A critical feature of development is the early commitment of cells to very specific courses of gene action, functional differentiation, and morphogenesis. Both maternal and embryonic programs are involved in these events. The objective of the proposal is to study molecular mechanisms which govern the determination and differentiation of one such cell lineage: the primary mesenchyme cells which arise from the four micromeres of the 16-cell sea urchin embryo. Shortly before gastrulation, primary mesenchyme cells migrate into the blastocoel and initiate a precise program which culminates in secretion of the larval skeleton. Micromeres become committed at the 4th cleavage, and if isolated and cultured in vitro differentiate into mesenchyme cells and produce a skeleton. In this proposal we challenge the general view that primary mesenchyme differentiation is a fully autonomous process, and argue that it instead consists of both autonomous and cell interaction-dependent phases. We have isolated mesenchyme-specific cDNA clones as well as a set of monoclonal antibodies to the major cell lineages of the embryo. These we will use for studying the molecular events of mesenchyme differentiation and the roles of particular gene products. There are three major specific aims. (1) We will use clones of gene sequences with mesenchyme-limited expression to examine gene regulation and the functions of these genes in the lineage. (2) We will use mesenchyme-specific probes to investigate autonomous and non-autonomous processes in cultured mesenchyme cells. (3) There are inductive restrictions as well as positive determinative controls in sea urchin embryos. In the absence of micromeres the neighboring macromeres partially change fate and give rise to primary mesenchyme cells. This determinative event occurs in culture in isolated macromeres, and thus will provide an in vitro model system to study the molecular and cellular events of mesenchyme determination.
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