EXPRESSION OF ENGRAILED IN IDENTIFIED NEURONS OF CNS

已识别的中枢神经系统神经元中 ENGRAILED 的表达

基本信息

批准号：
2271035
负责人：
MELODY V SIEGLER
金额：
$ 6.72万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1994
资助国家：
美国
起止时间：
1994-09-28 至 1997-08-31
项目状态：
已结题

项目摘要

The present work is part of a general effort to determine the role of homeobox genes in the development and maintenance of neuronal specificity in the central nervous system. Health related aspects of the work are readily recognized in the growing number of studies that show specific defects in brain and spinal cord, and in a number of non-neuronal tissues, when homeobox genes have been disrupted during development. Although the CNS defects resulting from homeobox disruption are typically described in rather broadly, the defects must ultimately reside in alterations in the phenotype of individual neurons, and further, result from the altered expression of particular structural genes. Homeobox genes are commonly regarded as developmental genes. Nonetheless, some continue expression into adulthood, and it is therefore speculated that they have a role in maintaining the integrity of neuronal structure, connectivity, or function. The first specific aim is to characterize the pattern of Engrailed expression in the adult CNS of Schistocerca. Engrailed is the protein product of the engrailed gene and will be detected using a monoclonal antibody (MAb 4D9) that recognizes the protein's homeodomain. Immunolabeled ganglia of the CNS will be examined to assess the stereotypy of the labeling, its extent and its location relative to landmarks. The second specific aim is to test the hypothesis that Engrailed expression is type-specific, with expression occurring interneurons but not efferents of lineally related neuronal groups. We will examine segmentally homologous midline groups of neurons which each comprise the progeny of an identified embryonic stem cell. A direct test of the hypothesis will be made by double labeling experiments: MAb 4D9 will be used together with retrograde labeling or intracellular injection of dye into identified neurons in the midline groups. We will assess the overlap between labels in different neuronal types. Corroborating evidence for type-speCificity will be obtained by a quantitative analysis of Engrailed-positive and -negative neurons within the lineage groups. The third specific aim is to characterize the Engrailed expression during embryonic development. We hypothesize that Engrailed expression evolves in a definable spatial and temporal pattern during embryogenesis to yield the mature pattern. Two levels of inquiry are proposed, one at population level: We will construct a population profile by counting immunolabeled and unlabeled cells in the midline groups at 5% increments from 30% to 100% of embryonic development. Our second level of inquiry will characterize expression with cellular resolution. We will trace expression within the stem cell and in a distinct anatomical figure that contains the most recently born progeny. These data together with estimates of cell cycle time will allow a characterization of the onset and offset of expression relative to time of birth.

目前的工作是确定作用的一般努力的一部分同源基因在开发和维持神经元特异性方面在中枢神经系统中。工作与健康相关的方面是在越来越多的研究表明特定的研究中很容易认识到大脑和脊髓的缺陷以及许多非神经组织中的缺陷，当同源基因在开发过程中被破坏时。虽然通常在相当广泛地，这些缺陷最终必须存在于单个神经元的表型，以及进一步的作用。特定结构基因的表达。同源基因通常是被视为发育基因。尽管如此，有些仍在继续表达成年后，据推测他们在保持神经元结构，连通性或功能。第一个具体目的是表征在Schistocerca的成年中枢神经系统中插入表达。插入了该基因的蛋白质产物，将使用识别蛋白质同源域的单克隆抗体（MAB 4D9）。将检查中枢神经系统的免疫标记的神经节，以评估刻板印象标签，其范围及其位置相对于地标。这第二个具体目的是检验插入表达的假设是类型特异性，表达出现的中间神经元，而不是线性相关的神经元组。我们将检查分段同源每个构成鉴定的后代的神经元的中线组胚胎干细胞。该假设的直接检验将由双重标记实验：MAB 4D9将与逆行一起使用将染料的标记或细胞内注射到确定的神经元中中线组。我们将评估不同标签之间的重叠神经元类型。佐证的类型特异性证据将是通过定量分析界面阳性和负分析获得谱系组中的神经元。第三个具体目的是表征胚胎发育过程中界面的表达。我们假设界面表达在可定义的空间和胚胎发生过程中的时间模式以产生成熟的模式。二提出了调查水平，一个人口层面：我们将构建通过计算免疫标记和未标记细胞的人口概况中线组以胚胎发育的30％增加到5％。我们的第二级查询将表征细胞的表达解决。我们将在干细胞和A中追踪表达独特的解剖图包含最近出生的后代。这些数据以及细胞周期时间的估计将允许相对于时间的表达的发作和表达的表征出生。