Fluid transport in inner ear development

描述（由申请人提供）：每1000名儿童中约有1名患有遗传性听力损失，其中SLC 26 A4突变是遗传性耳聋最常见的已知原因之一。高发病率提供了调查SLC 26 A4相关耳聋的病因学的必要性，最终目标是制定策略以恢复和保护患病个体的听力。人类基因SLC 26 A4和小鼠直系同源物Slc 26 a4编码pendrin。在小鼠中的研究表明，pendrin是耳蜗、前庭迷路和内淋巴囊中的Cl-/HCO 3-交换剂。在胚胎发育过程中缺乏pendrin会导致内淋巴酸化，前庭迷路中的液体分泌与内淋巴囊中pendrin依赖性液体吸收之间不匹配，导致膜迷路扩大和耳蜗发育受损。扩大的前庭水管，经常与SLC 26 A4突变有关，似乎是胚胎发育期间存在的这种扩大的化石记录。胚胎内耳中液体分泌和吸收的机制实际上是未知的。填补我们对内耳发育的理解中的这一空白对于开发治疗方法以保护患有SLC 26 A4突变的个体的听力至关重要。我们已经开发了四个特定目标，以解决小鼠模型中最突出的问题：目标1）-胚胎耳蜗和内淋巴囊内淋巴的离子组成是什么？这一目标将通过用离子选择性电极测量内耳液的组成来解决。目的2）什么机制介导液体分泌？目的3）什么机制介导液体吸收？这些目标将通过测试液体分泌和液体吸收的假设模型来解决。将通过qRT-PCR和免疫细胞化学或原位杂交确定候选通道和转运蛋白表达的起始和位置。将在化合物突变小鼠中测试选定的模型。与液体分泌有关的通道或转运蛋白的表达不足有望抑制耳蜗扩大，并可能在pendrin缺乏的情况下恢复正常的耳蜗发育。相反，在pendrin的存在下，与液体吸收有关的通道或转运蛋白的表达不足预计会导致耳蜗扩大。Aim 4）仅恢复内淋巴囊的pendrin表达是否足以预防耳蜗扩大和耳聋？这一目标将通过产生pendrin表达限于内耳内淋巴囊的小鼠来解决。研究将包括通过组织学监测耳蜗腔形成，用离子选择性电极测量耳蜗内电位和内淋巴pH值，并通过听觉脑干记录评估听力。这四个目标的完成将建立耳蜗发育中的流体稳态的理解，这有望揭示适合预防携带SLC 26 A4突变的个体耳聋的治疗策略。 公共卫生相关性：SLC 26 A4基因突变是幼儿听力损失的最重要原因之一，这种高发病率为研究SLC 26 A4相关耳聋的病因提供了必要条件，最终目标是制定恢复和保护受影响个体听力的策略。人类基因SLC 26 A4和小鼠直系同源基因Slc 26 a4编码蛋白质pendrin，该蛋白质有助于胚胎内耳中的液体运输，其方式对于听力的发育至关重要。这 该提案使用小鼠模型来解决有关胚胎内耳中液体运输的基本问题，这对于开发保护患有SLC 26 A4突变的个体听力的治疗至关重要。

DESCRIPTION (provided by applicant): About 1 in 1000 children has hereditary hearing loss with mutations of SLC26A4 being one of the most prevalent known causes of hereditary deafness. The high incidence provides an imperative to investigate the etiology of SLC26A4-related deafness with the ultimate goal to develop strategies to restore and preserve hearing in afflicted individuals. The human gene SLC26A4 and the mouse ortholog Slc26a4 code for pendrin. Studies in mouse have demonstrated that pendrin is a Cl-/HCO3- exchanger in the cochlea, vestibular labyrinth and the endolymphatic sac. Lack of pendrin during embryonic development causes an acidification of endolymph and a mismatch between fluid secretion in the vestibular labyrinth and pendrin-dependent fluid absorption in the endolymphatic sac that leads to an enlargement of the membranous labyrinth and an impairment of cochlear development. The enlarged vestibular aqueduct, frequently associated with mutations of SLC26A4, appears to be a fossil-like record of such an enlargement that was present during embryonic development. Mechanisms of fluid secretion and fluid absorption in the embryonic inner ear are virtually unknown. Filling this gap in our understanding of inner ear development is critical toward the development of treatments to protect hearing in individuals afflicted with mutations of SLC26A4. We have developed four Specific Aims that address the most salient questions in mouse models: Aim1) - what is the ionic composition of endolymph in the embryonic cochlea and the endolymphatic sac? This aim will be addressed by measuring the composition of inner ear fluids with ion-selective electrodes. Aim2) what mechanisms mediate fluid secretion? Aim3) what mechanisms mediate fluid absorption? These aims will be addressed by testing hypothetical models of fluid secretion and fluid absorption. The onset and location of expression of candidate channels and transporters will be determined by qRT-PCR and immunocytochemistry or in-situ hybridization. Selected models will be tested in compound-mutant mice. Deficient expression of channels or transporters implicated in fluid secretion is expected to curb cochlear enlargement and possibly restore normal cochlear development in the absence of pendrin. Conversely, deficient expression of channels or transporters implicated in fluid absorption is expected to cause cochlear enlargement in the presence of pendrin. Aim4) is restoration of pendrin expression solely to the endolymphatic sac sufficient to prevent cochlear enlargement and deafness? This aim will be addressed by generating mice with pendrin expression limited to the endolymphatic sac of the inner ear. Studies will include monitoring cochlear lumen formation by histology, measurements of the endocochlear potential and endolymphatic pH with ion-selective electrodes and evaluating hearing by auditory brain stem recordings. Completion of these four Aims will establish an understanding of fluid homeostasis in cochlear development which holds the promise to reveal treatment strategies that are suitable to prevent deafness in individuals that bear mutations of SLC26A4. PUBLIC HEALTH RELEVANCE: Mutations of the gene SLC26A4 are one of the most important causes of hearing loss in young children and this high incidence provides an imperative to investigate the etiology of SLC26A4-related deafness with the ultimate goal to develop strategies to restore and preserve hearing in afflicted individuals. The human gene SLC26A4 and the mouse ortholog Slc26a4 code for the protein pendrin that contributes to fluid transport in the embryonic inner ear in a way that is critical for the development of hearing. This proposal uses mouse models to address fundamental questions regarding fluid transport in the embryonic inner ear, which is critical for the development of treatments that protect hearing in individuals afflicted with mutations of SLC26A4.