Signals Regulating SAG Development

描述（由申请人提供）：神经元丧失，依赖或独立于感觉毛细胞丧失，对听力损失有重要影响，特别是在老龄化人群中。未来的治疗，特别是干细胞治疗，将需要详细了解静听神经节（SAG；第八脑神经）的发育过程。我们拟分析斑马鱼SAG发育过程中钙粘蛋白细胞粘附的分子和细胞机制。SAG前体细胞的行为将通过各种方法进行研究，包括先进的成像方法来分析增生、存活、成熟和SAG前体细胞从耳小泡到形成神经节的运动。这些实验特别符合NIDCD计划公告PA-07-127。总体假设：在SAG发育过程中，钙粘蛋白细胞粘附系统响应极性信号并调节形态发生细胞的运动。目的1：确定正常和敲除胚胎SAG发育过程中cadherin-2、-4、-6和-10（在SAG中表达的cadherin）的表达模式。Cdh2、Cdh4和Cdh6敲低实验（命名：基因名称为Cdh2、Cdh4、Cdh6和cdh10；蛋白质名称为Cdh2、Cdh4、Cdh6和cdh10）表明，这些钙粘蛋白是SAG发展所必需的。Cdh10敲低显示很少或没有SAG发育表型，但这种钙粘蛋白在SAG细胞的一个小亚群中表达。我们将研究钙粘蛋白-2、-4、-6和-10的时空表达模式。我们假设在内耳发育过程中，钙粘蛋白的表达调节SAG前体细胞运动、前体细胞存活和神经元分化。还将在Cdh2、4、6和10敲低胚胎中检测个体钙粘蛋白表达模式，以确定是否存在补偿性钙粘蛋白表达。目的2：确定钙粘蛋白信号在SAG发展中的作用。我们假设钙粘蛋白活性在SAG发育过程中调节特定的细胞表型（分化、生长、生存和迁移）。信号机制将比较使用分析的特异性，增殖和生存。此外，我们建议使用最先进的延时成像技术来评估SAG前体细胞的行为。将正常的SAG发展与钙粘蛋白功能丧失表型进行比较。此外，还将研究细胞极性信号分子在SAG发育过程中的作用。目标3。确定调节SAG细胞发育的特异性钙粘蛋白活性是细胞自主的还是非细胞自主的。我们推测，在SAG发育过程中，钙粘蛋白活性可以自主调节不同的细胞表型（如分化、生长、存活和迁移）和其他细胞表型（如细胞非自主调节）。细胞移植，基因镶嵌实验将进行，以确定是否分子和细胞机制的行为是细胞自主或细胞非自主。神经细胞的丧失会导致听力损失，尤其是在老年人群中。未来的听力损失治疗，特别是干细胞治疗的前景，将需要详细了解神经发育，包括听神经的生长和维持。我们提出了一种新的方法来研究这一过程，该方法适合NIDCD计划公告PA-07-127。

DESCRIPTION (provided by applicant): Neuron loss, either dependent or independent of sensory hair cell loss, contributes significantly to hearing loss, particularly in the aging population. Future treatments, particularly stem cell therapies, will require detailed understanding of statoacoustic ganglion (SAG; VIIIth cranial nerve) developmental processes. We propose to analyze molecular and cellular mechanism of cadherin cell adhesion during zebrafish SAG development. SAG precursor cell behaviors will be studied using various assays, including advanced imaging methods to analyze proliferation, survival, maturation and SAG precursor cell movements from the otic vesicle to the forming ganglion. These experiments specifically fit the NIDCD Program Announcement PA-07-127. Overall hypothesis: the cadherin cell adhesion system responds to polarity cues and regulates morphogenetic cell movements during SAG development. Aim 1: Determine the expression pattern for cadherin-2, -4, -6 and -10 (cadherins expressed in the SAG) during normal and knockdown embryo SAG development. Cdh2, Cdh4 and Cdh6 knockdown experiments (nomenclature: gene names cdh2, cdh4, cdh6 and cdh10; and protein names Cdh2, Cdh4, Cdh6 and Cdh10) showed that these cadherins are required for SAG development. Cdh10 knockdown showed little or no SAG development phenotype, but this cadherin is expressed in a much smaller subset of SAG cells. We will examine spatial and temporal expression patterns for cadherin-2, -4, -6 and -10. We hypothesize that cadherin expression regulates SAG precursor cell movements, precursor cell survival and neuronal differentiation during inner ear development. Individual cadherin expression patterns will also be examined in Cdh2, 4, 6 and 10 knockdown embryos to determine whether there is compensatory cadherin expression. Aim 2: Determine cadherin signaling effects on SAG development. We hypothesize that cadherin activity regulates specific cellular phenotype (differentiation, growth, survival and migration) during SAG development. Signaling mechanisms will be compared using assays for otic specification, proliferation and survival. In addition, we propose to evaluate SAG precursor cell behaviors using state-of-the-art time-lapse imaging. Normal SAG development will be compared with cadherin loss-of-function phenotypes. In addition, cell polarity signaling molecule functions during SAG development will be studied. Aim 3. Determine whether specific cadherin activity that regulates SAG cell development is cell autonomous or non-cell autonomous. We hypothesize that cadherin activity can regulate different cellular phenotypes (e.g., differentiation, growth, survival and migration) cell autonomously and other cellular phenotypes cell-non autonomously during SAG development. Cell transplantation, genetic mosaic experiments will be performed to determine whether the molecular and cellular mechanisms that were identified act cell autonomously or cell-non autonomously. Nerve cell loss contributes to hearing loss, particularly in the aging population. Future treatments for hearing loss, especially the promise of stem cell therapies, will require detailed understanding of nerve development, including growth and maintenance of the auditory nerve. We propose a novel approach to the study of this process, which fits NIDCD Program Announcement PA-07-127.