Signals Regulating SAG Development

描述(由申请人提供)：神经元丢失，依赖于或独立于感觉毛细胞丢失，对听力损失有很大的影响，特别是在老龄化人口中。未来的治疗，特别是干细胞治疗，将需要详细了解状态听神经节(SAG；第七脑神经)的发育过程。我们建议分析斑马鱼SAG发育过程中钙粘附素细胞黏附的分子和细胞机制。将使用各种分析方法研究SAG前体细胞的行为，包括分析增殖、存活、成熟和SAG前体细胞从耳泡到形成神经节的运动的先进成像方法。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，钙粘附素细胞黏附系统对极性提示做出反应，并调节形态发生细胞的运动。目的1：研究正常胚胎和敲除胚胎SAG发育过程中钙粘蛋白-2、-4、-6和-10的表达模式。CDH2、CDH4和CDH6基因敲除实验(命名法：基因名为CDH2、CDH4、CDH6和CDH10；蛋白质名为CDH2、CDH4、CDH6和CDH10)表明，这些钙粘附素是SAG发育所必需的。CDH10基因敲除显示SAG发育表型很少或没有，但这种钙粘附素在SAG细胞中表达的亚群要小得多。我们将研究钙粘蛋白-2、-4、-6和-10的空间和时间表达模式。我们推测，在内耳发育过程中，钙粘附素的表达调节SAG前体细胞的运动、前体细胞的存活和神经元的分化。在CDH2、4、6和10基因敲除的胚胎中，也将检测单个钙粘附素的表达模式，以确定是否存在代偿性钙粘附素表达。目的2：探讨钙粘素信号在SAG发生发展中的作用。我们假设，在SAG发育过程中，钙粘附素活性调节特定的细胞表型(分化、生长、存活和迁移)。信号机制将使用ONE规范、增殖和存活率的分析进行比较。此外，我们建议使用最先进的时间推移成像来评估SAG前体细胞的行为。正常的SAG发育将与钙粘素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。目的3.确定调节SAG细胞发育的特定钙粘附素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，钙粘附素活性可以自主地调节不同的细胞表型(如分化、生长、存活和迁移)，也可以调节其他细胞表型。在细胞移植方面，将进行基因镶嵌实验，以确定已确定的分子和细胞机制是自主作用于细胞还是非自主作用于细胞。神经细胞丢失会导致听力损失，特别是在老龄化人口中。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护。我们提出了一种新的方法来研究这一过程，该方法符合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.