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

在胚胎和胎儿阶段，肾脏发育出数以百万计的肾单位，产生高度专门化的 细胞。这些细胞确保流入肾脏的血液被过滤，所需的物质被重新吸收 而不需要的代谢物和溶质被引导到膀胱排泄。出生缺陷在老年人中很常见 肾脏，约1/100的新生儿患有所谓的先天性肾和尿路畸形(CAKUT)。在… 作为CAKUT最严重的终点，新生儿缺乏肾脏功能，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，基因变化缺乏背景。的确有 因此，迫切需要了解哪里会出现发育缺陷，并产生新的治疗方法 或者替换肾功能。在我们的工作中，我们使用了单细胞组学和分子表征 人类和小鼠的肾脏为肾单位的形成提供了蓝图，并绘制了在人类身上复制这种单位的地图 干细胞来源的肾脏有机体。在这样做的过程中，我们为基因提供了遗传和发育背景 在CAKUT患者中发现。在这份提案中，我们将遵循这些线索，并解决三个悬而未决的问题 在发育中的肾脏病。在目标1中，我们研究了远端肾小管段的胚胎起源。 我们将进行第一次将发育中的肾脏和成人肾脏联系起来的单细胞基因组分析。这提供了一个路线图 细胞是如何分化的。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 肾单位与成熟肾脏的功能细胞有关。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。我们将使用一种名为Cut&Run的技术来分析基因如何经常发生突变 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如何形成因果关系。 基因表达和调控之间的关系。我们将使用我们的新系统从 人类干细胞同步化的肾病。在这个系统中，肾单位的发育是同步的， 与体内不同的是，来自许多发育阶段的肾单位彼此形成很近。我们的系统提供 研究、操作和分离处于同一发育阶段的肾单位的细胞具有独特的优势。这个 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 肾脏学-肾单位最初是如何形成图案的？要做到这一点，我们将使用合成细胞组织器 分泌信号蛋白来塑造我们同步的肾小球。我们将研究信号配体如何控制 肾单位的形成和图案化。这也有实际的应用，因为我们可以获得对肾病的控制 图案化。我们的系统将为我们建立大量并行的肾小球阵列以进行替换提供信息 治疗和疾病模型。该提案的一个优点是人和人之间的独特专业知识 小鼠肾脏遗传学，干细胞衍生的肾脏有机化合物的新系统，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.