This renewal application follows on our success in generating a large-scale gene regulatory network (GRN) for development of the endomesoderm of the sea urchin embryo. Progress on this GRN from the beginning depended on computational tools and approaches developed under the auspices of this grant, as well as on system-level experimental approaches. When we originally applied for this grant, the endomesoderm GRN map was in a preliminary state of coalescence; now it is a well developed, experimentally testable, defined logic map of the genomic source code for embryonic specification. As described in Progress Report, we built the mode of presentation and analysis used for the GRN in the course of this project; and we developed from scratch an extremely effective computational approach to interspecific sequence comparison for the purpose of identifying cis-regulatory elements. This latter method is in wide use, and in our lab has permitted rapid identification of many cis-regulatory elements, thereby permitting test, at the DNA level, of the inputs produced in the GRN. We now propose to carry out computational development of the GRN to an entirely new level. We will produce an interactive GRN model that will: (i) display causal interrelations at any level from territorial functionalities (zoom out) to specific inputs into given cis-regulatory elements (zoom in); (ii) provide access to the interaction map, via given genes, via given times in development, via given embryonic spatial domains; (iii) permit incorporation into the GRN of kinetic relationships, as measurements become available; (iv) represent cis-regulatory elements as combinations of logic functions; and (v)provide links to relevant genomic sequence and to experimental evidence on which functional GRN linkages have been proposed and authenticated at the cis-regulatory level. We also propose to model and test many kinetic predictions by means of time course measurements, using approaches similar to those of our recently developed gene cascade kinetic simulations, described also in Progress Report. In addition we intend to complete an input/output kinetic analysis of a specific cis-regulatory system, as a demonstration project for mathematical representation of cis-regulatory operations within the GRN. Finally, we propose to identify given GRN circuit elements, produce synthetic cis-regulatory constructs in which normal operation of these elements will be altered, and reintroduce them into the embryo to determine whether our understanding of the GRN is sufficient to permit reengineering of the embryonic process.
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