This is an application for continuation and broadening of a Program Project now in its 9* year. Here we propose to build in many directions on the large success we have had in solving and authenticating a gene regulatory network (GRN) for development. GRNs provide causal explanations for developmental processes in the terms of the genomic regulatory code, where all species-specific developmental processes are ultimately programmed. A developmental GRN serves as a conceptual, system-level logic map, of direct predictive power. Thus GRNs bridge between functional genomic DNA sequence of regulatory significance and the biology of embryogenesis and body plan formation. They do this by specifying the regulatory interactions which causally drive the progression of regulatory states in diverse cellular territories. During recent years, this Program has been responsible for the experimental solution of the most advanced developmental GRN yet available for any developing animal organism. This is the GRN underlying the specification of the endomesodermal territories of the sea urchin embryo. Recently proof of the principle that as a GRN approaches completion it indeed provides explanation of all the observed biological functions has been obtained in this work. We now intend to capitalize on the growing suite of successful technological approaches to GRN analysis that we have developed, to confront challenges that heretofore were inaccessible, or could not even have been defined. The current sea urchin embryo GRN concerns about half of the embryo, that ultimately forming the gut, the skeletogenic cell lineages and the non-skeletogenic mesoderm, from the earliest zygotic genomic activity (at the beginning of cleavage) to just before gastrulation. A start on the oral and aboral ectodermal GRNs has also been made. We intend to expand this GRN in qualitatively distinct ways which will represent radical advances if successful. In the DAVIDSON COMPONENT the GRN will be expanded to include all regulatory genes predicted by genomic analysis, and observed to be expressed specifically in the endomesodermal territories, as well as in the oral and aboral ectoderm through gastrulation, to the point where the mesodermal cell types have appeared, and endodermal and ectodermal subdivisions (e.g. mouth, stomach, hindgut) have been territorially specified. This will amount to a several fold expansion of the GRN, but also produce a qualitatively new departure. Thus, except for the apical neurogenic domain, it would represent for the first time ever a global, (almost) whole embryo GRN that includes the mechanism for specification of almost all embryonic territories: it would enable us to see the dimensions and the organization of the genomic regulatory program for most of a whole developing embryo, something no one could have imagined until very recently. In the McCLAY COMPONENT the GRN will be extended in a different direction: to the downstream differentiation and morphogenesis genes that do the actual work of building tissues and expressing their particular functions: this will enable us to see how the transcriptional spatial regulatory states established during specification are used to control the actual """"""""jobs"""""""" of development, something of which we have but glimpses at present. We believe the conceptual and technological advances of the GRN analysis supported by the current POI are now sufficiently mature so that the time is ripe for transfer of these ideas and methods to developing amniote systems: thus in the BRONNER-FRASER COMPONENT a system-level GRN is to be constructed for chick neural crest. This is not only in its own right a fascinating, vertebrate specific, developmental feature, but due to extensive prior work, would appear ripe for application of many of the approaches emerging from the sea urchin GRN project. This Program has been designed in a heavily interdependent way so that each Component will enjoy and indeed will require close scientific interactions with other Components. These interactions are detailed in the following. No less crucial will be the general reliance of all components on two Core Units. These are the SPECIALIZED RESEARCH SUPPORT (SRC) CORE and the SCIENTIFIC AND ADMINISTRATIVE COORDINATION (SAC) CORE. The functions of the SRC CORE will be to provide special high tech services that will be essential to all three P01 Components, i.e., procedural and instrumental functions that would require a vast outlay of equipment and a vast effort to develop know-how were these functions to be duplicated in each independent lab. Among these, as detailed below, are: (1) construction of cis-regulatory expression vectors by BAC recombineering;(2) accurate amplification of ng quantity RNA preparations;(3) large scale arraying services;(4) bioinformatics services;(5) year round supply of sea urchins;(6) use of a newly on-line instrument, the NanoString nCounter System for direct measurement of large numbers of specific transcripts simultaneously and very quantitatively on small amounts of material. The SAC CORE will constitute the central managerial oversight of the whole project;will interface with individual University grants managers;will coordinate personnel, fiscal, and publication policies;will ensure maximally economic supply acquisitions;and will arrange for webcast conferences among Components as well as biannual meetings of POI personnel.
Showing the most recent 10 out of 163 publications
