Synthetic approaches to study cell polarity related kidney defects

摘要 先天性肾脏和泌尿道异常（CAKUT）约占儿童慢性 肾病病例。许多CAKUT缺陷涉及肾集合管尺寸或间距不精确 结构，如重复输尿管，囊性集合管和肾发育不全，这可能导致严重的 健康问题，包括末期肾病。因此，迫切需要了解如何 发育过程促进肾脏结构的适当大小、间距和定位， 缺陷是可以纠正的。肾脏的发育始于输尿管芽的树状生长 上皮（未来的集合管网络）变成疏松的结缔组织或间充质。精确 在该网络中定位输尿管芽小管需要严格控制Ret激酶信号传导。Ret回应 从周围的间充质细胞分泌胶质细胞源性神经营养因子（GDNF）， 影响Ret-GDNF信号传导导致CAKUT缺陷。虽然Ret-GDNF信号转导驱动增殖性 扩张的输尿管芽上皮细胞，新出现的证据表明，“平面细胞极性”（PCP）- a 细胞感知其在板和管内的平面位置的机制-控制了 集水管网值得注意的是，最近的研究结果表明，表达PCP的间充质细胞， Fat 4和Dchs 1基因阻止了输尿管芽的不适当分支，这些基因的突变导致CAKUT 老鼠和人类的缺陷。然而，目前还不清楚表达PCP的间充质细胞之间的界面是如何形成的。 细胞和表达Ret的上皮细胞加强了集合管小管的精确尺寸和间距， 避免缺陷。该提案的目标是在PCP表达之间创建受控的空间界面 细胞和表达Ret的细胞，并研究它们对Ret-GDNF信号传导水平的影响以及对 上皮结构的大小和形状。该提案的目的1建立了基于DNA的细胞模式化 该技术能够在工程组织中产生细胞界面。这种细胞图案化技术 将用于在表达PCP的细胞和表达Ret的细胞之间产生界面。细胞表达 基于荧光的激酶活性报告和数学建模将被用来研究这些 空间界面影响Ret-GDNF信号传导。本提案的目标2将在3D中对两种细胞类型进行图案化 组织支架，并研究界面对所得上皮结构的大小和形状的影响。的 该建议的中心假设是PCP表达细胞局部限制Ret-GDNF驱动的上皮细胞， 组织在界面处生长，从而产生限定尺寸和形状的结构。总之， 在这项提案中开发的将提高我们对引起CAKUT的细胞机制的理解 缺陷，并创造新的工具，以建立人类疾病的类器官模型定义的组织结构。

ABSTRACT Congenital abnormalities of the kidney and urinary tract (CAKUT) account for ~50% of childhood chronic kidney disease cases. Many CAKUT defects involve imprecise sizing or spacing of kidney collecting duct structures, such as duplicated ureters, cystic collecting ducts, and renal hypoplasia, which can lead to severe health problems including end-stage kidney disease. As such, there is a critical need to understand how developmental processes promote proper sizing, spacing, and positioning of kidney structures, so that CAKUT defects can be corrected. Kidney development begins through tree-like outgrowth of the ureteric bud epithelium (the future collecting duct network) into a loose connective tissue or mesenchyme. Precisely positioning ureteric bud tubules within this network requires tight control of Ret kinase signaling. Ret responds to secreted glial cell-derived neurotrophic factor (GDNF) from surrounding mesenchymal cells and mutations that affect Ret-GDNF signaling cause CAKUT defects. While Ret-GDNF signaling drives the proliferative expansion of ureteric bud epithelial cells, emerging evidence suggests that “planar cell polarity” (PCP) – a mechanism by which cells sense their planar positions within sheets and tubes – controls the shape of the collecting duct network. Significantly, recent findings indicate that mesenchymal cells expressing the PCP genes Fat4 and Dchs1 prevent improper ureteric bud branching and mutations in these genes cause CAKUT defects in mice and humans. However, it is unclear how interfaces between PCP-expressing mesenchymal cells and Ret-expressing epithelial cells enforce the precise sizing and spacing of collecting duct tubules and avoid defects. The objective of this proposal is to create controlled spatial interfaces between PCP-expressing cells and Ret-expressing cells and study their impact on Ret-GDNF signaling levels and the resultant effect on the size and shape of epithelial structures. Aim 1 of this proposal establishes a DNA-based cell patterning technology that enables the production of cell interfaces in engineered tissues. This cell patterning technology will be used to create interfaces between PCP-expressing cells and Ret-expressing cells. Cells expressing fluorescence-based kinase activity reporters and mathematical modeling will be used to study how these spatial interfaces influence Ret-GDNF signaling. Aim 2 of this proposal will pattern the two cell types in 3D tissue scaffolds and study the effects of interfaces on the size and shape of resulting epithelial structures. The central hypothesis of this proposal is that PCP-expressing cells locally restrict Ret-GDNF-driven epithelial tissue growth at interfaces, thereby producing structures of defined size and shape. Together, the approaches developed in this proposal will improve our understanding of the cellular mechanisms that cause CAKUT defects and create new tools to build defined tissue structures in organoid models of human disease.