CELL LINEAGE IN DEVELOPMENT

这项建议的总体目标是分析机制 哺乳动物细胞谱系决定和细胞承诺的调控 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 特定的胚胎血统。基因组印记，这发生在 哺乳动物中的配子发生，将不同的功能状态强加给 母系和父系衍生的基因组。因此，完成 正常的胚胎发育需要母体来源的和 父系起源的核。原核合子转移表明 滋养外胚层血统在雌核发育胚胎中不能发育，缺乏 雄性基因组，而内部细胞团衍生的谱系无法发育 在雄核发育胚胎中，只包含男性基因组。分析 雌核发育正常和雄核发育正常嵌合体已经证明 血统失败的细胞自主性。此外，还分析了 孤雌生殖正常嵌合体显然需要 形成骨骼肌的男性基因组。比细胞表面 在胚胎发育过程中出现在不同地点和时间的分子， 作为独特的细胞谱系标记，很可能也介导了细胞 形成血统所必需的相互作用。 为了研究印记在血统形成中的作用，我们提出了 从雄生殖、孤雌生殖和胚胎干细胞中分离胚胎干细胞 雌核发育囊胚，测试这些细胞对 嵌合体，并将其与雄性激素的发育能力进行比较 和雌核发育胚胎。通过这种方式，我们将解决稳定性和 在培养的干细胞系中的印迹程度并确定 印记中发生的变化改变了世系分配的能力 雌核发育胚胎和正常胚胎之间的嵌合体将在 利用原位杂交进行发育阶段的研究 确定肌肉谱系发育的细胞来自哪个阶段 孤雌生殖胚胎未能参与其中。肌成肌细胞培养 嵌合胚胎将被启动，克隆将来自于 分离出孤雌生殖和正常胚胎起源。基因比对 在正常v型孤雌生殖成肌细胞中的表达将定位这些基因 它们以不同方式表达(即印记)。为了接近 细胞表面分子在早期谱系分配中的作用 分析早期胚胎的合成调控和功能 存在于胚胎外胚层和内胚层的抗原。我们打算克隆 参与STAG合成的特异性岩藻糖基转移酶基因 特异性胚胎抗原1(SSEA-1)。这将通过表达来实现 使用哺乳动物表达载体和单抗进行克隆。这个 克隆的基因将被鉴定并确定其在发育中的功能 利用功能的获得和丢失诱导突变。

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.