Alessandra Livigni 1 , Lucy Jones 1 , Salvatore Pernagallo 2 , Fella Hammachi 1 , Alexei Sharov 3 , Gillian Morrison 1 , Minoru Ko 3 , Mark Bradley 2 , Joshua Brickman 1 1 MRC Centre for Regenerative Medicine – Institute for Stem Cell Research, University of Edinburgh, Edinburgh, United Kingdom 2 Chemical Biology Section, School of Chemistry, University of Edinburgh, Edinburgh, United Kingdom 3 National Institute on Aging, NIH, Baltimore, MD, United States The POU-V transcription factor Oct4 is a master regulator of self-renewal and pluripotency in embryonic stem (ES) cells as well as an important regulator of lineage commitment in embryonic development. We have shown that Oct4’s ability to regulate self- renewal in ES cells is related to a conserved function in regulating embryonic differentiation in certain vertebrates. During Xenopus laevis development three Oct4 homologues (Xlpou25, Xlpou60 and Xlpou91) are expressed and are thought to maintain a pool of undifferentiated multi-potent cells. One of these proteins, Xlpou91, is also capable of functionally substituting for Oct4 in ES cells. Here we explore the regulatory network downstream of the amphibian Oct4 homologues by using loss of function mor- pholino combinations and microarray analysis. A number of studies have analysed the downstream targets of mammalian Oct4 using ES cells as a model. By using the amphibian embryos our study aims to identify the targets essential for correct germ layer specification in addition to maintenance of the undifferen- tiated state. Based on in silico analysis of microarray data we selected a subset of 307 differentially regulated genes responsive to the amphibian Oct4 homologues. Known mammalian homo- logues exist for 202 of these targets and 66 of them have been identified as Oct4 targets in mammalian ES cells. We have also found that over expression of murine Oct4 activator and repressor fusion proteins regulate these targets in a manner consistent with the view that the activator function of PouV proteins plays the major role in the suppression of lineage specification. doi:10.1016/j.mod.2009.06.775 18-P009 Regulation and evolution of an extracellular glycoprotein inter- action network Stephen Martin 1 , Varodom Charoensawan 2 , Christian Soellner 1 , Boris Adryan 2 , Bernard Thisse 3 , Christine Thisse 3 , Sarah Teichmann 2 , Gavin Wright 1 1 Wellcome Trust Sanger Institute, Cambridge, United Kingdom 2 MRC Laboratory of Molecular Biology, Cambridge, United Kingdom 3 Department of Cell Biology, University of Virginia, Charlottesville, VA, United States Extracellular glycoprotein interactions are essential to initiate signaling pathways that orchestrate cellular behaviors within bio- logical systems. Despite their importance, for example in the development of the nervous system, the properties of extracellu- lar glycoprotein interaction networks are unknown because exist- ing high throughput protein interaction assays are unable to detect them. Here, we use our recent AVEXIS assay to build a net- work of 188 extracellular interactions including interactions for 18 orphan receptors. Network analysis reveals differences com- pared to intracellular networks: there are many more homophilic interactions, and secreted ligands are, on average, twice as con- nected as membrane-tethered receptors. The interaction network was supported and resolved into dynamic tissue and embryonic stage-specific networks by integrating developmental gene expression profiles. These data are organized into a database, ARNIE, which facilitates stage and orientation-matched compari- sons of detailed expression patterns for genes encoding interact- ing glycoproteins. Using these datasets, we show that creation of new signaling pathways is driven first by changes in receptor spa- tiotemporal expression and then by divergence in receptor cyto- plasmic signaling sequences, while extracellular sequences and interactions are more conserved. doi:10.1016/j.mod.2009.06.776 18-P010 Delay-driven oscillations in the Notch signalling network Nick Monk 1 , Hiroshi Momiji 2 1 University of Nottingham, Nottingham, United Kingdom 2 University of Sheffield, Sheffield, United Kingdom Intercellular signalling mediated by the Notch signalling net- work plays a central role in a wide range of developmental events. Activity of the Notch network depends on a set of interlocked positive and negative feedback loops, operating both within and between cells. Time delays resulting from processes such as tran- scription and translation can have a significant impact on net- work dynamics. Using delay models, I will illustrate the rich dynamics of the Notch network and discuss how these relate to oscillatory dynamics observed in vertebrate segmentation and stem cell differentiation. doi:10.1016/j.mod.2009.06.777 18-P011 High complexity, random-primed domain libraries for yeast two- hybrid analysis of human and model organism interactomes Vincent Collura , Petra Tafelmeyer, Maryline Masson, Emilie Vinolo, He ´le `ne Kiefer, Jean-Christophe Rain, Laurent Daviet, Etienne Formstecher Hybrigenics, Paris, France Yeast two-hybrid (Y2H) protein interaction mapping has pro- ven instrumental in the elucidation of protein function in model organisms and Homo sapiens, mostly thanks to pairwise testing or screening of oligo dT-primed cDNA libraries. However, interac- tion map completeness has been limited by the use of full-length proteins and C-terminal polypeptide fragments which result in significant false negative rates. To circumvent these limitations, we have used a domain-based strategy to construct dozens of highly complex, random-primed cDNA libraries relevant to developmental biology, prepared from human embryonic tissues or model organisms such as mouse, Dro- sophila, C. elegans, D. discoidum, zebra fish and medaka. The com- plexity of each library is greater than 10 million independent fragments in yeast, with an average fragment size of 800 bp. S287 MECHANISMS OF DEVELOPMENT 126 (2009) S285 – S290