The transcriptional control and function of proton-secreting cells

质子分泌细胞的转录控制和功能

• 批准号: 8389809
• 负责人: Ian K. Quigley
• 金额: $ 5.39万
• 依托单位: SALK INSTITUTE FOR BIOLOGICAL STUDIES
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8389809
• 关键词: Acid-Base Equilibrium; Acids; Address; Affect; Anions; Behavior; Beryllium; Bicarbonates; Biological Models; Cell membrane; Cells; Development; Distal; Duct (organ) structure; Embryo; Environment; Epidermis; Experimental Models; Family; Gene Expression; Gene Proteins; Genes; Homeostasis; Intercalated Cell; Ion-Selective Electrodes; Ions; Kidney; Life; Link; Mammals; Measures; Mediating; Modeling; Movement; Mus; Organ; Orthologous Gene; Physiological; Play; Property; Protein Isoforms; Proteins; Proton Pump; Proton-Motive Force; Proton-Translocating ATPases; Protons; Rana; Regulation; Role; Skin; Specialized Epithelial Cell; Testing; Time; Transcriptional Regulation; Transgenic Animals; Xenopus laevis; base; blastomere structure; cell type; mouse development; mouse genome; notch protein; novel; overexpression; pH Homeostasis; postnatal; programs; promoter; response; transcription factor

DESCRIPTION (provided by applicant): The intercalated cells of the mammalian kidney are critical to maintaining pH homeostasis in the body. To perform this function, alpha-type intercalated cells secrete acid and beta-types secrete bicarbonate. However, the specification and differentiation of these two subtypes of intercalated cells is challenging to study in the living organ. This application proposes to study a novel experimental model, the proton-secreting cells of the Xenopus laevis embryonic skin, which are thought to mediate pH homeostasis of the embryo. These cells are similar to intercalated cells of the kidney in that they are negatively regulated by Notch and require the transcription factor Foxi1 for their specification. X. laevis PSCs also come in two subtypes, alpha-PSCs and beta-PSCs, which resemble intercalated cell subtypes exactly in terms of gene expression and protein localization. Recently, I characterized a transcription factor in the grainyhead family, ubp1l, that mediates the developmental switch between alpha- and beta-PSCs. When over-expressed, it promotes the differentiation of beta-PSCs and represses the differentiation of alpha-PSCs. I hypothesize that ubp1l is essential for beta-PSC specification and function and that its role is conserved in the ICs in the kidney. I will explore the transcriptional mechanisms that underlie this fate decision as controlled by ubp1l with the following three aims. Aim 1. Examine the transcriptional control of PSC fate choice by ubp1l. In mediating the fate choice between alpha- and beta-PSCs, ubp1l must promote expression of the anion exchanger pendrin, a beta-PSC marker, while repressing expression of AE1, an alpha-PSC marker. I will examine the transcriptional control of these genes by ubp1l by manipulating ubp1l levels, timing of activity, and making activator and repressor fusion constructs. I will also examine the pendrin and AE1 promoters with GFP-transgenic animals. Aim 2. Determine the transcriptional and functional plasticity of X. laevis PSCs. Intercalated cells of the kidney change their proton secretory properties and the proportion of alpha- to beta-intercalated cells in response to pH challenge. To tie the regulation of PSCs to secretory function, I will directly measure proton flux from pH-challenged embryos with self-referencing ion-selective electrodes. Next, I will determine if there are changes in the proportions of PSC subtypes and examine if ubp1 lies upstream of such changes. Aim 3. Is ubp1l specifically expressed in beta-ICs of the mammalian kidney? ubp1l is conserved, yet unannotated, in the mouse genome. First, I will examine the timing of intercalated cell subtype fate decisions over the course of early postnatal mouse development in order to compare the expression dynamics with orthologous genes observed in the X. laevis skin. Next, I will determine if ubp1l is expressed in the mouse intercalated cells, and if it is expressed at an appropriate time to affect cell fate choice, and if it is co-expressed with beta-intercalated cell markers.

描述（由申请人提供）：哺乳动物肾脏的嵌入细胞对于维持体内 pH 稳态至关重要。为了执行此功能，α 型嵌入细胞分泌酸，β 型嵌入细胞分泌碳酸氢盐。然而，在活体器官中研究这两种闰细胞亚型的规格和分化具有挑战性。本申请旨在研究一种新的实验模型，即非洲爪蟾胚胎皮肤的质子分泌细胞，该细胞被认为可以介导胚胎的 pH 稳态。这些细胞与肾脏的闰细胞相似，它们受到 Notch 的负调控，并且需要转录因子 Foxi1 来实现其规范。 X. laevis PSC 也有两种亚型：α-PSC 和 β-PSC，它们在基因表达和蛋白质定位方面与闰细胞亚型完全相同。最近，我鉴定了粒状头家族中的一个转录因子 ubp1l，它介导 α-PSC 和 β-PSC 之间的发育转换。当过度表达时，它促进 β-PSC 的分化并抑制 α-PSC 的分化。我假设 ubp1l 对于 β-PSC 的规范和功能至关重要，并且其作用在肾脏的 IC 中是保守的。我将探索由 ubp1l 控制的这一命运决定背后的转录机制，其目标如下：目标 1. 检查 ubp1l 对 PSC 命运选择的转录控制。在介导 α-PSC 和 β-PSC 之间的命运选择时，ubp1l 必须促进阴离子交换剂 pendrin（一种 β-PSC 标记物）的表达，同时抑制 AE1（一种 α-PSC 标记物）的表达。我将通过操纵 ubp1l 水平、活动时间以及制作激活子和阻遏子融合结构来检查 ubp1l 对这些基因的转录控制。我还将用 GFP 转基因动物检查 pendrin 和 AE1 启动子。目标 2. 确定 X. laevis PSC 的转录和功能可塑性。肾脏的闰细胞响应 pH 挑战而改变其质子分泌特性以及 α 与 β 嵌入细胞的比例。为了将 PSC 的调节与分泌功能联系起来，我将使用自参考离子选择电极直接测量来自 pH 挑战胚胎的质子通量。接下来，我将确定 PSC 亚型的比例是否发生变化，并检查 ubp1 是否位于此类变化的上游。目标 3. ubp1l 是否在哺乳动物肾脏的 β-IC 中特异性表达？ ubp1l 在小鼠基因组中是保守的，但尚未注释。首先，我将检查出生后早期小鼠发育过程中插入细胞亚型命运决定的时间，以便将表达动态与在非洲爪蟾皮肤中观察到的直系同源基因进行比较。接下来，我将确定 ubp1l 是否在小鼠闰细胞中表达，是否在适当的时间表达以影响细胞命运选择，以及是否与 β 嵌入细胞标记物共表达。