Early cardiac progenitors

J. David Gladstone Institutes, San Francisco CA

Investigators

Linked publications, trials & patents

Abstract

PROJECT SUMMARY Transcription factor (TF) mutation underlie many birth defects, including congenital heart defects (CHD). Many of the transcription factors mutated in CHD are active very early in development, in some cases prior to organogenesis. For example, NKX2-5, TBX5, and GATA4 . These transcription factors, along with their partner TFs, such as MEF2C, operate to control broad gene regulatory network (GRNs) that control precisely the timing, levels, and location of multiple genes. Our primary goal is to comprehensively decode TF function in early heart development, and to discover underpinnings of how they control their respective GRNs. We aim to shed light on fundamental aspects of heart development, and the molecular basis of human CHDs. We hypothesize that early cardiac TFs affect cardiac morphogenesis and quantitative gene expression at specific loci and/or structures of the developing heart, and that this fine GRN regulation is based on motif grammar and binding co-factors. Aim 1 is to define chamber-specific gene regulatory networks in early cardiac progenitors. This will be accomplished using multiomics (paired single nucleus RNAseq and ATACseq) and data analysis with new machine learning-based analyses. Investigating mouse embryos lacking MEF2C or TBX5 we will uncover the GRNs that quantitatively and spatially control chamber formation. We will discover several candidate regulatory elements, which we will test at medium scale in transgenic zebrafish embryos. For aim 2, we will use quantitative live imaging of cardiac lineages in mouse embryos. To label specific lineages, we will image embryos harbouring lineage reporters for the first heart field (Tbx5CreERT2), anterior second heart field (Mef2cAHF::Cre or Mef2cAHF::DreERT2), or both simultaneously. We will examine these lineages, their behaviours and relative allocation in control embryos, and in embryos lacking MEF2C or TBX5. For Aim 3, we will turn to human cardiac progenitors to study a new CHD locus that is predicted to affect the earliest decisions in cardiogenesis. We have identified a de novo 58kb deletion in a child with hypoplastic left heart syndrome, which encompasses MESP1 and MESP2. In preliminary data we have found that human iPS cells engineered to be heterozygous for the deletion when differentiated into mesoderm have disrupted gene expression, indicating haploinsufficiency. We will characterize this human CHD model using chamber-specific organoids (“cardioids”), as this will allow us to investigate the function of this locus in atrial, left ventricular, and right ventricular cardioids. We will dissect the locus with CRISPR-based genome engineering, to determine which genes within this locus contribute to its haploinsufficiency. In summary, this project will generate foundational new results that will be applicable to development in general, and to CHDs in particular.