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

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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.