GATA6 mutations in hiPSCs inform mechanisms for maldevelopment of the heart, pancreas, and diaphragm
Abstract
Damaging GATA6 variants cause cardiac outflow tract defects, sometimes with pancreatic and diaphragmic malformations. To define molecular mechanisms for these diverse developmental defects, we studied transcriptional and epigenetic responses to GATA6 loss of function and missense variants during cardiomyocyte differentiation of isogenic human induced pluripotent stem cells. We show that GATA6 is a pioneer factor in cardiac development, regulating SMYD1 that activates HAND2, and KDR that with HAND2 orchestrates outflow tract formation. Loss of function variants perturbed cardiac genes and also endoderm lineage genes that direct PDX1 expression and pancreatic development. Remarkably, an exon 4 GATA6 missense variant, highly associated with extra-cardiac malformations, caused ectopic pioneer activities, profoundly diminishing GATA4, FOXA1/2 and PDX1 expression and increasing normal retinoic acid signaling that promotes diaphragm development. These aberrant epigenetic and transcriptional signatures illuminate the molecular mechanisms for cardiovascular malformations, pancreas and diaphragm dysgenesis that arise in patients with distinct GATA6 variants.
Data availability
All data generated or analyzed during this study are included in the manuscript.
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GATA6 ChIP-seq in differentiated cellsNCBI Gene Expression Omnibus, GSM575227.
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GATA6 ChIP-seqNCBI Gene Expression Omnibus, GSM1151694.
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GATA6 ChIP-seq in proliferating cellsNCBI Gene Expression Omnibus, GSM575226.
Article and author information
Author details
Funding
National Institutes of Health (UM1HL128711)
- George Porter
- Martin Tristani-Firouzi
- Deepak Srivastava
- Jonathan G Seidman
- Christine E Seidman
Howard Hughes Medical Institute
- Tarsha Ward
National Institutes of Health (UM1HL128761)
- Christine E Seidman
National Institutes of Health (UM1HL098147)
- Daniel M DeLaughter
National Institutes of Health (U01-HL098153)
- Jonathan G Seidman
- Christine E Seidman
National Institutes of Health (U01-HL098163)
- Jonathan G Seidman
- Christine E Seidman
National Institutes of Health (U01-HL098123)
- Jonathan G Seidman
- Christine E Seidman
National Institutes of Health (U01-HL098162)
- Jonathan G Seidman
- Christine E Seidman
National Science Foundation (EEC-1647837)
- Jonathan G Seidman
- Christine E Seidman
National Institutes of Health (T32HL116273)
- Arun Sharma
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Human subjects: CHD subjects were recruited to the Congenital Heart Disease Network Study of the Pediatric Cardiac Genomics Consortium (CHD GENES: ClinicalTrials.gov identifier NCT01196182) after approval from Institutional Review Boards as previously described (Pediatric Cardiac Genomics et al., 2013; Jin et al., 2017). Written informed consent was received from subjects or their parents prior to inclusion in the study. Clinical diagnoses were standardized based on review of medical data and family interviews.
Reviewing Editor
- Edward E Morrisey, University of Pennsylvania, United States
Publication history
- Received: November 1, 2019
- Accepted: October 14, 2020
- Accepted Manuscript published: October 15, 2020 (version 1)
- Version of Record published: October 28, 2020 (version 2)
Copyright
© 2020, Sharma et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Further reading
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During mammalian development, the left and right ventricles arise from early populations of cardiac progenitors known as the first and second heart fields, respectively. While these populations have been extensively studied in non-human model systems, their identification and study in vivo human tissues have been limited due to the ethical and technical limitations of accessing gastrulation stage human embryos. Human induced pluripotent stem cells (hiPSCs) present an exciting alternative for modeling early human embryogenesis due to their well-established ability to differentiate into all embryonic germ layers. Here, we describe the development of a TBX5/MYL2 lineage tracing reporter system that allows for the identification of FHF- progenitors and their descendants including left ventricular cardiomyocytes. Furthermore, using single cell RNA sequencing (scRNA-seq) with oligonucleotide-based sample multiplexing, we extensively profiled differentiating hiPSCs across 12 timepoints in two independent iPSC lines. Surprisingly, our reporter system and scRNA-seq analysis revealed a predominance of FHF differentiation using the small molecule Wnt-based 2D differentiation protocol. We compared this data with existing murine and 3D cardiac organoid scRNA-seq data and confirmed the dominance of left ventricular cardiomyocytes (>90%) in our hiPSC-derived progeny. Together, our work provides the scientific community with a powerful new genetic lineage tracing approach as well as a single cell transcriptomic atlas of hiPSCs undergoing cardiac differentiation.
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