INBRE: KSU: MECHANISMS OF DORSOVENTRAL PATTERNING DURING NEURAL DEVELOPMENT

INBRE：KSU：神经发育过程中背腹模式的机制

• 批准号: 7610217
• 负责人: TONIA L VON OHLEN
• 金额: $ 5.27万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7610217
• 关键词: Address; Amino Acid Sequence; Anterior; Binding; Binding Sites; Biological Assay; Cell division; Complement; Complex; Computer Retrieval of Information on Scientific Projects Database; Conserved Sequence; Cues; Cultured Cells; Data; Development; Dorsal; Drosophila genus; Elements; Embryo; Funding; Gene Expression; Gene Targeting; Generations; Genes; Genetic Enhancer Element; Genetic Transcription; Goals; Grant; Homeobox; Homeodomain Proteins; Insecta; Institution; Interneurons; Length; Link; Motor Neurons; Muscle; Nervous system structure; Neuraxis; Neuroglia; Neurons; Organism; Pattern; Peptide Sequence Determination; Phenotype; Process; Proteins; Regulatory Element; Reporter Genes; Research; Research Personnel; Resources; Role; Signal Pathway; Site; Site-Directed Mutagenesis; Source; Specific qualifier value; Stereotyping; Testing; To specify; Transcript; Transcription Repressor/Corepressor; Transgenes; Transgenic Organisms; United States National Institutes of Health; Whole Organism; Work; fly; gain of function; homeodomain; in vivo; loss of function; mutant; nerve stem cell; neuroblast; neurodevelopment; novel; research study; transcription factor

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The Drosophila central nervous system (CNS) is composed of a complex array of motoneurons, interneurons and glia. Generation of the mature CNS involves a two step process. First, positional cues across the anterior-posterior and dorsoventral (DV) axes act to specify neural stem cells called neuroblasts. Second, neuroblasts undergo stereotypes cell divisions to generate the precise complement of neuronal and glial cells that populate the mature CNS. Our work focuses on how these positional cues specify the formation of neuroblasts across the DV axis, and how these neuroblasts in turn generate neuronal and glial diversity. Specifically we have shown that three signaling pathways, Dorsal, Egfr and Dpp are required to subdivide the neurectoderm in to three columns across the DV axis, these correspond to the domains of expression of three homeodomain proteins. From ventral to dorsal these are Ventral nervous system defective (Vnd), Intermediate neuroblasts defective (Ind) and Muscle segment homeobox (Msh). Each of these proteins is required for proper formation and/or specification of the corresponding neuroblasts from their respective column. The goal of my research is to establish the link between the dorsoventral restricted homeodomain proteins and the development of the complex array of neurons and glia that compose the Drosophila CNS. The mechanism by which these dorsoventral transcription factors induce different types of neurons and glia is currently unknown in any organism. In order to address this question we have chosen to focus on the role of the Ind homeodomain protein. My lab is currently focused on the characterizing the function of the Ind protein in regulating gene expression. To do this we have identified conserved domains within the protein and are characterizing the function of these domains by testing the function modified versions of the protein in vivo and in cell culture. An alignment on Ind protein sequences from three insect species revealed three domains of highly conserved sequence similarity, including the homeodomain. Preliminary data in cell culture and invivo suggest that the two domains outside the homeodomain are critical for the ability of Ind to act as a transcriptional repressor. We are currently developing an in vivo expression assay that will allow us to characterize the function of these domains in the whole organism. In addition, we have data suggesting that Ind can positively regulate its own expression. To this end, we have shown that in a transcript positive ind mutant ind expression is initiated normally but not maintained. Furthermore we have identified a novel ind regulatory element that contains putative Ind binding sites. We have shown that expression of a reporter gene from this enhancer element is dependent on ind activity, in ind mutant embryos we fail to detect expression. We are currently in the process of further characterization of this element and testing the ability of Ind to regulate transcription from it. To do this we are performing site directed mutagenesis of the putative ind binding sites in order to demonstrate that these sites are required for expression from this construct and that ind binding is necessary. Ultimately we intend to use microarrays to identify novel targets of Ind and Vnd in effort to determine how Ind regulates formation of the intermediate column in the CNS. To this end we have built TAPtagged Ind and Vnd expression constructs we are currently in the process of testing these constructs for their ability to duplicate Ind and Vnd gain of function phenotypes respectively. Preliminary experiments suggest that tagged Ind constructs do function in a manner similar to untagged full length Ind in their ability to repress the known Ind target gene Msh. f the previous projects we are moving forward on identification of Vnd targets with development of new TAPtagged Vnd transgenes. We are currently in the process of characterizing these transgenic fly lines now. We are also attempting to obtain a loss of function phenotype for the CG10479 gene.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 果蝇中枢神经系统（CNS）是由运动神经元、中间神经元和胶质细胞组成的复杂系统。 成熟CNS的产生涉及两步过程。首先，前后和背腹（DV）轴的位置线索作用于指定称为神经母细胞的神经干细胞。其次，神经母细胞经历定型细胞分裂以产生填充成熟CNS的神经元和神经胶质细胞的精确补充。我们的工作重点是如何这些位置的线索指定整个DV轴的成神经细胞的形成，以及这些成神经细胞如何反过来产生神经元和神经胶质的多样性。具体地说，我们已经表明，三个信号通路，Dorsal，Egfr和Dpp需要细分的神经外胚层在DV轴上的三列，这些对应于三个同源结构域蛋白的表达域。从腹侧到背侧依次为中枢神经系统缺陷型（Vnd）、中间神经母细胞缺陷型（Ind）和肌节同源异型盒（Msh）。 这些蛋白质中的每一种都是从它们各自的柱中正确形成和/或指定相应的成神经细胞所必需的。 我的研究目标是建立背腹侧限制性同源结构域蛋白与构成果蝇中枢神经系统的复杂神经元和神经胶质细胞阵列的发育之间的联系。这些背腹侧转录因子诱导不同类型的神经元和神经胶质的机制目前在任何生物体中都是未知的。为了解决这个问题，我们选择专注于Ind同源结构域蛋白的作用。 我的实验室目前专注于表征Ind蛋白在调节基因表达中的功能。为此，我们已经确定了蛋白质内的保守结构域，并通过在体内和细胞培养中测试蛋白质的功能修饰版本来表征这些结构域的功能。对三种昆虫的Ind蛋白序列进行比对，发现三个结构域具有高度保守的序列相似性，包括同源结构域。细胞培养和体内的初步数据表明，同源结构域外的两个结构域对于Ind作为转录抑制因子的能力至关重要。我们目前正在开发一种体内表达试验，这将使我们能够表征这些结构域在整个生物体中的功能。 此外，我们有数据表明Ind可以积极调节其自身的表达。为此，我们已经表明，在一个转录阳性ind突变ind表达启动正常，但不维持。此外，我们已经确定了一种新的ind调控元件，其中包含推定的Ind结合位点。我们已经表明，从这个增强子元件的报告基因的表达是依赖于ind的活性，在ind突变体胚胎中，我们无法检测到表达。我们目前正在进一步表征这个元素的过程中，并测试的能力，从它的Ind. To做到这一点，我们正在执行的推定ind结合位点的定点诱变，以证明这些网站是从这个构建表达所需的，ind的结合是必要的。 最终，我们打算使用微阵列来确定新的目标的Ind和Vnd的努力，以确定如何在中枢神经系统中的Ind调节中间柱的形成。为此，我们已经构建了TAP标记的Ind和Vnd表达构建体，我们目前正在测试这些构建体分别复制Ind和Vnd功能表型的能力。初步实验表明，标记的Ind构建体在抑制已知Ind靶基因Msh的能力方面确实以与未标记的全长Ind类似的方式起作用。 在以前的项目中，我们正在通过开发新的TAP标记的Vnd转基因来鉴定Vnd靶标。我们目前正在对这些转基因果蝇品系进行鉴定。我们还试图获得CG 10479基因的功能丧失表型。