Investigating the role of NR2F2 in fibroblast during human heart development

Abstract

Congenital heart disease (CHD) is responsible for significant morbidity and mortality, but the aetiologies are poorly understood despite the impact of cardiac anomalies. Previous evidence shows that genetic mechanisms play a role in CHD, and recent data shows that a key gene involved in embryonic development, NR2F2, has a role in human disease. It was hypothesised that NR2F2 is part of a molecular network with a role in cardiogenesis, and its investigation could elucidate NR2F2 networks associated with heart development. Previous unpublished data showed that fibroblasts and endothelial cells from developing human hearts express NR2F2 mRNA. Transcriptomic analysis in genome editing models enables the investigation of gene expression patterns and transcription factors as regulators of pathways and genomic networks. We aimed to investigate the role of NR2F2 by knocking out its transcription in primary cardiac fibroblasts and further analyse the consequent global transcriptomic changes by single-cell RNA sequencing. We isolated primary migrating cells from human foetal heart samples and characterized them by flow cytometry and single-cell RNA sequencing. We also assessed the expression of both NR2F2 transcript and protein in those cells, and then protocols were optimized for CRISPR/Cas9. We knocked down NR2F2 in fibroblasts using the ribonucleoprotein (RNP) system and then a droplet-based single-cell RNA-sequencing was performed in knocked-down and control samples to compare the perturbations caused by the absence of NR2F2. Bioinformatic pipelines were applied to similar publicly available datasets of cardiac cells for comparison. These pipelines comprised known bulk and single-cell RNA sequencing tools for gene expression analysis, including limma and Scanpy. Thus, we could conclude that reduced activity of NR2F2 in human foetal fibroblasts interfere with several cardiac gene expression important for cardiogenesis, e.g. GATA6 and HEY1, and may also influence the activity of other transcription factors, e.g. NKX2-5, that were previously associated with CHD phenotypes.

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Identifiers

ORCID for Maria Gabriela De Oliveira Barbeta: orcid.org/0000-0001-6848-8581

ORCID for Tilman Sanchez-Elsner: orcid.org/0000-0003-1915-2410

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