Cardiac progenitor cells

The heart is composed of diverse myogenic and non-myogenic cell lineages: cardiomyocytes, endocardial endothelial cells, cardio-fibroblasts, smooth muscle cells, valvular components and connective tissue. During heart development, differentiation of these multiple cardiac lineages is spatially and temporally controlled, resulting in the coordinated formation of the distinct tissue components of the heart (Harvey, 2002). In this regard, tissue ablation, genetic ablation, and lineage labeling experiments have demonstrated the dynamic nature of heart tube formation, and the existence of two myocardial sources in the mouse embryo (Buckingham et al., 2005). According to this model, growth of the embryonic heart occurs by progressive addition of progenitor cells of the second heart field (SHF), localized in the pharyngeal mesoderm, to the poles of the heart tube, itself derived from the cardiac crescent (first heart field). Cells from the SHF give rise to a large part of the heart including atria, atrio-ventricular septum, right ventricle, and outflow tract (Buckingham et al., 2005). The discovery of the SHF has had a major impact on our understanding of heart development and it is now well established that perturbation of SHF development results in a spectrum of common congenital heart defects (CHD) (Kelly RG, 2023).

Our main objective is to identify the molecular and cellular mechanisms regulating cardiac development to better understand the etiology of CHD. To address this aim we are using different approaches including genetics, lineage tracing, in vitro culture system and single cell transcriptomic analysis. The goal of our work is to establish relationships between signaling pathways and transcription factors that control the cardiac development.

Our previous studies clearly demonstrated that retinoic acid (RA), the active derivative form of the vitamin A, plays a critical role for SHF development and subsequent cardiac morphogenesis (Ryckebusch et al., 2008; Ryckebusch et al., 2010; El Robrini et al., 2015). RA acts as a diffusible molecule regulating transcriptional activation of target genes in the pharyngeal region through the anterior posterior axis. RA signaling is strictly regulated during embryogenesis and its dysregulation is associated with severe CHD (see Zaffran & Niederreither, 2015). Using several approaches to examine the contribution of the SHF to heart development in RA-deficient mouse embryos, we have shown that RA is required to restrict the SHF posteriorly (Ryckebusch et al., 2008). In collaboration with Dr. Kelly lab (IBDM, Marseille) we used in vivo culture system to show that RA signaling is required to activate Tbx5 in the posterior SHF resulting in the formation of a sharp transcriptional boundary between arterial and venous pole progenitor populations (De Bono et al., 2018).

CHD are the most common class of birth defect and about 30% affects the conotruncal region (also called the outflow tract), which gives rise to the great arteries. Using genetic lineage tracing analysis, we revealed that anterior Hox genes including Hoxa1, Hoxa3 and Hoxb1, are expressed in a sub-population of the SHF contributing to the outflow tract (Bertand et al., 2011). We also reported that Hoxa1 and Hoxb1 are required for correct outflow tract development. Indeed, deletion of Hoxb1 leads to outflow tract defect and ventricular septal defects (VSD) in mouse embryos (Roux et al., 2015). In a recent study we have characterized for the first time the accessibility of chromatin as well as the transcriptome of the anterior and posterior SHF subpopulations. Our analysis showed significant enrichment of the HOX factor binding motif in open chromatin regions of the posterior SHF subpopulation. In vitro and in vivo approaches allowed us to unravel that Hoxb1 represses differentiation in the posterior SHF (Stefanovic*, Laforest* et al., 2020). Indeed, ectopic expression of Hoxb1 in the anterior SHF of mouse embryo leads to the formation of a hypoplastic right ventricle. The use of an inducible embryonic stem cell model confirmed that Hoxb1 activation blocks cardiomyocyte differentiation. Furthermore, analysis of Hoxb1-deficient mouse embryos revealed premature cardiac differentiation of SHF progenitors. Finally, premature differentiation in the posterior SHF of embryos mutant for Hoxb1 and its paralog Hoxa1 results in abnormalities of the atrioventricular septum. In conclusion, our results showed that the Hoxb1 factor plays a key role in the differentiation of cardiac progenitor cells which contribute to the formation of the heart providing new information on the etiology of CHD.

These works were supported by the AFM-Telethon, the FRM, and the ANR

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