ANALYSES OF FOUNDER CELLS IN THE EPIBLAST AND SOMITES

乔治-温斯坦实验室已经证明， 从减数分裂前节板中胚层和上胚层能够 分化成骨骼肌和成软骨细胞时，分离， 胚胎周围组织，并在无激素和蛋白质的环境中培养 介质 MyoD和Pax-1的mRNA，调节 节段板中可检测到肌生成和硬结发育 通过RT-PCR，MyoD在上胚层的细胞中是可检测的。 博士 George-Weinstein将测试以下假设：（1）稳定编程的创始人 鸡的骨骼肌细胞和软骨细胞 在原肠胚形成开始之前的胚胎;和，（2）肌原性和 软骨形成的创始细胞在体节中招募它们未定型的邻居 形成肌节和硬结。 后一种情况可能发生在 在接收到来自周围结构的信号之后 还有脊索 信号可能是分泌音刺猬和 Wnt 1、3和4。 现有数据表明，差异化可能是 受组织相互作用、直接细胞-细胞相互作用以及 影响转录的核蛋白。 例如，当分段 将平板培养为单细胞悬浮液， 与作为完整组织培养时相比， 为了解决许多相互矛盾的数据，并回答有关问题， 未定型细胞的性质和时间，位置和出现 稳定定向的前体细胞，乔治-温斯坦博士提出了四个 具体目标。 在目标1中，将确定Pax-1，一个特定的因子， 对于成软骨细胞分化，在分离的上胚层中表达 来自前条纹阶段胚和来自原始条纹阶段胚。 这 将证实成软骨细胞的创始细胞位于 上胚层 在具体目标2中，表达MyoD和MyoD的细胞的数量和位置被确定。 Pax-1 mRNA将通过组织切片上的原位杂交测定 不同阶段的胚胎。 这些实验预计将在 由于探头的灵敏度增加（高达100倍）， 使用荧光标记的树枝状聚合物， Polyprobe，Inc.开发的信号网络分子。另一种方法 增加灵敏度的方法是原位PCR，然后用 适当的树枝修剪器。 对于具体目标3，外胚层和节段性细胞亚群的命运 将测定平板细胞。 该实验室此前已开发出5 单克隆抗体在未固定的细胞中产生不同的标记模式， 条纹前和原始条纹阶段的胚胎，每个结合到一个有限的 外胚层内的细胞数量。 抗体将用于 确定它们是否真的是肌细胞和软骨细胞的标记 以及与这些细胞结合的上胚层细胞 抗体负责在体节内建立组织。 的 金标记的抗体将被（阶段2）胚胎内吞， 然后在接下来的30小时内培养胚胎并取样。 一 第二个实验涉及抗体与胶体的共定位 金和荧光树枝状聚合物对MyoD和Pax-1的反应。 具体目标4类似于目标3进行，除了那些结合 抗体将通过补体介导的裂解而被消除。 治疗的整体 然后将胚胎培养长达30小时， 将对皮肌节和硬结形成进行形态学分析， 与肌节肌球蛋白抗体，并通过原位杂交与 MyoD和Pax-1的探针。

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.