IMMUNOLOGIC STUDIES OF C ELEGANS--EARLY DEVELOPMENT

线虫的免疫学研究——早期发育

• 批准号: 3485074
• 负责人: WILLIAM B WOOD
• 金额: $ 25.41万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-12-01 至 1990-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3485074
• 关键词: Caenorhabditis elegans; antibody formation; antibody specificity; cell differentiation; developmental genetics; early embryonic stage; embryogenic cleavage; flow cytometry; fluorescence microscopy; gel electrophoresis; gene expression; genetic manipulation; growth /development; histogenesis; hybridomas; immunochemistry; immunofluorescence technique; immunoprecipitation; laboratory mouse; laboratory rabbit; laboratory rat; membrane proteins; messenger RNA; microinjections; monoclonal antibody; morphology; mutant; nonmammalian vertebrate embryology; nucleic acid hybridization; oogenesis; protein structure; radiotracer; ribonucleoproteins; surface antigens

The goal is to understand the mechanisms by which cells become committed to specific developmental fates in early embryogenesis. Using embryos of the nematode Caenorhabditis elegans, and taking a combined genetic, cellular, and molecular approach, we shall continue to investigate the nature of early cytoplasmic influences on cell fate. We plan to test the validity of two possible models for the mechanism of determination. Model A is the classical view that several distinct, lineage-specific, maternally derived cytoplasmic deteminants in the form of informational macromolecules are segregated during cleavage to the various embryonic founder cells, whose fates are thereby determined prior to the onset of embryonic gene expression. Model B is an alternative view that gradients of only one or two less specific, maternally derived determinants dictate these fates by regulating early differential expression of selector genes in the different founder cells. In evaluating Model A, we shall biochemically and functionally characterize the germ-line-specific P granules, which segregate in the manner of cytoplasmic determinants, to establish whether or not they are, in fact, determinative for the germ line. Biochemical characterization will include analysis of their components by immunoprecipitation and islolation of cloned genes coding for the major P-granule antigens. Experiments to ascertain their function(s) will include microinjection of anti-P-granule antibodies into early embryos and continued screening for P-granule-defective mutants. To evaluate Model B we shall characterize the earliest embryonic gene expression after fertilization, by radioactive labelling of RNA in early embryos as well as generation of monoclonal antibodies against the first newly synthesized embryonic antigens. As byproducts of these experiments we hope to expand our library of monoclonal antibodies to lineage-specific antigens and to identify additional components of the cellular machinery responsible for generating asymmetry and for accomplishing asymmetric segregation in the early embryo.

目的是了解细胞致力于的机制 早期胚胎发生中的特定发育命运。 使用胚胎 线虫秀丽隐杆线虫，并采用遗传，细胞的组合 和分子方法，我们将继续研究 早期细胞质对细胞命运的影响。 我们计划测试 确定机制的两个可能的模型。 A模型是 经典观点，这些观点是几种不同的，谱系特异性的母体衍生的观点 信息大分子的形式的细胞质替代因子是 在裂解过程中分离到各种胚胎创始人细胞，他们的细胞 因此，在胚胎基因发作之前确定命运 表达。 B是一种替代观点，只有一个或 两个不太具体的母体得出的决定因素决定了这些命运 调节不同选择基因的早期差异表达 创始人细胞。 在评估A模型A时，我们应在生化和功能上表征 种系特异性的P颗粒，该颗粒以隔离的方式分离 细胞质决定因素，以确定它们是否实际上是 种系的决定性。 生化特征将包括 通过免疫沉淀和异位分析其成分 编码主要P颗粒抗原的克隆基因。 实验 确定其功能将包括抗P颗粒的显微注射 抗体进入早期胚胎并继续筛选 P颗粒缺陷突变体。 为了评估模型B，我们将表征 受精后最早的胚胎基因表达，通过放射性 早期胚胎和单克隆的产生RNA的标记 针对第一个新合成的胚胎抗原的抗体。 作为 这些实验的副产品我们希望扩大单克隆图书馆 对谱系特异性抗原的抗体，并确定其他 负责产生不对称性的细胞机制的组件 并用于在早期胚胎中完成不对称的隔离。