GATA6 regulates WNT and BMP programs to pattern precardiac mesoderm during the earliest stages of human cardiogenesis (CUT&RUN)

Haploinsufficiency for GATA6 is associated with congenital heart disease (CHD) with variable comorbidity of pancreatic or diaphragm defects, although the etiology of disease is not well understood. Here, we used cardiac directed differentiation from human embryonic stem cells (hESCs) as a platform to study GATA6 function during early cardiogenesis. GATA6 loss-of-function hESCs had a profound impairment in cardiac progenitor cell (CPC) specification and cardiomyocyte (CM) generation due to early defects during the mesendoderm and lateral mesoderm patterning stages. Profiling by RNA-seq and CUT&RUN identified genes of the WNT and BMP programs regulated by GATA6 during early mesoderm patterning. Furthermore, interactome analysis detected GATA6 binding with developmental transcription factors and chromatin remodelers suggesting cooperative regulation of cardiac lineage gene accessibility. We show that modulating WNT and BMP inputs during the first 48 hours of cardiac differentiation is sufficient to partially rescue CPC and CM defects in GATA6 heterozygous and homozygous mutant hESCs. This study provides evidence of the regulatory functions for GATA6 directing human precardiac mesoderm patterning during the earliest stages of cardiogenesis to further our understanding of haploinsufficiency causing CHD and the co-occurrence of cardiac and other organ defects caused by human GATA6 mutations. CUT&RUN was performed on control H1-GATA6-hESCs (GATA6+/+) using a GATA6 antibody and IgG control antibody at day 2 of in vitro cardiac directed differentiation.

GATA6单倍体剂量不足（haploinsufficiency）与先天性心脏病（congenital heart disease, CHD）相关，该疾病可伴随胰腺或膈肌缺陷的可变共病表现，但目前其具体致病机制尚未完全明确。本研究以人类胚胎干细胞（human embryonic stem cells, hESCs）的心脏定向分化体系作为研究平台，探究GATA6在早期心脏发生过程中的生物学功能。GATA6功能缺失型hESCs在中内胚层与侧中胚层模式形成阶段即出现早期发育缺陷，进而导致心脏祖细胞（cardiac progenitor cell, CPC）特化与心肌细胞（cardiomyocyte, CM）生成受到严重损伤。通过RNA-seq与CUT&RUN测序分析，我们鉴定出GATA6在早期中胚层模式形成阶段调控的WNT与BMP信号通路相关基因。进一步的相互作用组分析显示，GATA6可与发育相关转录因子及染色质重塑因子结合，提示其通过协同调控心脏谱系基因的染色质可及性发挥功能。本研究证实，在心脏分化的最初48小时内调控WNT与BMP信号输入，可部分挽救GATA6杂合及纯合突变hESCs中的CPC与CM生成缺陷。本研究揭示了GATA6在心脏发生最早期阶段指导人类前心中胚层模式形成的调控功能，有助于进一步理解GATA6单倍体剂量不足导致CHD的分子机制，以及人类GATA6突变引发的心脏与其他器官缺陷共现现象。此外，本研究在体外心脏定向分化第2天，使用GATA6抗体与IgG对照抗体，对对照H1-GATA6-hESCs（GATA6+/+）样本开展了CUT&RUN实验。