play a phenotype which recapitulates the clinical condition while hemizygous male mutants die by E12 and display neural tube defects, exencephaly and left–right asymmetry. Analysis of het- erozygous female mutants revealed a severe brain phenotype including expansion of the ventral telencephalon in the dorsal part of the brain, reduction of the cortex and defects in the forma- tion of dorsomedial telencephalic structures from E12.5. Morpho- logical staining of mutant brains showed disorganization of brain architecture with a progressive disappearance of the olfactory bulbs. In situ hybridization revealed ectopic expression of tran- scription factors critical for development of ventral telencephalon including Nkx2.6, Dlx2, Mash, Gsh2 while markers strongly expressed in the dorsal cortex, such as Ngn2 and Pax6, displayed a normal pattern of expression. Defective brain patterning was found associated with abnormal Shh signaling and impairment of the processing of Gli3. In addition, we observed an upregula- tion of canonical Wnt signaling in the cortex and the caudal fore- brain. Preliminary data also revealed cilia abnormalities within affected structures of the CNS. Altogether, these data indicate that Ofd1 is a patterning factor essential for development and specification of the telencephalon. Ongoing studies will clarify the contribution of cilia to the CNS defects observed in these mutants and the molecular basis of the brain abnormalities observed in OFDI syndrome. doi:10.1016/j.mod.2009.06.714 16-P024 Functional characterization of the ciliary proteins lebercilin and MKS1 Gabrielle Wheway , Carmel Toomes, Chris Inglehearn, Colin Johnson Leeds Institute of Molecular Medicine, University of Leeds, Leeds, United Kingdom LCA5, which encodes the ciliary protein lebercilin, is mutated in type 5 Leber’s Congenital Amaurosis (LCA), a severe form of hered- itary blindness. MKS1, which encodes the basal body protein MKS1, is mutated in type 1 Meckel–Gruber syndrome (MKS), a lethal devel- opmental disorder. Both conditions are ciliopathies, since the pro- teins play a role at the primary cilium, the non-motile organelle responsible for chemosensation of the cell environment, mechanosensation and numerous other roles in signaling. To determine the functional roles of lebercilin and MKS1, we have confirmed known and determined novel interacting partners for these proteins. Previous findings suggest that lebercilin is only involved in retrograde intraflagellar transport (IFT) along the ciliary axoneme. However, our work demonstrates that lebercilin is also an anterograde IFT components since: (i) it interacts strongly with IFT88; (ii) siRNA knockdown of LCA5 gives a characteristic failure of ciliogenesis; and (iii) live cell imaging visualizes lebercilin in both anterograde and retrograde movement. Yeast 2-hybrid screening with the B9 domain of MKS1 has identified several components of the ubiquitin pathway as MKS1 interactants that have been con- firmed by biochemical interaction assays. Immunocytostaining of ciliated cell lines shows clear colocalization of MKS1 with these proteins. Identification of another ubiquitin pathway protein as a putative lebercilin interactant suggests a link between lebercilin and MKS1, which provides scope for further study. We are currently characterizing the involvement of MKS1 and lebercilin in the ubiq- uitin pathway by studying mutated patient fibroblasts and siRNA knockdowns in ciliated cell lines. doi:10.1016/j.mod.2009.06.715 16-P025 The role of cilia during pectoral fin formation Susana Pascoal , Leonor Sau ´ de Instituto de Medicina Molecular e Instituto de Histologia e Biologia do Desenvolvimento, Faculdade de Medicina de Lisboa, Lisbon, Portugal Instituto Gulbenkian de Cie ˆncia, Oeiras, Portugal Cilia are microtubule-based organelles that protrude from the surface of most cells in the vertebrate body. The assembly, main- tenance and function of motile and sensory cilia, is dependent on intraflagellar transport (IFT), a motor-dependent movement of IFT particles along the microtubules. Polaris is a core component of the mammalian IFT machinery and is required for the formation of all cilia and flagella. In the mouse, primary cilia play an essential role in Hedgehog signal transduction and are required for both the activator and repressor activities of Gli proteins during limb bud development. The disrup- tion of polaris in mouse limbs gives rise to an expansion of the ante- rior–posterior axis, leading to the formation of extra digits. We show that similarly to the mouse, zebrafish pectoral fins express polaris and possess cilia. In order to study the function of polaris/cilia in fin formation in zebrafish we made used of the antisense morpholino technology to disrupt polaris transla- tion. In polaris morphants, pectoral fins are smaller or absent in clear contrast to the polydactyly phenotype observed in polaris mouse mutants. In addition, shh expression and its downstream target patched1 are not affected in the developing pectoral fin in the absence of polaris. However, we found that polaris controls the expression of fgf8 and wnt5b in the pectoral fin. These findings reveal that the use of cilia by different signalling pathways in appendage formation might not be conserved among vertebrates. doi:10.1016/j.mod.2009.06.716 16-P026 The role of ciliary genes in Shh signaling Ben Chih , Andrew Peterson Genentech, South San Francisco, United States The Shh signaling pathway is essential for the proper develop- ment of the nervous system. Despite its importance, how the molecular signal is transmitted from the cell surface receptor Ptched to downstream effectors is not clearly understood. Recently, it has been shown that the long forgotten cellular organelle, cilia are required for the signaling. In fact, the cell membrane proteins Ptched and SMO, and the Gli transcription factors are localized to the cilia. This suggests that the Shh signal is transduced within the cilia and ciliary genes may regulate Shh signaling. A focused RNAi screen was conducted for ciliary genes that can disrupt Shh signaling. We identified a novel gene, B9D1, which is required for S269 MECHANISMS OF DEVELOPMENT 126 (2009) S262 – S270