Establishing and Mimicking Patterning Mechanisms in the Distal Nephron Tubule and Kidney Organoid

在胚胎和胎儿阶段，肾脏发育出数百万个肾单位，产生高度专业化的肾单位 细胞。这些细胞确保流入肾脏的血液被过滤并重新吸收所需的物质 而不需要的代谢物和溶质则被引导至膀胱排泄。出生缺陷常见于 肾脏，约 1/100 的新生儿患有所谓的肾脏和尿路先天性异常 (CAKUT)。在 CAKUT 最严重的结局是，新生儿丧失肾功能，并且他们的预期寿命低于 一年。大多数异常目前没有有效的干预措施，并且基因变化缺乏背景。有 因此，迫切需要了解发育缺陷出现的位置，并制定新的疗法来恢复发育缺陷。 或替代肾功能。在我们的工作中，我们使用了单细胞组学和分子特征 人类和小鼠肾脏，为肾单位如何形成提供蓝图，并绘制在人类中复制这一点的蓝图 干细胞衍生的肾脏类器官。在此过程中，我们为基因提供了遗传和发育背景 在 CAKUT 患者中发现。在本提案中，我们将遵循这些线索并解决三个悬而未决的问题 在发育肾病学中。在目标 1 中，我们研究远端肾单位小管段的胚胎起源。 我们将进行首次连接发育中肾脏和成年肾脏的单细胞组学分析。这提供了一个路线图 了解细胞如何分化。我们将使用新的小鼠遗传谱系追踪工具来测试早期远端细胞如何 肾单位与成熟肾脏中的功能细胞有关。这些实验将绘制出需要基因的位置： 肾单位发育。在目标 2 中，我们将研究开启和关闭基因的蛋白质如何控制 远端肾单位的发育。我们将使用一种称为 Cut&Run 的技术来分析基因如何经常突变 在 CAKUT 中，控制 DNA 和基因表达。我们还将激活信号通路并改变表达 与 CAKUT 相关的基因。这将使我们能够直接研究远端肾单位细胞的形成方式并提供因果关系 基因表达与调控之间。我们将使用我们的新系统生成数百个肾单位 同步肾病中的人类干细胞。在这个系统中，肾单位同时和同步发育， 与体内不同，来自多个发育阶段的肾单位彼此靠近形成。我们的系统提供 具有在同一发育阶段研究、操作和分离肾单位细胞的独特优势。的 我们收集的数据将显示基因是如何被激活的。在目标 3 中，我们解决了发展中的一个基本问题 肾脏病学 - 肾单位最初是如何形成的？为此，我们将使用合成细胞组织者 分泌信号蛋白来模拟我们的同步肾病。我们将研究信号配体如何控制 肾单位的形成和模式。这也有实际应用，因为我们可以控制肾病 图案化。我们的系统将为我们构建大规模并行肾细胞阵列以进行替换的努力提供信息 疗法和疾病建模。该提案的一个优势是独特的专业知识交叉人类和 小鼠肾脏遗传学、干细胞衍生的肾脏类器官的新系统以及合成生物学。

During embryonic and fetal stages, the kidneys develop millions of nephrons that generate highly specialized cells. These cells ensure that blood flowing into the kidney is filtered and required substances are reabsorbed while unwanted metabolites and solutes are led to the bladder for excretion. Birth defects are common in the kidney, ~ 1/100 of all births have a so-called Congenital Anomaly of the Kidney and Urinary Tract (CAKUT). At the most severe end of CAKUT, newborns are missing kidney functionality, and their life expectancy is less than one year. Most abnormalities have no current effective interventions and genetic changes lack context. There is thus a critical need to understand where developmental defects arise and to generate new therapies restoring or replacing kidney function. In our work we have used single cell omics and molecular characterizations of human and mouse kidneys to provide a blueprint for how nephrons form and maps for to replicate this in human stem cell-derived kidney organoids. In doing so we provide a genetic and developmental context to genes identified in CAKUT patients. In this proposal we will follow these leads and address three outstanding questions in developmental nephrology. In Aim 1, we investigate the embryonic origins of distal nephron tubule segments. We will perform the first single cell omic analysis linking developing and adult kidneys. This provides a roadmap for how cells differentiate. We will use new genetic mouse lineage-tracing tools to test how cells in the early distal nephron relate to functional cells in mature kidneys. These experiments will map where genes are required as the nephron develops. In Aim 2, we will investigate how proteins that turn genes on and off control the development of the distal nephron. We will use a technique called Cut&Run to analyze how genes often mutated in CAKUT, control DNA and gene expression. We will also activate signaling pathways and alter the expression of genes linked to CAKUT. This will allow us to directly study how distal nephron cells form provide causality between gene expression and regulation. We will use our new system to generate hundreds of nephrons from human stem cells in - synchronized nephroids. In this system, nephrons develop at the same time and pace, unlike in the body where nephrons from many developmental stages form near each other. Our system provides a unique advantage to study, manipulate, and isolate cells from nephrons at the same developmental stage. The data we collect will show how genes are activated. In Aim 3 we address a fundamental question in developmental nephrology - how is the nephron initially patterned? To do this we will use synthetic cellular organizers that secrete signaling proteins to pattern our synchronized nephroids. We will study how signal ligands control nephron formation and patterning. This also has a practical application as we can gain control over nephroid patterning. Our system will inform our efforts to build massive parallel arrays of nephroids for replacement therapies and disease modeling. A strength of the proposal is the unique expertise intersecting human and mouse kidney genetics, a novel system of stem cell derived kidney organoids, and synthetic biology.