Herramientas genéticas basadas en el enhancer neural de Nestina para la caracterización de las poblaciones endoteliales del corazón: papel del Sox17 en el desarrollo de la vasculatura coronaria

Abstract

The study of the developmental origin of the coronary vasculature in mammals has been subject of extensive research in recent years, and yet the signals regulating the formation of the primary plexus, remain poorly understood. In this Doctoral Thesis the murine genetic tools Nes-Gfp and Nes-CreERT2 lines have been used for studying coronary endothelium formation during cardiac development, with special emphasis on the stages of remodeling and arteriovenous maturation. The strong characterization of the Nes-Gfp allele during the intermediate stages of cardiogenesis indicated that it is expressed in mesenchymal strains of the cardiac bulb, pericytes and mural cells associated with the vessels (analogously to the pattern already described in other organs), and unexpectedly and dynamic, its expression is also induced robustly in the nascent coronary endothelium. By taking advantage of this pattern of vascular expression and using whole-tissue clarification imaging and confocal microscopy, we aim to characterize the process of sprouting of the coronary vessels and arterial remodeling at unprecedented resolution. In addition, thanks to the relative intensity of expression of the GFP reporter, in combination with the Endomucin marker, it allowed us to discriminate between ventricular endocardial cells (where GFP is barely expressed) and the coronary vascular plexus. By cytometry, we were able to isolate both primary endothelial populations with great purity, from the same hearts and obtain the corresponding transcriptional profiles, in different phases of their development: the initial stage of angiogenic expansion (E13.5) and during the process of active remodeling of the primary plexus (E17.5). Moreover, lineage tracing using the Nes-CreERT2 driver (which shares the presence of regulatory sequences of the neural enhancer of the Nestin gene with the Nes- Gfp line) allows us to selectively label the intramyocardial coronary plexus enriched in capillary and arteriolar endothelium. Therefore, the combination of both genetic tools has allowed us to isolate and define transcriptomically the three subpopulations of blood vascular cells present in the heart (endocardium, intramyocardial arteries and subepicardial veins). This strategy allowed us to identify new enriched factors in the arterial branch. Among the differentially expressed genes, we have found several members of the SOX transcription factors family, and especially Sox17. Interestingly, we detected nuclear expression of Sox17 in the first endothelial cells of the plexus that migrated through the subepicardium. Later on, its expression is progressively restricted in the domain of intramural Nes-GFP+ vessels, with an arterial phenotype. Due to the robust co-localization observed between the Sox17+ population and the GFP+ cells, we wonder if this transcription factor could be a direct transcriptional activator of the neural enhancer present in the regulatory region of the Nes-Gfp allele, verifying it with a series of assays in vitro. For all these reasons, we propose that the Nestin neural enhancer could also act as a coronary arterial enhancer in certain phases of cardiac development, and whose continued study could help to discover new molecular bases in this important and relevant process for human health, given the high incidence of coronary disease worldwide.