The question of how differential gene expression is established in the embryo is at the heart of developmental biology. Gene regulatory networks, which describe the interconnections between regulatory genes and signaling components at different stages of development, are powerful tool to understand how distinct territories of gene expression are established within the embryo. A few system-level approaches of early developmental processes have been successful in vertebrates, but these are made difficult by the large number of cells present in a vertebrate embryo, their high diversity, the complex morphogenetic movements that occur during vertebrate development as well as by the partial redundancy of their genomes. Here we propose to attempt a large scale analysis of the processes responsible for regionalization of the embryo along the dorso-ventral axis in a model that is much simpler than vertebrates: the sea urchin embryo. Sea urchins are non chordate deuterostomes and this position among the invertebrates makes them an interesting group to study the mechanism of axis specification and the evolution of developmental mechanisms. Unlike vertebrates, sea urchin embryos have a relatively small number of cells, are fully transparent and the morphogenetic movements that accompany gastrulation are easy to follow. A rich panoply of techniques is available for functional analysis including microinjection of morpholino oligonucleotides for gene loss of function, overexpression of mRNA and pharmacological treatments for gain of function, transgenesis by microinjection of plasmidic DNA, etc. Finally, the sea urchin embryo has a prolific history of experimental embryology and a wealth of biological knowledge is available on its development. The genome of Strongylocentrotus purpuratus has been sequenced, revealing that echinoderms have a rich genetic repertoire and a low level of genetic redundancy. For all these reasons, the sea urchin embryo has turned out to be a model of choice for the analysis of gene regulatory networks during early development. The project builds on previous work from our laboratory in identifying key elements of the gene regulatory network driving dorsal-ventral patterning such as Nodal and BMP2/4 and in identifying key transcription factors and signaling factors acting upstream and downstream of these signals. However, until recently, our studies focused on small scale analyses and on the roles of individual genes in cell fate specification. This project instead proposes to use a large scale approach to understand several key features of an important developmental process common to all bilateria : the establishment of dorso-ventral polarity. The first aim of this project is to assemble a gene expression atlas of all the regulatory factors involved in dorso-ventral axis formation then a map describing the regulatory interactions between the most important players. This will be achieved by using a combination of expression studies (in situ screen, micro array screens), perturbation analysis (loss and gain of function), and promoter analysis of the most important genes (identification of cis-regulatory modules by phylogenetic footprinting). The second aim of this project is to understand how Nodal and BMP2/4 work to organize the whole dorso-ventral axis of the embryo. While Nodal has long range organizing activity in rescue experiments, preliminary experiments indicate that Nodal does not work as a morphogen but suggest instead that it requires a relay to specify dorsal fates. The third aim of this project is to understand the molecular mechanisms and to identify the maternal factors that activate the dorso-ventral gene regulatory network. While redox gradients and spatially restricted activation of p38 MAP kinase signaling have been implicated in the early steps of dorso-ventral axis formation upstream of Nodal, the link between these events and the transcriptional machinery responsible for initiating nodal expression remain to be established. We propose to characterize the activating transcription factors that initiate nodal expression and to characterize the function and regulation of a transcriptional repressor that we identified and which appears to be a major determinant of the spatial expression of nodal and an excellent candidate to integrate signals from Redox./MAP kinase signaling. The long term goal is to draw a detailed picture of which genes are regulated by which transcription factors, how extracellular signals regulate the activity of these transcription factors and how these genes work together to form a transcriptional network that drives morphogenesis of the embryo. This work is important since not only it will improve our undestanding of a complex developmental process, but it will also illuminate the evolution of mechanisms regulating nodal expression, and more generally, the mechanisms responsible for dorsal-ventral patterning in deuterostomes.