Defining the downstream genetic networks regulated by GATA6 during human cardiogenesis using iPSC and hESC models

摘要 模式生物的遗传功能丧失研究已经阐明了转录因子的加塔家族 调节心脏形态发生的关键方面，包括早期胚层模式和心脏祖细胞 规格和差异化。重要的是，人类心脏发生因子GATA 6的杂合突变 与各种形式的先天性心脏病（CHD）有关，如流出道和间隔缺损。 在含有这些GATA 6突变的患者中，CHD的表型多样性可能是由于未知的 修饰或相互作用基因中的变体的组合，其会聚以影响心脏表型。一 因此，对控制人类心脏发育的GATA 6基因调控网络有了更深入的了解， 对于推进治疗至关重要。在这里，我建议利用心脏定向分化人类多能 干细胞（hPSC）作为研究GATA 6功能的系统。初步证据显示，GATA 6-/-hPSC 不能产生心肌细胞或表达心脏祖细胞的标记物，并且心脏祖细胞的数量减少。 与野生型（WT）对照相比，在心脏定向分化期间中胚层标志物表达。 此外，GATA 6 +/-hPSC产生心肌细胞的效率较低，并且具有减少的心脏祖细胞， 标记基因表达与WT细胞相比，揭示了GATA 6杂合子的表型， 在以前的动物研究中也有报道。基于这些数据，我的目的是研究GATA 6在心脏 中胚层模式和GATA 6单倍不足通过两个特定的目的在心脏发生。在目标1中， 我计划在人类心脏发生的最早阶段发现GATA 6的功能。两个候选 将首先研究从RNA-seq数据中鉴定的GATA 6活性的下游靶标LGR 5和RIPPLY 1 通过基因功能获得和丧失的研究。GATA 6 ChIP-seq和ATAC-seq也将在 GATA 6-/-和WT细胞在心脏分化的早期阶段，以鉴定GATA 6和WT细胞的直接靶标。 受染色质不可及性影响的调控基因。在目标2中，我建议界定 在心脏发生过程中的人GATA 6单倍不足的后果。诱导多能干细胞（iPSC） 从含有GATA 6杂合突变（c.1071delG）的CHD患者产生细胞系， 与WT iPSC系相比，其分化为心肌细胞的效率较低。在此，工程化的GATA 6 +/-hESC和 患者来源的GATA 61071 delG/+ iPSC将分化为心肌细胞，并在心肌细胞分化的不同阶段进行检查。 心脏发生以仔细定义心脏表型。GATA 6 +/-细胞具有增加的视黄酸表达， 酸（RA）信号相关基因，这可能是心脏表型的基础。因此，RA信号传导将被抑制 目的是挽救GATA 6单倍不足的表型。最后，区分 将使用RNA-seq分析GATA 6 +/- hESC和患者来源的iPSC系，以发现新的修饰基因。 可能与CHD相关的基因。总的来说，这些研究将扩大我们对遗传学的理解。 并可能为未来CHD治疗奠定基础。

ABSTRACT Genetic loss-of-function studies in model organisms have elucidated that the GATA family of transcription factors regulate crucial aspects of heart morphogenesis including early germ layer patterning and cardiac progenitor cell specification and differentiation. Importantly, heterozygotic mutations of the cardiogenic factor GATA6 in humans are associated with various forms of congenital heart disease (CHD) such as outflow tract and septal defects. The phenotypic diversity of CHD in patients containing these GATA6 mutations is likely due an unknown combination of variants in modifying or interacting genes that converge to influence the cardiac phenotype. A greater understanding of the GATA6 genetic regulatory network that controls human heart development is thus essential for advancing therapies. Here, I propose utilizing cardiac-directed differentiating human pluripotent stem cells (hPSCs) as a system to study GATA6 function. Preliminary evidence showed that GATA6-/- hPSCs failed to generate cardiomyocytes or express markers of cardiac progenitors, and had reduced cardiac mesoderm marker expression during cardiac-directed differentiation compared to wild-type (WT) controls. Furthermore, GATA6+/- hPSCs generated cardiomyocytes less efficiently and had reduced cardiac progenitor marker gene expression compared to WT cells, revealing a phenotype in GATA6 heterozygotes that has not been reported in previous animal studies. Based on these data, I aim to study GATA6 function during cardiac mesoderm patterning and GATA6 haploinsufficiency during cardiogenesis through two Specific Aims. In Aim 1, I plan to discover the function of GATA6 during the earliest stages of human cardiogenesis. Two candidate downstream targets of GATA6 activity identified from RNA-seq data, LGR5 and RIPPLY1, will first be studied through genetic gain- and loss-of-function studies. GATA6 ChIP-seq and ATAC-seq will also be performed in GATA6-/- and WT cells during early stages of cardiac differentiation to identify direct targets of GATA6 and regulatory genes affected by chromatin inaccessibility, respectively. In Aim 2, I propose to define the consequence of human GATA6 haploinsufficiency during cardiogenesis. An induced pluripotent stem cell (iPSC) line was created from a CHD patient containing a heterozygous mutation in GATA6 (c.1071delG) that differentiates to cardiomyocytes less efficiently than a WT iPSC line. Here, engineered GATA6+/- hESCs and patient-derived GATA61071delG/+ iPSCs will be differentiated to cardiomyocytes and examined at distinct stages of cardiogenesis to carefully define the cardiac phenotype. GATA6+/- cells have increased expression of retinoic acid (RA) signaling related genes, which may underlie the cardiac phenotype. RA signaling will thus be inhibited with the goal of rescuing the phenotype of GATA6 haploinsufficiency. Finally, the transcriptomes of differentiating GATA6+/- hESC and patient-derived iPSC lines will be analyzed using RNA-seq to discover novel modifying genes that may be relevant to CHD. Overall, these studies will expand our understanding of the genetics of human heart development and potentially lay a foundation for future CHD therapies.