Gene Expression Changes in Early 3D Renal Tubulogenesis

描述（由申请人提供）：许多上皮器官，如肾脏，主要由分支管状结构组成。我们的目标是了解一般的小管形成的生物学，特别是肾小管形成。小管的形成是一个涉及多种因素和受体的鲜为人知的过程。由于器官发生的复杂性和短暂性，在体内研究微管发生是非常困难的。我们和其他人已经成功地使用二维（2D）体外细胞培养（在塑料或可渗透过滤器上生长的细胞）来研究小管上皮细胞的生长因子依赖性调节。然而，越来越多的证据表明，上皮细胞受细胞微环境的不同调控，强烈主张使用适当的三维（3D）模型来研究小管形成，例如在3D胶原基质中研究Madin-Darby犬肾（MDCK）细胞生长直到囊肿阶段，并使用肝细胞生长因子（HGF）诱导小管形成的实验。我们已经使用这个系统来鉴定对小管形成至关重要的蛋白质。我们的基本假设是，3D培养MDCK细胞中HGF诱导后的基因表达变化比HGF刺激前2D培养MDCK细胞的基因表达变化更具生理性，因此与肾小管形成有关。为了通过无偏倚的方法定义参与MDCK细胞微管形成的新基因和途径，我们提出了一个利用3D MDCK/HGF检测结合新可用的犬DNA微阵列的系统，我们最近描述并验证了（Aim 1.1）。我们将具体确定HGF刺激肾小管MDCK细胞的基因表达如何根据微环境（即2D与3D培养）而不同（目的1.2）。最有希望的候选“微管基因”将根据mRNA折叠变化、途径放置和机制合理性进行选择（Aim 2.1）。这些候选基因将在体外肾小管形成的不同阶段通过其蛋白产物的定位和突变分析进行表征，以确定其在小管形成中的作用（Aim 2.2）。接下来将提出进一步的研究，包括建立体内模型来测试假定的小管基因在肾脏发育中的作用，最终目标是调节这些小管基因和修复小管形成缺陷的途径。

DESCRIPTION (provided by applicant): Many epithelial organs, such as the kidney, are composed largely of branching tubular structures. Our goal is to understand the biology of tubulogenesis in general and renal tubulogenesis in particular. Tubule formation is a poorly understood process involving multiple factors and receptors. Due to the complexity and transitory nature of organogenesis, it is exceedingly difficult to study tubulogenesis in vivo. We and others have successfully used two-dimensional (2D) in vitro cell culture (cells grown on plastic or permeable filters) to study growth factor dependent regulation of tubular epithelial cells. However, a growing body of evidence indicates that epithelial cells are differentially regulated depending upon the cellular microenvironment, arguing strongly for the study of tubulogenesis using an appropriate three-dimensional (3D) model, such as the well-studied assay involving growth of Madin-Darby canine kidney (MDCK) cells in a 3D collagen matrix until the cyst stage and induction of tubule formation with hepatocyte growth factor (HGF). We have used this system to identify proteins critical for tubulogenesis. Our underlying hypothesis is that gene expression changes following HGF induction in MDCK cells grown in 3D culture is more physiologic than gene expression changes in MDCK cells grown in 2D culture prior to stimulation with HGF, and, therefore, is relevant to renal tubulogenesis. To define novel genes and pathways involved in tubulogenesis of MDCK cells by an unbiased approach, we propose a system utilizing the 3D MDCK/HGF assay in combination with a newly available canine DNA microarray, which we recently described and validated (Aim 1.1). We will specifically determine how gene expression differs in renal tubular MDCK cells stimulated with HGF depending on the microenvironment (i.e. in 2D versus 3D culture) (Aim 1.2). The most promising candidate "tubulogenes" will be selected based on mRNA fold change, pathway placement, and mechanistic plausibility (Aim 2.1). These candidate genes will be characterized during the different stages of in vitro renal tubule formation by localization of their protein products and mutational analysis, to define their role in tubulogenesis (Aim 2.2). Further studies will then be proposed that involve generation of in vivo models to test the role of the putative tubulogenes in renal development, with the ultimate goal of modulating these tubulogenes and pathways to repair defects in tubule formation.