The Role of GATA3 and SATB2 in Early Colonic Patterning

后肠产生结直肠和尿路上皮/膀胱的命运。这两个词的生成 命运需要分割后肠。后肠不正确的分割导致 肛门直肠畸形。我们已经产生了一种强大的新的体外模型，它允许 人诱导多能干细胞分化为结直肠和尿路上皮类器官 通过骨形态发生蛋白(BMP)信号的瞬时激活来激活细胞(IPSCs)。 使用这个体外模型，我们已经确定WNT信号是一个关键的外在信号， 指定尿路上皮祖细胞并抑制结直肠上皮祖细胞。此外，使用 CRISPR-Cas9介导的人IPSCs编辑，我们发现尿路上皮规范是 在GATA3缺陷细胞中受到干扰。基于这些观察，我们制定了 假设BMP和WNT两条发育途径汇聚在一起诱导 随后抑制CDX2的GATA3在尿路上皮祖细胞中的表达。 这一假设将在两个具体目标中得到检验： 目的1：探讨BMP和WNT信号转导机制。 尿路上皮细胞中GATA3的激活。 目的2：确定CDX2被抑制的分子机制 尿路上皮炎。 使用我们的大肠和尿路上皮分化的体外模型，我们将确定BMP如何 WNT信号融合诱导GATA3的表达。此外，我们将确定是否 GATA3通过抑制CDX2来驱动尿路上皮规范。这些研究将 阐明后肠分割的关键机制，这将使发展 改进的结肠和尿路上皮有机化合物，可用于疾病建模和 器官移植。

The hindgut gives rise to colorectal and urothelial/bladder fates. The generation of these 2 fates requires the partitioning of the hindgut. Improper partitioning of the hindgut causes anorectal malformations. We have generated a powerful new in vitro model that allows for the generation of both colorectal and urothelial organoids from human induced pluripotent stem cells (IPSCs) through the transient activation of Bone Morphogenetic Protein (BMP) signaling. Using this in vitro model, we’ve identified WNT signaling as a critical extrinsic signal that specifies urothelial progenitors and represses colorectal progenitors. In addition, using CRISPR-Cas9 mediated editing of human IPSCs, we found that urothelial specification is perturbed in GATA3 deficient cells. Based on these observations we formulated the hypothesis that two developmental pathways, BMP and WNT, converge to induce expression of GATA3 which subsequently represses CDX2 in urothelial progenitors. This hypothesis will be tested in 2 specific aims: Aim 1: Determine the mechanism of cooperative BMP and WNT signaling in the activation of GATA3 in the urothelium . Aim 2: Determine the molecular mechanisms through which CDX2 is repressed in the urothelium. Using our in vitro model of colorectal and urothelial differentiation, we will determine how BMP and WNT signaling converge to induce expression of GATA3. In addition, we will determine if GATA3 drives urothelial specification through the inhibition of CDX2. These studies will elucidate key mechanisms of hindgut partitioning which will enable the development of improved colonic and urothelial organoids which can be used for disease modeling and for organ replacement.