RPG: Preliminary Functional Studies of Hro-twi Protein in the Leech Embryo

本研究的重点是研究水蛭胚胎中胚层形成和分化过程中相关基因的作用。形态学信息，以及从最近的核酸和遗传分析中获得的信息表明，无脊椎动物和脊椎动物物种的许多发育过程非常相似，表明这些过程可能是进化保守的。原肠胚形成的一个基本过程包括中胚层、内胚层和外胚层的形成。外胚层或外层产生上皮和神经系统。中胚层或中间层产生结缔组织、肌肉、血细胞、血管和一些器官。内胚层，或内层，产生消化道和一些器官的内层。确定是什么调节了这些层的形成，对于理解正常和异常的胚胎发育具有明确的意义。本研究的主要目的是在细胞和分子水平上了解水蛭胚胎这一简单生物的原肠胚形成过程。两种不同的方法将用于研究水蛭的原肠形成事件。第一个将涉及一种分子方法，其中PCR和DNA克隆将被用来识别与生长过程有关的基因。第二种方法是利用细胞谱系分析来鉴定参与原肠胚形成过程的细胞，因此受到DNA方法鉴定的基因的影响。在此计划拨款期间，研究将集中在水蛭胚胎中中胚层测定的候选物Hro-twi （Soto等人，出版中）。作为计划活动的一部分，将纯化针对Hro-twi蛋白的多克隆抗体并测定蛋白表达。此外，我们还将进行初步的功能分析，以确定反义/过表达方法研究水蛭胚胎中重要发育基因功能的可行性。

9727760 Soto The main focus of this research is to study the action of genes involved in the process of mesoderm formation and differentiation in the leech embryo. Morphological information, in addition to information obtained from more recent nucleic acid and genetic analysis, has shown that many developmental processes in invertebrate and vertebrate species are very similar, indicating that these processes may be evolutionarily conserved. An example of one of these fundamental processes gastrulation, involves the formation of mesodermal, endodermal and ectodermal embryonic layers. The ectoderm, or outer layer, gives rise to the epithelium and the nervous system. The mesoderm, or middle layer, gives rise to connective tissue, muscles, blood cells, blood vessels, and some organs. The endoderm, or inner layer, gives rise to the digestive tract and the interior lining of some organs. Determining what regulates the formation of these layers has clear implications for the understanding of both normal and abnormal embryonic development. The main objective of this research is to understand the process of gastrulation at both the cellular and molecular level in a simple organism, the leech embryo. Two different approaches will be used to study gastrulation events in the leech. The first will involve a molecular approach where PCR and DNA cloning will be used to identify genes involved in the process of grastrulation. The second approach is the utilization of cell lineage analysis to identify cells involved in the process of gastrulation and therefore affected by the genes identified by the DNA approach. During the period covered by this planning grant, studies will focus on a candidate of mesodermal determination in the leech embryo, Hro-twi (Soto et al., in press). As part of the planning activities, polyclonal antibodies against Hro-twi protein will be purified and protein expression determined. In addition, preliminary functional analysis will be performed, which will determine th e feasibility of antisense/overexpression approaches to study the function of developmentally-important genes in the leech embryo.