The transcriptional control and function of proton-secreting cells

描述（由申请人提供）：哺乳动物肾脏的嵌入细胞对维持体内pH稳态至关重要。为了实现这一功能，α型插层细胞分泌酸，β型细胞分泌碳酸氢盐。然而，这两种插层细胞亚型的分化和分化在活体器官中的研究具有挑战性。本研究提出了一种新的实验模型，即非洲爪蟾胚胎皮肤的质子分泌细胞，该细胞被认为是调节胚胎pH稳态的细胞。这些细胞类似于肾的插层细胞，它们受Notch负调控，需要转录因子fox1来规范。在基因表达和蛋白定位方面，天牛的PSCs也分为α -PSCs和β -PSCs两种亚型，它们与插层细胞亚型完全相似。最近，我在粒头家族中发现了一个转录因子ubp1l，它介导α -和β - psc之间的发育转换。当过表达时，它促进β - pscs的分化，抑制α - pscs的分化。我假设ubp1l对于β - psc的规范和功能是必不可少的，并且它的作用在肾脏的ic中是保守的。我将通过以下三个目标来探索ubp11控制的这种命运决定的转录机制。目的1。研究ubp1l对PSC命运选择的转录控制。在α - psc和β - psc之间的命运选择中，ubp1l必须促进阴离子交换剂pendin （β - psc标记物）的表达，同时抑制α - psc标记物AE1的表达。我将通过操纵ubp1l水平、活动时间以及制造激活子和抑制子融合结构来研究ubp1l对这些基因的转录控制。我还将用gfp转基因动物研究pendrin和AE1启动子。目标2。确定白杨PSCs的转录和功能可塑性。肾插层细胞改变其质子分泌特性和α -到β -插层细胞的比例以响应pH挑战。为了将PSCs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了变化，并检查ubp1是否位于这种变化的上游。目标3。ubp1l在哺乳动物肾脏β - ic中特异性表达吗？Ubp1l在小鼠基因组中是保守的，但未被注释。首先，我将研究在小鼠出生后早期发育过程中插入细胞亚型命运决定的时间，以便将其表达动态与在X. laevis皮肤中观察到的同源基因进行比较。接下来，我将确定ubp1l是否在小鼠插层细胞中表达，它是否在适当的时间表达以影响细胞命运选择，以及它是否与β插层细胞标记共表达。

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.