ANALYSES OF FOUNDER CELLS IN THE EPIBLAST AND SOMITES

外胚层和体节中始祖细胞的分析

• 批准号: 2665706
• 负责人: MINDY GEORGE-WEINSTEIN
• 金额: $ 10.11万
• 依托单位: PHILADELPHIA COLLEGE OF OSTEOPATHIC MED
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2665706
• 关键词: cartilage; cell differentiation; cell population study; chick embryo; early embryonic stage; embryo /fetus tissue /cell culture; gene expression; in situ hybridization; messenger RNA; polymerase chain reaction; striated muscles; transcription factor; vertebrate embryology

DESCRIPTION: The George-Weinstein laboratory has demonstrated that cells from the presomitic segmental plate mesoderm and epiblast are able to differentiate into skeletal muscle and chondroblasts when isolated from surrounding tissues of the embryo and cultured in hormone and protein free medium. mRNA for MyoD and Pax-1, transcription factors that regulate myogenesis and sclerotome development, are detectable in the segmental plate by RT-PCR, and MyoD is detectable in the cells of the epiblast. Dr. George-Weinstein will test the hypothesis that (1) stably programmed founder cells for skeletal muscle and chondrogenic lineages are present in the chick embryo prior to the initiation of gastrulation; and, (2) that myogenic and chondrogenic founder cells recruit their uncommitted neighbors in the somite to form the myotome and the sclerotome. This latter event happens perhaps after receiving signals from surrounding structures such as the neural tube and the notochord. The signals are probably secreted sonic hedgehog and Wnts 1, 3, and 4. Existing data indicates that differentiation may be inhibited by tissue interactions, direct cell-cell interactions, and by nuclear proteins that affect transcription. For instance, when segmental plates are cultured as single cell suspensions, differentiation occurs readily, compared to when they are cultured as intact tissues. In order to resolve much conflicting data and to answer questions concerning the nature of uncommitted cells and the timing, location, and emergence of stably committed precursor cells, Dr. George-Weinstein proposes four specific aims. In aim 1, it will be determined if Pax-1, a factor specific for chondroblast differentiation, is expressed in the epiblasts isolated from prestreak stage embryos and from primitive streak stage embryos. This will substantiate that founder cells for chondroblasts are localized in the epiblast. In specific aim 2, the numbers and locations of cells expressing MyoD and Pax-1 mRNAs will be determined by in situ hybridization on tissue sectioned from various stage embryos. These experiments are expected to be informative because of the increased sensitivity (up to 100x) of probes using fluorescently labeled dendrimers, a high complexity nucleic acid signal network molecule developed by Polyprobe, Inc. An alternative approach of increased sensitivity will be in situ PCR followed by probing with the appropriate dentrimer. For specific aim 3, the fate of subpopulations of epiblast and segmental plate cells will be determined. The lab has previously developed 5 monoclonal antibodies that give distinct labeling patterns in unfixed prestreak and primitive streak stage embryos, each binding to a limited number of cells within the epiblast. The antibodies will be used to determine if they are, in fact, markers for myogenic and chondrogenic cells within the epiblast, and whether the epiblast cells that bind these antibodies are responsible for establishing tissues within the somite. The gold-labeled antibodies will be endocytosed by the (stage 2) embryos and then the embryo will be cultured and sampled over the next 30 hours. A second experiment involves the co-localization of antibodies with colloidal gold and fluorescent dendrimers to MyoD and Pax-1. Specific aim 4 proceeds similarly to aim 3, except those cells which bind antibody will be ablated by complement mediated lysis. The treated whole embryo will then be cultured up to 30 hours and the fate of tissue formation will be analyzed morphologically for dermomyotome and sclerotome formation, with antibodies to sarcomeric myosin, and by in situ hybridization with probes for MyoD and Pax-1.

描述：乔治 - 温斯坦实验室已经证明了细胞 从前节段板中的中胚层和epipiblast能够 与 周围的胚胎组织，并在无激素和无蛋白质中培养 中等的。 MYOD和PAX-1的mRNA，调节的转录因子 在节板中可检测到肌发生和硬化核心的发育 由RT-PCR和MYOD在层细胞的细胞中可检测到。 博士 乔治·韦恩斯坦（George-Weinstein）将检验（1）稳定编程的创始人的假设 雏鸡中存在用于骨骼肌和软骨谱系的细胞 胃开始之前的胚胎； （2）肌原和 软骨的创始人细胞招募了他们在各个体内的邻居 形成肌瘤和硬化症。 后一个事件也许发生了 从周围结构（例如神经管）收到信号后 和努力。 这些信号可能是分泌的声音刺猬， WNTS 1、3和4。现有数据表明差异化可能是 被组织相互作用，直接细胞 - 细胞相互作用和 影响转录的核蛋白。 例如，当分段 将板培养为单细胞悬浮液，发生分化 与将其作为完整组织培养的何时相比，很容易。 为了解决大量矛盾的数据并回答有关的问题 单元格的性质以及时间，位置和出现 稳定的前体细胞，乔治·温斯坦博士提出了四个 具体目标。 在AIM 1中，将确定PAX-1是否是特定因素的PAX-1 对于软骨细胞的分化，在分离的层细胞中表达 来自Prestreak阶段胚胎和原始条纹阶段胚胎。 这 将证实是软骨细胞的创始人细胞位于 epiblast。 在特定的目标2中，表达myod和的单元格的数量和位置 PAX-1 mRNA将通过在组织截面上的原位杂交确定 来自各个阶段的胚胎。 这些实验预计将是 信息丰富，因为探针的敏感性（最高100倍） 使用荧光标记的树突聚合物，高复杂性核酸 Polyprobe，Inc。开发的信号网络分子。一种替代方法 敏感性提高将是原位PCR，然后探测 适当的牙二聚体。 对于特定的目标3，层细胞和分段的亚群的命运 将确定板单元。 实验室以前已经开发了5 单克隆抗体在未固定的情况下提供不同的标记模式 普雷斯特龙和原始条纹阶段胚胎，每个胚胎都与有限 层细胞中的细胞数量。 抗体将用于 确定它们是否是肌原性和软骨基细胞的标记 在整个细胞中，以及是否结合这些细胞 抗体负责在体内建立组织。 这 金标记的抗体将被（第2期）胚胎和 然后，将在接下来的30小时内对胚胎进行培养和采样。 一个 第二个实验涉及与胶体的抗体共定位 金和荧光树状聚合物的MYOD和PAX-1。 特定的目标4与AIM 3相似，除了那些结合的单元 补体介导的裂解将消化抗体。 被治疗的整体 然后将胚胎培养长达30小时，并形成组织的命运 将在形态学上分析皮肤病组和硬化菌组的形成， 抗肉类肌球蛋白的抗体，并与原位杂交 探测Myod和Pax-1。