Abstracts Cell–cell signaling Program/Abstract # 183 The effect of Notch signaling on neurotransmitter phenotype specification in Xenopus Michael S. Harper, Stephanie Byers, Margaret S. Saha Department of Biology, College of William and Mary, Williamsburg, VA, USA The development of a functioning nervous system depends on individual neurons acquiring the correct neurotransmitter pheno- type. In the developing spinal cord, neurons often display different fates in a "salt and pepper" pattern. The mechanism by which this non-random dispersed patterning occurs remains unknown. How- ever, given the role of Notch signaling in other cell fate decisions, the Notch pathway is a possible mediator because of its role in lateral inhibition. It has been shown that blocking Notch signaling by targeting the main downstream effector of Notch, Hes1, resulted in primarily GABAergic differentiation in human neural stem cells (Kabos et al., 2002 J Biol Chem 277: 8763). Based upon this finding we hypothesize that Notch antagonizes the acquisition of inhibitory neurotransmitter phenotypes. To test this hypothesis, we have up- regulated Notch signaling in developing Xenopus laevis embryos by injecting synthetic mRNA for the intracellular domain (ICD) of Notch. We have also blocked Notch signaling with the injection of mRNA for Su(H) DNA Binding Mutant, an inactive form of the transcription factor Su(H). Injected embryos were assayed using in situ hybridization for GABAergic, glycinergic, glutamatergic, and pan-neural markers following the completion of primary neurogen- esis. Initial results show that embryos injected with either Notch ICD or Su(H) DBM exhibit decreased density and organization of neurons in the CNS. We are currently performing double in situ hybridizations for excitatory and inhibitory markers to determine how the Notch signaling pathway affects the ratios of excitatory to inhibitory neurons. doi:10.1016/j.ydbio.2008.05.196 Program/Abstract # 184 Decoding the in vivo Notch targetome Christina Morgenstern, David Ish-Horowicz Developmental Genetics Laboratory, Cancer Research UK London Research Institute, London, UK The conserved Notch signalling pathway is involved in regulating many cellular processes throughout development and renewal of adult tissue in metazoans such as cell proliferation, stem cell maintenance, cell fate specification and apoptosis. So far, the readout of the pathway has been limited to a handful of targets, with the Hes family being the most prominent group of downstream effectors. Using the high-affinity of the biotin-streptavidin system combined with Chromatin Immunoprecipitation (ChIP) we aim to find novel target genes of the Notch intracellular domain (NICD), the transcriptional activator that results from cleavage of the Notch receptor. We have successfully generated transgenic mice carrying Biotin Acceptor Peptide (BAP)-tagged versions of Notch1 . These mice are crossed to mice expressing the E. coli biotinylase BirA, which leads to biotinylation of the BAP-tagged protein in vivo. Cross-linked and fragmented chromatin from different tissues will be precipi- tated with streptavidin and subsequent genome-wide analysis of bound DNA using high-throughput sequencing will reveal novel targets during development and throughout oncogenic transforma- tion. Similarly, we have established a transgenic mouse line expressing biotinylated Hes7, a transcriptional repressor and down- stream target of Notch. It has been shown that the Notch pathway and its target Hes7 play a key role during vertebrate segmentation. Using these transgenic mice we aim to identify novel targets of Hes7, which are implicated in regulating the periodic formation of somites. doi:10.1016/j.ydbio.2008.05.197 Program/Abstract # 185 Fgf signaling during cerebellar morphogenesis Yuichiro Yaguchi a , Tian Yu a , Mary Gait b , Ivor J. Mason b , M. Albert Basson a a Department of Craniofacial Development, King's College London, UK b MRC Centre for Developmental Neurobiology, King's College London, UK The cerebellum is derived from dorsal rhombomere 1 in the embryo. A large number of studies have defined important roles for FGF8 signaling during early development of rhombomere 1. However, the major classes of cerebellar neurons are only born and differentiate after embryonic day (E) 12.5 in the mouse, when expression of Fgf8 has been reported to cease. A key process required for normal cerebellar development is the extensive proliferation of granule cell precursors in the external granule cell layer during the early postnatal period, a process driven by Sonic Hedgehog (SHH). After a period of proliferation, the cells exit the cell cycle, start differentiating and migrate inwards to form the internal granule cell layer, where final maturation takes place. To investigate whether Fgf signaling is required during these later stages of cerebellar morphogenesis, we Developmental Biology 319 (2008) 521–523 Contents lists available at ScienceDirect Developmental Biology journal homepage: www.elsevier.com/developmentalbiology 0012-1606/$ – see front matter