Fluid transport in inner ear development

描述（由申请人提供）：大约每1000名儿童中就有1名患有遗传性听力损失，SLC26A4基因突变是遗传性耳聋最普遍的已知原因之一。slc26a4的高发病率为研究slc26a4相关耳聋的病因提供了必要的条件，最终目标是制定恢复和保护患者听力的策略。人类基因SLC26A4和小鼠同源基因SLC26A4编码pendin。小鼠实验表明，pendrin在耳蜗、前庭迷路和内淋巴囊中具有Cl-/HCO3-交换作用。胚胎发育过程中缺乏penddrin会导致内淋巴酸化，前庭迷路的液体分泌与内淋巴囊中penddrin依赖性液体吸收之间的不匹配，从而导致膜性迷路扩大和耳蜗发育受损。增大的前庭导水管，通常与SLC26A4突变有关，似乎是胚胎发育期间出现的这种增大的化石样记录。胚胎内耳中液体分泌和吸收的机制几乎是未知的。填补我们对内耳发育理解的这一空白，对于开发保护SLC26A4突变患者听力的治疗方法至关重要。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？这一目标将通过离子选择电极测量内耳液体的组成来解决。目的2)什么机制介导液体分泌？目标3)什么机制介导流体吸收？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。候选通道和转运体的表达开始和位置将通过qRT-PCR和免疫细胞化学或原位杂交确定。选定的模型将在化合物突变小鼠中进行测试。参与液体分泌的通道或转运蛋白表达不足有望抑制耳蜗增大，并可能在缺少pendin的情况下恢复正常的耳蜗发育。相反，与液体吸收有关的通道或转运蛋白表达不足，预计会在存在pendin时引起耳蜗增大。仅在内淋巴囊内恢复penddrin表达是否足以预防耳蜗增大和耳聋？这一目标将通过产生内耳淋巴囊内表达penddrin的小鼠来解决。研究将包括通过组织学监测耳蜗腔的形成，使用离子选择电极测量耳蜗内电位和内淋巴pH值，以及通过听觉脑干记录评估听力。完成这四个目标将建立对耳蜗发育中的流体稳态的理解，从而有望揭示适合于预防SLC26A4突变个体耳聋的治疗策略。

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.