CELL LINEAGE IN DEVELOPMENT

发育中的细胞谱系

• 批准号: 3323374
• 负责人: DAVOR SOLTER
• 金额: $ 20.7万
• 依托单位: WISTAR INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-09-01 至 1993-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3323374
• 关键词: cell cell interaction; cell differentiation; cytogenetics; early embryonic stage; embryo /fetus cell /tissue; gel electrophoresis; gene expression; genetic manipulation; genetic markers; genetically modified animals; genome; growth factor; histogenesis; hybrid cells; in situ hybridization; laboratory mouse; mammalian embryology; molecular cloning; morphology; myoblasts; nucleic acid probes; nucleic acid sequence; parthenogenesis; tissue mosaicism; transfection

The overall objective of this proposal is to analyze the mechanisms regulating cell lineage determination and cell commitment during mammalian development. Specifically, we propose to explore the role of imprinted genes and the role of cell surface molecules in initiation and maintenance of specific embryonic lineages. Genomic imprinting, which occurs during gametogenesis in mammals, imposes different functional states upon maternally and paternally derived genomes. Accordingly, completion of normal embryogenesis requires the presence of both a maternally derived and paternally derived nucleus. Zygotic transfer of pronuclei shows that the trophectodermal lineages fail to develop in gynogenetic embryos, which lack the male genome, while the inner cell mass derived lineages fail to develop in androgenetic embryos, which only contain the male genome. Analysis of gynogenetic <-> normal and androgenetic <-> normal chimeras has demonstrate the cell autonomous nature of lineage failure. In addition, analysis of parthenogenetic <-> normal chimeras has demonstrated an apparent need for the male genome in formation of skeletal muscle. Specific cell surface molecules, which appear in a distinct place and time during embryogenesis, serve as unique cell lineage markers and, very likely, also mediate cellula interactions necessary for lineage formation. In order to examine the role of imprinting in lineage formation we propose to isolate embryonic stem (ES) cells from androgenetic, parthenogenetic, an gynogenetic blastocysts, test the ability of these cells to contribute to chimeras, and compare this with the developmental capacity of androgenetic and gynogenetic embryos. In this way we will address the stability and extent of imprinting in cultured stem cell lines and determine whether changes occur in imprinting which alter the capacity for lineage allocation Chimeras between gynogenetic and normal embryos will be analyzed at progressive developmental stages using in situ hybridization in order to determine at which stage of muscle lineage development cells from parthenogenetic embryos fail to participate. Myoblast cultures from chimeric embryos will be initiated and clones derived from cells of parthenogenetic and normal embryonic origin isolated. Comparison of gene expression in normal v. parthenogenetic myoblasts will pinpoint the genes which are differentially expressed (i.e. imprinted). In order to approach the role of cell surface molecules in early lineage allocation we will analyze the control of synthesis and the function of early embryonic antigens present in embryonic ectoderm and endoderm. We propose to clone the gene for the specific fucosyl transferase involved in synthesis of stag specific embryonic antigen 1 (SSEA-1). This will be achieved by expression cloning using mammalian expression vectors and a monoclonal antibody. The cloned gene will be characterized and its function in development determine using gain and loss of function induced mutations.

本提案的总体目标是分析 在哺乳动物发育过程中调节细胞谱系确定和细胞定型 发展 具体地说，我们建议探索印记的作用， 基因和细胞表面分子在启动和维持中的作用 特定的胚胎谱系。 基因组印记，发生在 哺乳动物的配子发生，将不同的功能状态强加于 母系和父系的基因组。 因此，完成 正常的胚胎发生需要母源性和 父源核 原核的合子转移表明， 滋养外胚层谱系未能在雌核发育胚胎中发育， 男性基因组，而内细胞团衍生谱系未能发展 只含有雄性基因组的雄性胚胎中。 分析 雌核发育<->正常和雄核发育<->正常嵌合体已经证明 细胞谱系失败的自主性。 此外，分析 单性生殖<->的正常嵌合体已经证明， 男性基因组在骨骼肌的形成。 特异性细胞表面 分子，在胚胎发生过程中出现在不同的地方和时间， 作为独特的细胞谱系标记，很可能也介导细胞分化， 形成谱系所必需的相互作用。 为了研究印记在谱系形成中的作用，我们提出 从雄核发育的、孤雌发育的和 雌核发育囊胚，测试这些细胞的能力，以促进 嵌合体，并将其与雄激素发生的发育能力进行比较， 和雌核发育胚胎。 这样，我们将解决稳定和 在培养的干细胞系中印迹的程度，并确定是否 改变发生在印记，改变能力的血统分配 雌核发育和正常胚胎之间的嵌合体将在 进行性发育阶段的原位杂交， 确定肌肉谱系发育细胞的哪个阶段 孤雌胚胎不能参与。 成肌细胞培养物来自 将启动嵌合胚胎，并从 孤雌生殖和正常胚胎来源分离。 基因比较 在正常和孤雌生殖成肌细胞中的表达将精确定位基因 它们是差异表达的（即印记的）。 为了接近 细胞表面分子在早期谱系分配中的作用， 分析早期胚胎发育的合成控制和功能 存在于胚胎外胚层和内胚层的抗原。 我们建议克隆 该基因的特定岩藻糖基转移酶参与合成stag 特异性胚胎抗原1（SSEA-1）。 这将通过表达 使用哺乳动物表达载体和单克隆抗体进行克隆。 的 克隆的基因将被表征，其在发育中的功能将被确定。 使用功能获得和丧失诱导的突变。