The superfamily of TGF-ß ligands represents one of the most prominent families of morphogens. These factors have profound effects on many aspects of embryonic development, cell behaviour and homeostasis and malfunction of the pathways associated with these cytokines can lead to a variety of pathologies. Despite intensive research, there are still large gaps in our knowledge regarding the specificity of these ligands, the regulation of the activity of their receptors and the interactions between the TGF-ß pathways and other signalling pathways. In particular, how sources of TGF-ß morphogens are generated and how the resulting morphogen gradients can be translated into patterns of gene expression during early development remain central questions in current developmental biology. This proposal attempts to fill these gaps in our knowledge by characterising novel regulators of dorsal-ventral axis formation upstream and downstream of Nodal and by modelling the gene regulatory network (GRN) activated by this factor. We address this question within the sea urchin model, an organism phylogenetically close to vertebrates but with many advantages for the analysis of regulatory networks in early development. Our first aim is to characterize the early events that shape the Nodal gradient and initiate the downstream GRN that controls dorsal-ventral (D/V) axis formation. Our laboratory has recently discovered several key factors involved in D/V axis formation. We identified a maternal TGF-ß ligand, a transmembrane protein, an ETS domain transcriptional repressor and the JNK kinase as factors critically required to restrict the spatial expression of nodal. The similarity of the phenotypes caused by inactivation of either this maternal TGF-ß ligand, the transmembrane protein or this ETS factor strongly suggests that they act in the same pathway to specify the D/V axis. However, the relationships between these factors and the mechanisms by which they antagonize nodal expression are presently completely enigmatic. To clarify the relationships between these factors we will identify the binding partners of the TGF-ß ligand and perform biochemical analyses and epistasis experiments. In this first aim, we will also determine whether Nodal and/or BMP2/4 work as morphogens, i.e. as long-range, concentration-dependent signalling factors in the sea urchin embryo by using a combination of treatments with recombinant Nodal and BMP2/4 proteins and ectopic expression of mRNAs encoding these ligands or the activated forms of their receptors. The second aim of this project is to extend and model the gene regulatory network activated by Nodal. We recently identified and validated about fifteen novel genes regulated by Nodal encoding a variety of regulatory proteins including transcription factors, cytokines, and secreted proteins most of which have never been characterized. The expression patterns of these genes identify novel regulatory domains and boundaries along both the animal-vegetal and dorsal-ventral axes, revealing an unsuspected complexity in patterning of the ectoderm. We propose to dissect the regulatory mechanisms establishing these new domains, to characterize these novel Nodal target genes and to analyze their function and position in the GRN. Finally, to further test the role of individual components of this network and understand how it achieves both robustness to environmental perturbations such as regulative development, and plasticity to evolutionary scenarios, we will start to construct a logical model of this GRN. Our third and last aim is to start dissecting the mechanisms that allow responding cells to read different levels of Nodal or BMP2/4. We will start to investigate how thresholds of response are encoded in the genome. We will perform detailed bioinformatics analyses on an extended set of Nodal and BMP2/4 target genes to identify and dissect the architecture of the cis-regulatory modules of these selected target genes.