Pathways governing survival of neural crest derivatives

描述(由申请人提供)：在发育过程中促进细胞存活的遗传途径还没有明确的定义，但却是抗癌药物的潜在靶点。促进黑素细胞存活的基因，特别是那些独立于由受体酪氨酸激酶C-KIT领导的具有良好特征的抗凋亡途径的基因，在很大程度上是未知的。通过对斑马鱼的正向和反向遗传学研究，我们已经确定了两个独立于C-KIT调控胚胎黑素细胞数量的基因。一种是TCT，它似乎可以防止黑素细胞的坏死细胞死亡，但其分子身份尚不清楚。另一个是转录因子AP-2pha，人们早就知道它在神经脊中表达，但由于冗余，其在那里的功能仍不清楚。有趣的是，我们有证据表明，TCT和AP-2也是神经脊源性Rohon-Beard感觉神经元(RBS)所必需的。我们的总体假设是，TCT和AP-2调节神经脊衍生物的存活，并且它们可能作为单一调节途径的一部分来实现这一点。在目标1中，为了深入了解可能的新的TCT途径，我们建议通过定位克隆来鉴定TCT基因，并识别新的等位基因。在目标2中，为了了解AP-2活性在神经脊和黑素细胞中的功能，我们提出了细胞类型特异性启动子对AP-2活性的时间和空间调节。在目的3中，为了确定TCT和AP-2在RBS中的功能，我们提出了TCT突变体中RBS的超微结构分析，以及Rb特异性启动子对AP-2类型活性的时空调控。最后，我们建议通过双突变实验来测试TCT和AP-2类型活性可能的相互作用。这些实验利用斑马鱼模型的优势来剖析知之甚少的神经脊调节通路。这些通路是治疗恶性黑色素瘤和其他神经脊疾病的潜在切入点。

DESCRIPTION (provided by applicant): Genetic pathways that promote cell-survival during development are poorly defined, yet represent potential targets for anti-cancer drugs. The genes that promote survival of melanocytes, in particular those that operate independently of the well-characterized anti-apoptotic pathway headed by the receptor tyrosine kinase, C-kit, are largely unknown. Through forward and reverse genetic studies in zebrafish, we have identified two genes that regulate the number of embryonic melanocytes independently of C-kit. One is Touchtone (Tct), which appears to prevent necrotic cell death in melanocytes, but whose molecular identity is unknown. The other is transcription factor AP-2alpha, which has long been known to be expressed in neural crest, but whose function there has remained obscure because of redundancy. Interestingly we have evidence that Tct and AP-2 are also required within neural crest-derived Rohon-Beard sensory neurons (RBs). Our overall hypothesis is that Tct and AP-2 regulate survival of neural crest derivatives, and that they may do so as part of a single regulatory pathway. In Aim 1, to gain insight into the possibly novel Tct pathway, we propose to identify the Tct gene by positional cloning and identify new alleles. In Aim 2, to learn the function of AP-2-type activity in neural crest and melanocytes, we propose temporal and spatial modulation of AP-2-type activity with cell-type-specific promoters. In Aim 3, to determine the function of Tct and AP-2 in RBs, we propose ultrastructural analysis of RBs in the Tct mutant, and temporal and spatial modulation of AP-2-type activity with RB-specific promoters. Finally we propose to test for possible interaction of Tct and AP-2 type activity with double mutant experiments. These experiments exploit the advantages of the zebrafish model to dissect poorly understood neural crest regulatory pathways. These pathways are potential entry points for therapies against malignant melanoma and other neural crest diseases.