Genetic Regulation of Rhombomere Formation

描述（由申请人提供）：将脊椎动物胚胎后脑分割成斜脑(r)，确保后脑衍生物（例如网状脊髓中间神经元和脑神经的运动核）的适当空间定位，因此对正常的神经活动至关重要。这一分割过程受到越来越多基因的调控，但其中许多基因的功能以及它们之间的调控关系尚不清楚。我们已经确定了12个在后脑中表达的新基因，我们假设这些基因在一个控制菱形体形成的调节网络中起作用。我们有两个目标来验证我们的假设：首先，我们将确定几个新的后脑基因在菱形体4/5形成中的功能。我们特别关注3-4个基因，我们预测这些基因与菱形球边界的细胞分选有关。我们将使用功能缺失方法（morpholino介导的敲除，锌指核酸酶介导的靶向缺失）以及错误表达方法（mRNA注射，GAL4:UAS转基因）来确定这些基因的功能。其次，我们将描述控制菱形体4/5形成的转录调控途径。许多已知的r4/r5基因编码转录因子，但尚不清楚它们调节哪些基因。我们已经生成了几种这些转录因子的抗血清，并将使用染色质免疫沉淀（ChIP）测定来确定作用于r4/r5的基因之间的直接调节关系。我们将采用候选方法，其中我们测试特定转录因子与预测目标启动子的结合，以及全局方法，其中我们设计一个后脑启动子拼接阵列，允许识别由给定转录因子结合的所有后脑启动子。我们的实验很重要，因为发育中的后脑对各种因素（如环境毒素、感染因子和遗传条件）的干扰很敏感，这些因素会导致一系列出生缺陷——运动控制问题，如共济失调，认知缺陷，如自闭症和颅面缺陷。因此，对后脑形成的更好理解将适用于广泛的生物过程和人类疾病状况。我们的实验还在斑马鱼中新颖地使用了几种技术- ChIP， GAL4:UAS转基因。

DESCRIPTION (provided by applicant): Segmentation of the embryonic vertebrate hindbrain into rhombomeres (r) ensures proper spatial positioning of hindbrain derivatives (e.g. reticulospinal interneurons and motornuclei of the cranial nerves) and is therefore essential for normal neurological activity. This segmentation process is regulated by a growing number of genes, but the function of many of these genes, as well as the regulatory relationships among them, remains unclear. We have identified 12 novel genes expressed in the hindbrain and we hypothesize that these genes act in a regulatory network controlling rhombomere formation. We have developed two aims to test our hypothesis: First we will determine the function of several novel hindbrain genes in rhombomere 4/5 formation. We are focusing particularly on 3-4 genes that we predict are involved in cell sorting at rhombomere boundaries. We will use loss of function approaches (morpholino-mediated knock-down, zinc-finger nuclease-mediated targeted deletions), as well as misexpression approaches (mRNA injections, GAL4:UAS transgenesis), to determine the function of these genes. Second, we will delineate transcription regulatory pathways controlling formation of rhombomere 4/5. Many known r4/r5 genes encode transcription factors, but it is not clear which genes they regulate. We have generated antisera to several of these transcription factors and will use chromatin immunoprecipitation (ChIP) assays to identify direct regulatory relationships among genes acting in r4/r5. We will take both a candidate approach, where we test binding of a specific transcription factor to a predicted target promoter, and a global approach, where we design a hindbrain-promoter tiling-array that will permit identification of all hindbrain promoters bound by a given transcription factor. Our experiments are important because the developing hindbrain is sensitive to disruptions by a variety of factors (e.g. environmental toxins, infectious agents and genetic conditions) that give rise to a range of birth defects - motor control problems such as ataxia, cognitive defects such as autism and craniofacial defects. A better understanding of hindbrain formation will therefore be applicable to a broad set of biological processes and human disease conditions. Our experiments also make novel use of several techniques - ChIP, GAL4:UAS transgenics - in zebrafish. PUBLIC HEALTH RELEVANCE: The proposed experiments are aimed at understanding hindbrain formation during embryogenesis. The embryonic hindbrain (and associated neural crest cells) gives rise to many essential structures - sensory ganglia and branchiomotor neurons of the nervous system, as well as bone, cartilage and muscle of the head. The developing hindbrain is sensitive to disruptions by a variety of factors (e.g. environmental toxins, infectious agents and genetic conditions) that give rise to a range of birth defects - motor control problems such as ataxia, cognitive defects such as autism and craniofacial defects. In addition, the genes studied in this proposal regulate other aspects of neural development (e.g. dorsoventral patterning of the neural tube), and other aspects of embryogenesis (e.g. hematopoiesis). The results from our proposed experiments will therefore be applicable to a broad set of biological processes and human disease conditions.