with the EOMs, which do not arise exclusively from neural crest mesenchyme as previously thought. Using 3D imaging approaches, we established an integrative blueprint for the EOM functional unit. Collectively, our results highlight how global and site-specific programs are deployed for the assembly of muscle functional units with precise definition of muscle shapes and topographical wiring of their tendon attachments.

Partner 2 discovered that the retinoic acid (RA) intercellular signaling pathway is required for myogenic fate acquisition in posterior cardiopharyngeal mesoderm. Blocking RA signaling during a defined early time window leads to venous pole cardiac septation defects (De Bono et al., 2018). Pharmacological inhibition of RA signaling during this developmental window also blocks development of the trapezius neck muscle anlagen. Camille Dumas (Myohead funded PhD student) has shown that RA is required for trapezius development upstream of activation of myogenic determination factors. While muscle fibers of the trapezius are CPM-derived, the connective tissue is derived from the somitic lineage. These results demonstrate that CPM derived muscles have somite derived connective tissue.
The phenotype of embryos in which the dominant negative RA receptor is activated using Pax3-Cre suggest that this leads to a striking and selective disruption of trapezius muscle development, in addition to craniofacial, including extraocular muscle, and cardiac defects. Detailed phenotyping of embryos where RA signal reception is blocked in the Pax3 lineage is underway. These results enabled us to draw up a new model by which RA signaling indirectly regulates trapezius development via signal reception in the Pax3 lineage. This model implicates a second signaling event, downstream of RA signal reception, from somitic mesoderm to CPM that would promote growth of the trapezius anlagen.

The MyoHead project exploits state-of-the-art genetic approaches, live imaging, and single cell transcriptomic studies to address these crucial questions. First, an unbiased genome-wide single-cell RNA-seq/ATACseq strategy will be used to define transcriptional hierarchies, regulatory enhancers, and developmental trajectories controling myogenic fates in CPM (Aim1). A spatiotemporal 4-D fate map of CPM contributions will be generated to define morphogenetic dynamics of head muscle formation (Aim2). This data will be combined with mouse genetic studies and ex vivo approaches to decipher the genetic mechanisms regulating head and neck myogenic fates in emerging CPM, and ontogenetic links with their postnatal derivatives (Aim3). MyoHead will synergise expertise of the Tajbakhsh (skeletal muscle regulatory hierarchies and stem cells) and Kelly (craniofacial and cardiac myogenesis) teams to investigate congenital pathologies of the head. We will leverage our preliminary scRNAseq data and extensive published and preliminary evidence from our groups to extend these studies and provide a high-resolution spatiotemporal roadmap of transcriptional diversity at the time of myogenic fate acquisition. By providing a mechanistic understanding of cell fate choices in the embryo and functional links with postnatal muscle stem cells (MuSCs), MyoHead will generate clinically relevant insights into the molecular defects underlying congenital craniofacial anomalies.

Grimaldi A, and S Tajbakhsh (2021). Diversity in cranial muscles: Origins and developmental programs. Curr. Opin. Cell Biol. Sep 6;73:110-116. doi: 10.1016/j.ceb.2021.06.005.

Grimaldi A, G Comai, S. Mella and S Tajbakhsh (2022). Identification of bipotent progenitors that give rise to myogenic and connective tissues in mouse. www.biorxiv.org/content/10.1101/2021.05.26.445757v1. Elife. 2022 Feb 28;11:e70235. doi: 10.7554/eLife.70235.

Comai G*, Tesarova M, Dupé V, Rhinn M, Vallecillo Garcia P, da Silva F, Feret B, Exelby K, Dollé P, Carlsson L, Pryce B, Spitz F, Stricker, S, Zikmund T, Kaiser J, Briscoe J, Schedl, A Ghyselinck NB, Schweitzer R and Tajbakhsh S* (2020). Local retinoic acid directs emergence of the extraocular muscle functional unit. doi: doi.org/10.1101/2020.01.07.897694 *co-corresponding. PLOS Biology. 2020 Nov 17;18(11):e3000902. doi: 10.1371/journal.pbio.3000902.

Protocols for Investigating the Epithelial Properties of Cardiac Progenitor Cells in the Mouse Embryo.
Cortes C, De Bono C, Thellier C, Francou A, Kelly RG. Methods Mol Biol. 2022;2438:231-250. doi: 10.1007/978-1-0716-2035-9_15. PMID: 35147946

Emergence of heart and branchiomeric muscles in cardiopharyngeal mesoderm.
Lescroart F, Dumas CE, Adachi N, Kelly RG. Exp Cell Res. 2022 Jan 1;410(1):112931. doi: 10.1016/j.yexcr.2021.112931. Epub 2021 Nov 16. PMID: 34798131

Single cell multi-omic analysis identifies a Tbx1-dependent multilineage primed population in murine cardiopharyngeal mesoderm.
Nomaru H, Liu Y, De Bono C, Righelli D, Cirino A, Wang W, Song H, Racedo SE, Dantas AG, Zhang L, Cai CL, Angelini C, Christiaen L, Kelly RG, Baldini A, Zheng D, Morrow BE. Nat Commun. 2021 Nov 17;12(1):6645. doi: 10.1038/s41467-021-26966-6. PMID: 34789765

Over 60 oral presentations by members of the two labs where ANR MyoHead was cited, as well as meetings attended

Craniofacial malformations are among the most common human birth defects and have multiple etiologies that affect diverse tissues. Some progress has been made in understanding the embryological origins of these disorders, yet the underlying genetic, developmental and clinical causes of these malformations are largely unknown. It is therefore of high interest to identify all of the cell types that give rise to craniofacial structures, and the molecular regulators that lead to the establishment and morphogenesis of the various cell lineages in the head. We and others have shown that skeletal muscles in the body and head are governed by distinct transcription factors, demonstrating and unexpected diversity in founder stem cell populations that establish diverse muscles. Further, clinical data point to "escaper" muscles that do not succumb to disease, whereas other muscles are non-functional, in a variety of myopathies. Therefore, we hypothesized that the embryonic blueprint that leads to distinct phenotypic outcomes in postnatal skeletal muscles contributes, at least in part, to disease susceptibility. Accordingly, some key transcription factors that play critical roles in embryonic craniofacial muscle development are directly implicated in craniofacial disease conditions.

Unlike the trunk where skeletal muscle lineages arise from paraxial mesoderm, evolutionarily conserved cardiopharyngeal mesoderm (CPM) contributes progenitors to craniofacial striated muscles and cardiac lineages of the second heart field. In addition, the head/trunk interface where progenitors of distinct embryological origin establish different muscle masses (neck, esophagus and larynx) highlights the complex ontogeny of this tissue. Specifically, skeletal myogenic progenitors segregate within CPM to give rise to branchiomeric muscles involved in mastication, facial expression, some neck and extraocular muscles (EOMs). Our recent genetic studies identified esophageal (ESM), laryngeal (LSM), and some neck muscles as novel CPM myogenic derivatives, a first step in establishing a roadmap of craniofacial morphogenesis. However, precisely where and when myogenic fate decisions are made with CPM and the regulatory pathways driving divergent fates remain unknown. We propose here to define the gene regulatory networks, cell lineages, and morphogenetic events that govern the emergence and fate of craniofacial muscle progenitors, information critical for a full understanding of the etiology and diagnosis of craniofacial congenital defects. Significantly, adult cranial-derived muscle stem cells (ex. EOMs) have a more robust growth and engraftment potential compared to those from trunk and limbs. We have defined conditions that distinguish these properties and aim to identify the underlying regulatory mechanisms to inform on therapeutic strategies. Our results will also have important implications for dissecting the connection between craniofacial and cardiovascular congenital defects observed in a number of genetic syndromes.

The MyoHead project exploits state-of-the-art genetic approaches, live imaging, and single cell transcriptomic studies to address these crucial questions. First, an unbiased genome-wide single-cell RNA-seq/ATACseq strategy will be used to define transcriptional hierarchies, regulatory enhancers, and developmental trajectories controling myogenic fates in CPM (Aim 1). A spatiotemporal 4-D fate map of CPM contributions will be generated to define morphogenetic dynamics of head muscle formation (Aim 2). This data will be combined with mouse genetic studies and ex vivo approaches to decipher the genetic mechanisms regulating head and neck myogenic fates in emerging CPM, and ontogenetic links with their postnatal derivatives (Aim3). MyoHead will synergise expertise of the Tajbakhsh (skeletal muscle regulatory hierarchies and stem cells) and Kelly (craniofacial and cardiac myogenesis) teams to investigate congenital pathologies of the head.

Project coordination

Shahragim Tajbakhsh (INSTITUT PASTEUR)

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.

Partner

IP INSTITUT PASTEUR
CNRS DR12_IBDM Centre National de la Recherche Scientifique Délégation Provence et Corse _Institut de Biologie du Développement de Marseille

Help of the ANR 460,430 euros
Beginning and duration of the scientific project: December 2019 - 36 Months

Useful links

Explorez notre base de projets financés

 

 

ANR makes available its datasets on funded projects, click here to find more.

Sign up for the latest news:
Subscribe to our newsletter