Diversité fonctionnelle du facteur de transcription Tbx5 dans le coeur

The vertebrate heart is a modular organ, which requires the complex patterning of the morphogenetic heart fields and the coordinated convergence of the diverse subpopulations of cardiogenic progenitors. At least 7 transcription factors of the T-box family cooperate within these numerous subpopulations of cardiogenic progenitors to regulate the morphogenesis and the layout of multiple structures along the primordial heart tube, which explains that the human mutations of these genes induce various congenital heart defects (CHDs). One of these T-box genes, Tbx5, whose haploinsufficiency generates the Holt-Oram syndrome (HOS), intervenes in a wide variety of gene regulatory networks (GRNs) that orchestrate the morphogenesis of the atria, the left ventricle, the mitral valve, the inter-atrial and inter-ventricular septa, as well as the cardiac conduction system. The diversity of GRNs involved in the formation of these cardiac structures suggests that Tbx5 holds a profusion of functions which will be identifiable only by indexing its molecular activities in each separately examined cardiac lineage. To address this problem, a conditional knockout of Tbx5 in the endocardium was generated. This experiment demonstrated a crucial role of Tbx5 in the survival of the endocardial cells lining the septum primum and the cardiomyocytes within this embryonic structure, which will contribute to the morphogenesis of the inter-atrial septum. Moreover, this study revealed a crosstalk between the Tbx5-positive endocardial cells subpopulation and the myocardium at the level of the septum primum to ensure the survival of cardiomyocytes, and ultimately to guarantee the maturation of the inter-atrial septum. Our results also confirmed the importance of genetic interdependence (Tbx5 and Gata4 as well as Tbx5 and Nos3) between different loci in the morphogenesis of the inter-atrial septum, and particularly the influence that the environment can have on the penetrance and the expressivity of atrial septal defects (ASDs) in the HOS. Besides, since the functions of a gene usually depend on the different isoforms it can generate, a second study focused more on the transcriptional aspect of Tbx5. This approach led to the discovery of 6 alternative transcripts exhibiting both common and specific functions. The characterization of 2 of these isoforms revealed the role of the long isoform (Tbx5_v1) in the regulation of cardiomyocytes growth during cardiogenesis, whereas the short isoform (Tbx5_v2), preferentially expressed in the mature heart, represses cell growth. It is thus entirely conceivable that TBX5 mutations leading to a C-terminal truncation increase the concentration of a mutated protein, which, like Tbx5_v2, interferes with the growth of certain cardiac structures. On the other hand, the divergence of functions of these isoforms, characterized by the disparities of subcellular localization and interaction with other cardiac cofactors, suggests that mutations affecting more one isoform would favor the emergence of a particular type of CHD. Finally, a last objective was to identify one or several molecular mechanism(s) by which Tbx5 regulates its main target gene, Nppa, and to extract clues that might clarify its transcriptional function. This objective required in a first place to identify the various cis-regulatory modules (CRMs) coordinating the transcriptional regulation of Nppa and Nppb, two natriuretic genes whose tandem organization and expression pattern during cardiogenesis are preserved in most vertebrates. The phylogenetic footprint approach employed to scan the Nppb/Nppa locus allowed the identification of three CRMs evolutionary conserved between different mammals species, one of which (US3) is specific to eutherians. This study confirmed that the regulation of the tandem genes Nppb/Nppa required the transcriptional activity of enhancers in complement to Nppa and Nppb promoters. The almost perfect concordance between the expression profiles of Tbx5 and these two natriuretic genes in mammals, suggests that the ventricular expression gradient of Tbx5 is interpreted by the recruitment of this factor to the identified enhancers. Altogether, the studies presented in this thesis allowed clarifying the profusion of Tbx5 cardiac functions. Some of these functions emanate from the alternative splicing of Tbx5, which favors the synthesis of isoforms endowed with specific properties. The diverse combinatorial interactions between these isoforms and other cardiac factors within the various cardiogenic progenitor subpopulations contribute to the emergence of distinct cardiac RRGs.

Keyphrases: Tbx5, Nppa, Nppb, Gata4, Nos3, Cardiogenesis, Endocardium, Atrial septal defect, Congenital heart defect, Holt-Oram syndrome, Alternative splicing, Enhancer