We will determine the mechanisms of lineage segregation of the cardiopharyngeal mesoderm and investigate defective lineage segregation in syndromes with head and heart defects, with simultaneous lineage and transcriptomic approaches in vivo and in a novel in vitro model. The cardiopharyngeal mesoderm is a cell population that participates in several lineages of the head and heart, with the existence of bi-potent progenitors contributing to muscles of the head and heart. The early steps of cardiopharyngeal mesoderm specification have been poorly explored and a certain number of questions remains to be addressed: Is there a multipotent progenitor that contributes to all cardiopharyngeal mesoderm lineages? When, where and how do cell fate decisions occur? Nobody has investigated precisely what happens during the early steps of cardiopharyngeal specification and how these steps are affected in the velocardiofacial syndrome, a rare congenital disease where both the head and heart are affected. This is however critical to improve the diagnosis and care of patients with these rare diseases. To determine the mechanisms of cardiopharyngeal mesoderm specification in congenital diseases, HEARTIST aims to: 1) Model normal cardiopharyngeal mesoderm lineage segregation, 2) Test cardiopharyngeal mesoderm lineage segregation in syndromic models. HEARTIST will go beyond the state of the art by using simultaneous lineage and transcriptomic approaches in mouse models and in a novel in vitro model of gastrulation derived from mouse embryonic stem cells. HEARTIST is built on solid preliminary results. This ambitious project will enhance our knowledge on cardiopharyngeal mesoderm specification and the mechanisms of congenital diseases that affect multiple organs and improve the diagnosis and care of patients. HEARTIST is built on 2 teams with complementary and solid expertise on cardiopharyngeal mesoderm lineages and gastrulation.

Congenital heart defects affect 1% of live births and have a major impact on mortality and morbidity. The most frequent anomalies affect essential barriers known as septa that separate pulmonary and systemic blood flow within the heart. Despite this clinical imperative, the mechanisms of septum morphogenesis are poorly understood. Insights have come from the realisation that cardiac septa arise at the boundary between early differentiating myocardium of the first heart field (FHF) and cells derived from the second heart field (SHF). New evidence reveals that transient coexpression of FHF and SHF regulators, followed by downregulation of SHF genes, precisely defines the sites of both atrial and ventricular septa. These findings indicate that the heart field interface is a boundary organiser that orchestrates cardiac septation. However, downstream effector genes driving boundary formation and initiating septal morphogenesis have yet to be identified. The overall objective of our project is to uncover how cells at the heart field interface establish a boundary and direct septum morphogenesis. Using in vivo mouse genetic lineage and functional analyses, quantitative imaging, spatial transcriptomics, together with in vitro embryo and gastruloid culture, we will dissect the genetic and cellular events regulating 1) boundary formation at the heart field interface and 2) morphogenesis of the muscular interventricular septum. Our experimental approach builds on new findings that dynamic changes in progenitor cell transcription, including downregulation of Hox gene expression, demarcate septal primordia along a common heart field boundary and that retinoic acid signaling is required for morphogenesis of the muscular ventricular septum, implicating a novel zipper mechanism initiating at the heart field interface. Our results will provide fundamental insights into mechanisms of organogenesis and a new paradigm for understanding the origins of congenital heart defects.

Type 2 diabetes (T2D) is a major public health issue increasingly affecting adolescents. While glycemia control has significantly improved the quality of life for diabetic patients, some complications persist, particularly those associated with the progressive degeneration of the sensory nervous system. This process often leads to neuropathic pain, frequently associated with chronic skin ulcers that can result in amputations. A better understanding of the underlying mechanisms leading to diabetic neuropathies (DN) and chronic wounds will permit the development of effective treatments. Congenital absence of sensory neurons can lead to chronic ulcers, resembling T2D complications. Recently, we have identified a new neuro-immune axis in which subsets of c-fibers sensory neurons, including nociceptors and C-low threshold mechanoreceptors (C-LTRMs), are capable of activating the pro-reparative functions of dermal macrophages. The progression of the disease is poorly understood, but obesity is often a triggering factor for T2D. Hence, we are currently investigating the impact of obesity on these pro-regenerative neuroimmune mechanisms. We have identified specific sensory neurons, innervating the subcutaneous adipose tissue and responding to their "metabolic state." We have demonstrated that the activation of these neurons accelerated the phagocytic properties of adipose tissue macrophages. This regulatory loop is crucial for the healing process upon skin injury, but obesity could lead to the overactivation and potentially the exhaustion of this mechanism. Understanding how these sensory neurons maintain adipose tissue homeostasis could help prevent the acceleration of metabolic syndrome in overweight adolescents and treat chronic wounds in T2D patients.

Controlling cytokinesis geometry by regulating asymmetric cleavage furrow ingression has just started to emerge as a key event to couple cell division to specific developmental processes. In the context of an epithelium, controlled cytokinesis geometry is essential to ensure the integrity of proliferative tissues. However, most studies have focused so far on cytokinesis geometry in isolated cells, one cell stage embryos or on Drosophila two-dimensional (2D) epithelia. Importantly, very little is known on the contribution of cytokinesis geometry to 3D epithelial tissue architecture. Indeed, how basal-to-apical cytokinesis asymmetry is achieved in 3D epithelia and whether tissue topology impacts on cytokinesis is not yet understood. Moreover, the mechanical impact of cytokinesis geometry on 3D epithelial arrangement has never been directly addressed. Epithelial tubules which are composed of a curved polarized monolayer of epithelial cells surrounding a central luminal cavity, constitute the functional units of many organs among which the kidney. Maintenance of a proper tubular organization is essential to ensure organ function. Impaired tubule organization and loss of lumen size control are characteristic of renal pathologies including polycystic kidney diseases. Recent results from the consortium show that a rigorous control of cytokinesis geometry is required for proper lumen organization in 3D culture of kidney cells. Understanding how asymmetric cytokinesis is achieved in tubular structures and whether and how random cytokinesis positioning impacts on tubular epithelia is thus of the utmost importance. However, with more than a million of nephrons, the mammalian kidney poses significant technical challenges to study dynamically the cellular processes contributing to kidney tubule organization. Indeed, live monitoring of cytokinesis events during the formation of tubular epithelia still remains a critical issue. To overcome the complexity of the mammalian kidney and determine the contribution of cytokinesis geometry to epithelial tubule homeostasis, we propose to take advantage of complementary approaches: an in vitro approach with bioengineered 3D cultures of renal epithelial cell lines using microfabrication techniques, and an in vivo approach using zebrafish a powerful model to study kidney tubule organization. More specifically, combining cell biological and biophysical approaches, our main objectives will be: (1) to study how cytokinesis geometry is controlled in a 3D environment, (2) to assess whether and how local perturbations of cytokinesis geometry affect large scale tissue behavior and (3) to define the involvement of cytokinesis geometry to epithelial tubule structure and lumen size/shape maintenance. Overall, by linking the cellular to the tissue scale and thanks to the complementarity of the consortium, this project will unravel the molecular and mechanical mechanisms controlling cytokinesis geometry in 3D epithelia, will allow to determine the impact of cytokinesis to large scale tissue organization and will shed light on its impact on kidney tubule organization.

In epithelia, at tricellular contacts where three cells meet, bicellular junctions are disjointed and specialized structures called tricellular junctions (TCJs) are assembled. In addition to ensure epithelial barrier functions, TCJs just emerged as hot spots for integrating epithelial tension. In this context, we aim to understand how TCJ components are assembled and regulate actomyosin cytoskeleton to control forces at cell-cell contacts. We will compare symmetric and asymmetric cell division models within Drosophila and mammalian epithelia that have a distinct organization of junction domains and molecular components, using approaches that include advanced imaging, genetic, mechanical perturbations and modelling. This multidisciplinary project will provide a comprehensive understanding on how TCJs control the geometry and force applied on cell-cell contacts, hence the strength of cell communication, to regulate the epithelium organization and homeostasis of progenitors and stem cells