The transcriptional control and function of proton-secreting cells

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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. PUBLIC HEALTH RELEVANCE: To maintain the acid/base balance of the body, the kidney requires cell types called intercalated cells that regulate acid or bicarbonate. We do not know much about how these cells are regulated or how they develop, and their behavior is difficult to study inside the living organ of a developing mammal. This study proposes to examine the development of easily accessible cells in the frog epidermis which perform a similar function as, and strongly resemble, the intercalated cells of the kidney.