Localization and Identification of Cytoplasmic Determinants

早期胚胎发育被认为是由存在于卵细胞浆中的决定因素介导的，但这些物质尚未被表征。在本研究中，我们继续对海鞘内胚层碱性磷酸酶（AP）、肌肉和胚胎轴发育的细胞质决定因素进行研究。AP决定因素由母体AP mrna组成的可能性，这些mrna定位于卵细胞中，在分离到内胚层细胞后被翻译激活，将通过克隆AP探针进行原位杂交来检验。肌肉和轴向决定因素的空间分布将通过卵子的紫外线微束照射和早期胚胎细胞的显微手术删除来检查。肌肉和轴向决定因子所属的一般分子类别将使用紫外作用光谱分析和生物测定相结合的方法进行研究，该方法涉及将正常卵子的分离细胞质成分微量注射到因局部紫外线照射而发育轴向或肌肉的能力受损的卵子中。编码25 kd蛋白的母体mRNA在轴确定中的潜在作用，这一成分在轴缺陷胚胎中缺失，也将通过紫外线照射和体外翻译进行研究。预计将获得关于涉及三种不同发展决定的决定因素的定位和特性的新信息。调控早期胚胎发生的最重要的机制之一是发育信息，即“决定因素”或“形态形成因子”在胚胎早期发育过程中向不同区域的分离。据信，这些形态原决定了特定细胞系的命运，并引导它们分化成最终的组织，如肌肉、神经和肠道。这些决定因素的分子性质尚未确定。Jeffery博士在这一领域做出了重要贡献，现在他即将确定肌肉和肠道发育以及背腹轴本身建立所必需的分子。

Early embryonic development is thought to be mediated by determinants present in the egg cytoplasm, but these substances have yet to be characterized. In this proposal we continue our studies on cytoplasmic determinants involved in endodermal alkaline phosphatase (AP), muscle and embryonic axis development in the ascidian Styela. The possibility that AP determinants consist of maternal AP mRNAs, which are localized in the egg and translationally activated after segregation to the endodermal cells will be examined by in situ hybridization with a cloned AP probe. The spatial distribution of muscle and axial determinants will be examined by UV microbeam irradiation of the egg and microsurgical deletion of cells from the early embryo. The general class of molecules to which the muscle and axial determinants belong will be investigated using a combination of UV action spectrum analysis and a bioassay involving microinjection of fractionated cytoplasmic components of normal eggs into eggs whose ability to develop axis or muscle has been impaired by localized UV irradiation. The potential role in axis determination of a maternal mRNA encoding a 25 kd protein, a component that is absent in axis deficient embryos, will also be investigated by UV irradiation and in vitro translation. It is anticipated that new information will be obtained concerning the localization and identity of determinants involved in three different developmental decisions. %%% One of the most important mechanisms regulating the control of early embryogenesis is the segregation of developmental information, "determinants" or "morphogens," to various regions of the embryo during early development. It is believed that these morphogens determine the fate of specific cell lineages and guide them to differentiate into their final tissue such as muscle, nerve and gut. The molecular nature of these determinants has not yet been determined. Dr. Jeffery has made important contributions to this area and is now on the verge of identifying those molecules necessary for muscle and gut development as well as for the establishment of the dorsal- ventral axis itself.