This proposal examines fundamental mechanisms that underlie the patterning of early animal embryos, using the sea urchin as a model system. Specifically, the proposal examines a key transcriptional gene regulatory network (GRN) in early sea urchin development. This GRN drives the specification and differentiation of the large micromere-primary mesenchyme cell (PMC) lineage. The large micromeres and their descendants transmit critical inductive signals early in development and later execute a dramatic sequence of morphogenetic behaviors, including epithelial-mesenchymal transition, direction cell migration, cell fusion, and secretion of a biomineralized skeleton.
The proposal also addresses the long-standing problem of embryonic regulation. How can early differential gene expression in embryos, entrained in the unfertilized egg, be reconciled with labile blastomere fates during early development? Sea urchin embryos are famous for their regulative properties and some of the most spectacular examples involve ectopic specification of PMCs. Although the PMC GRN is normally activated only in the large micromeres, any blastomere of the early cleavage stage embryo can give rise to PMCs under appropriate experimental conditions. Recent progress in identifying gene networks and signaling pathways that control early sea urchin development provides a new opportunity to address the historic problem of embryonic regulation in a modern context.
Specific Aim 1 examines the PMC GRN as it is deployed during normal development. 1) Dr. Ettensohn will identify mechanisms that normally restrict activation of the GRN specifically to the large micromere lineage, focussing on the roles of beta-catenin levels and unequal cell division. Using molecular biological and pharmacological methods, we will manipulate levels of nuclear beta-catenin and patterns of cell division in early blastomeres and determine whether this causes ectopic activation of the PMC GRN. 2) He will use fluorescent, multiplex in situ hybridization and intron probes to determine the temporal order of activation of genes in the network. This information will be critical in elucidating potential regulatory interactions between genes. 3) He will expand the PMC GRN by identifying new components, including downstream "morphoregulatory" genes. This will be accomplished by scaling-up a successful pilot PMC EST project which identified several critical genes in the GRN. The overarching goal is to develop a complete picture of this GRN that links the earliest molecular patterning events in the embryo to specific morphogenetic cell behaviors during gastrulation.
Specific Aim 2 examines the PMC GRN as it is deployed during regulative development. Using molecular probes and morpholinos, he will analyze the 1) expression, 2) function, and 3) regulatory interactions of the known upstream components of the PMC GRN under several different experimental scenarios that induce ectopic activation of the GRN.
The broader impact of the proposed research is derived from the training of undergraduate students, graduate students, and postdoctoral fellows. In addition, the work will contribute to the development and analysis of a large-scale PMC "gene catalogue" that will represent the most complete picture of the program of gene expression in a specific embryonic cell type in any developing embryo. This data set will be a valuable community resource.
