The transcriptional control and function of proton-secreting cells

描述（由申请方提供）：哺乳动物肾脏的嵌入细胞对于维持体内pH稳态至关重要。为了执行这一功能，α型插入细胞分泌酸，β型分泌碳酸氢盐。然而，这两种亚型的闰细胞的特化和分化在活体器官中的研究具有挑战性。本申请提出研究一种新的实验模型，非洲爪蟾胚胎皮肤的质子分泌细胞，其被认为介导胚胎的pH稳态。这些细胞类似于肾脏的闰细胞，因为它们受到Notch的负调控，并且需要转录因子Foxi 1用于其特化。X.子宫动脉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似。最近，我在grainyhead家族中鉴定了一种转录因子ubp 1 l，它介导了α-和β-PSC之间的发育转换。当过表达时，它促进β-PSC的分化并抑制α-PSC的分化。我假设ubp 1 l是必不可少的β-PSC的规范和功能，它的作用是保守的IC在肾脏。我将探索由ubp 1 l控制的这种命运决定的转录机制，有以下三个目标。目标1。检查ubp 1 l对PSC命运选择的转录控制。在介导α-和β-PSC之间的命运选择时，ubp 1 l必须促进阴离子交换剂pendrin（β-PSC标记物）的表达，同时抑制AE 1（α-PSC标记物）的表达。我将研究这些基因的转录控制ubp 1 l操纵ubp 1 l水平，活动的时间，并使激活剂和阻遏物融合结构。我还将检查pendrin和AE 1启动子与GFP转基因动物。目标2.确定X的转录和功能可塑性。光滑的PSC。肾脏的嵌入细胞改变其质子分泌特性和响应pH挑战的α-至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亚型的比例是否有变化，并检查ubp 1是否位于这种变化的上游。目标3：ubp 1 l是否在哺乳动物肾脏的β-IC中特异性表达？在小鼠基因组中，ubp 1 l是保守的，但未注释。首先，我将研究在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，以比较在X染色体中观察到的直向同源基因的表达动态。光滑皮肤接下来，我将确定ubp 1 l是否在小鼠嵌入细胞中表达，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-嵌入细胞标记物共表达。

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.