Slit axon guidance in C. elegans

Poster abstracts / Int. J. Devl Neuroscience 24 (2006) 495–603

migration, RGC axons need to cross the midline, travel dorsalwards through the diencephalon, and recognize the midbrain target, the optic tectum. The growing axons find their way by interpreting guidance cues presented to the growth cones. Using the optic pathway of zebrafish, we are investigating the role of semaphorins (Semas) in patterning. Semaphorins make up a large family of mainly repulsive guidance cues that signal through plexin receptors. Class three Semas (Sema3s) are secreted and play a role in patterning the developing nervous system. Sema3s are unique in that they require neuropilin as a coreceptor with plexin. It has recently been shown that neuropilin mRNA is expressed in zebrafish RGCs at a time when their axons are extending (Liu et al., 2004). The presence of Sema3-selective receptors in RGCs suggests that Sema3s may be actively directing the development of the optic pathway. As a first step to identifying Sema3s important in RGC axon guidance we performed in situ hybridization with digoxygenin-labeled antisense mRNA probes for several sema3 genes (sema3aa, sema3fa, sema3fb, sema3ga, sema3gb) over the time when zebrafish RGCs first leave the eye (34 h post fertilization (hpf)), and reach and fully innervate the optic tectum (48–60 hpf). In the differentiating retina, sema3fa is expressed at early stages (30 hpf) in the RGC layer. Sema3aa is expressed in the telencephalon, apparently just anterior to the route RGC axons take through the diencephalon. Several sema3s were expressed either in broad (sema3aa, sema3ga, sema3fa) or highly restricted expression (sema3fb, sema3gb) patterns in the optic tectum at the time of RGC axon entry. These data suggest that several different sema3s are expressed in or near the optic pathway and could guide RGC axons. We are currently testing this idea by loss-offunction approaches.

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hydroxyeicosatetraenoic acid [12(S)- and 15(S)-HETE], (ii) activation of protein kinase C epsilon (PKC␧), (iii) phosphorylation of the myristoylated, alanine-rich C kinase substrate (MARCKS), an adhesion site protein, and (iv) detachment of adhesion sites, followed by growth cone turning or collapse. We have shown that growth factors as well as a linoleic acid derivative, 13(S)-hydroxyoctadecadienoic acid [13(S)-HODE], can inhibit repellent-induced growth cone collapse. Preliminary data also show that growth factors (e.g., IGF-1 and HGF) stimulate 13(S)-HODE synthesis. Furthermore, 13(S)-HODE inhibits 12(S)-HETE-stimulated PKC␧ activity in vitro. In the growth cone, this is likely to inhibit MARCKS phosphorylation and may thus prevent growth cone collapse. This may be the mechanism by which growth factors inhibit repellent action, and PKC␧ seems to be the point at which their signals converge. Acknowledgements Supported by National Institutes of Health Grants R01 NS041029 (to K.H.P.) and Ruth Kirschstein Fellowship F31 NS048710 (to S.D.S.). Keywords: Pathfinding; Growth cone; Growth factors; Repellents doi:10.1016/j.ijdevneu.2006.09.305 [P248] A novel transmembrane protein involved in SLT-1/Slit axon guidance in C. elegans K. Fujisawa 2,∗ , J.G. Culotti 1,2

Acknowledgements CIHR Training Grant in Genetics, Child Health and Development, AHFMR, CIHR. Keywords: Retinal ganglion cell; Semaphorin; Axon guidance; Zebrafish doi:10.1016/j.ijdevneu.2006.09.304 [P247] Modulation of growth cone repellent responses S. Sanford ∗ , K. Pfenninger University of Colorado Health Sciences Center, USA The molecular mechanisms by which the developing nervous system directs axon growth are complex processes that require strict temporal and spatial regulation of adhesion and the cytoskeleton. The Pfenninger lab has characterized a repellent-activated signaling pathway that regulates adhesion in rat neurons (de la Houssaye, 1999; Mikule et al., 2002). Its stimulation results in (i) increased synthesis of arachidonic acid (AA) derivatives, the eicosanoids 12(S)- and 15(S)-

1 University

of Toronto, Canada; 2 Samuel Lunenfeld Research Institute of Mt. Sinai Hospital, Canada

The SLT-1/Slit diffusible guidance cue is required for axon guidance along the dorsal-ventral axis in C. elegans. SAX3/ROBO, mediates repulsive responses to dorsally expressed SLT-1/Slit, one of the ligands for SAX-3, to orient axons in a ventral direction (Hao et al., 2001). For example, the pioneer axon of the lateral AVM sensory neuron in the anterior body extends its axon toward the ventral nerve cord (VNC) in wildtype animals, but in sax-3 and slt-1 mutants, the AVM axon frequently grows longitudinally along the basal surface of the lateral epidermis toward the head (Hao et al., 2001; Zallen et al., 1998). Although only one sax-3/robo gene and only one slt1/slit gene are present in the C. elegans genome, surprisingly, slt-1 mutants do not exhibit the nerve ring and epithelial defects of sax-3/robo mutants, suggesting that SAX-3/Robo has both Slit-dependent and Slit-independent functions in development (Hao et al., 2001). Here we identify a transmembrane protein named EVA-1 (enhancer of ventral axon guidance defects of unc-40), which is required for guidance of the AVM pioneer axon to the VNC. Our genetic data suggest that EVA-1 acts in the SLT-1 signaling pathway and that it is involved in mediating all, or nearly

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Poster abstracts / Int. J. Devl Neuroscience 24 (2006) 495–603

all, pioneer axon guidance effects of SLT-1 acting through the SAX-3/Robo receptor, but does not appear to be involved in SLT1-independent signaling by SAX-3/Robo. The more restricted set of axons affected by eva-1 and slt-1 mutations compared to sax-3 mutations and the more limited expression of EVA-1 compared to SAX-3, suggests that in neurons expressing EVA-1 and SAX-3 (e.g., AVM) EVA-1 causes SAX-3 to respond to SLT-1 as a guidance cue. Consistent with this, genetic mosaic analysis and rescue of AVM guidance defects by cell-specific expression indicate that EVA-1, like SAX-3, acts in AVM to perform its guidance functions.

the brain results in abundant projections to the dorsal IPN, as well as ectopic axonal projections that extend beyond it. The data support a model whereby Sema3D acts as an attractant in concert with Nrp1A to guide left habenular axons to innervate the dorsal IPN. Ectopic expression of Nrp1a in neurons of the right habenula is underway to determine whether this is sufficient to misdirect their axons to the dorsal target. Keywords: Left-right asymmetry; Semaphorins; Habenula; Axon guidance doi:10.1016/j.ijdevneu.2006.09.307

Keywords: Axon guidance; SAX-3/robo; SLT-1/slit; Galactose References Hao, J.C., Wu, T.W., Fujisawa, K., Culotti, J.G., Gengyo-Ando, K., Mitani, S., Moulder, G., Barstead, R., Tessier-Lavigne, M., Bargmann, C.I., 2001. Neuron 32, 25–38. Zallen, J.A., Yi, B.A., Bargmann, C.I., 1998. The conserved immunoglobulin superfamily member SAX-3/Robo directs multiple aspects of axon guidance in C. elegans. Cell 92, 217–227.

doi:10.1016/j.ijdevneu.2006.09.306 [P249] Class III semaphorin signaling is required for differential target recognition by the left and right habenular nuclei of the zebrafish brain Y.-S. Kuan 1,∗ , H.-H. Yu 2 , C.B. Moens 2 , M.E. Halpern 1 1 Carnegie

Institution of Washington, Department of Embryology, USA; 2 Howard Hughes Medical Institute and Division of Basic Science, Fred Hutchinson Cancer Research Center, USA The zebrafish dorsal diencephalon is serving as a useful model to explore how left–right (L–R) asymmetry can arise in a developing vertebrate brain. The bilateral habenular nuclei (HB) exhibit L–R differences in neuroanatomy, gene expression, and axonal projections to their unpaired midbrain target, the interpeduncular nucleus (IPN). Previous studies indicate that Nodal signaling biases the L–R position of the parapineal, which, in turn, influences the identity of the neighbouring habenula. Normally, efferents from the left habenula project throughout the dorsoventral extent of the IPN, while right habenular axons only project to the ventral region. How the asymmetric HB–IPN connection is established and the factors that are involved in target recognition are unknown. We have found that Neuropilin1a (Nrp1a), a receptor for class III Semaphorins (Sema), is selectively expressed in the left habenula prior to IPN innervation. The directional asymmetry of Nrp1a expression relies on Nodal signaling and parapineal position. Antisense morpholino (MO) depletion of Nrp1a disrupts left habenular axonal projections to the dorsal IPN. MO-depletion of the putative Nrp1a ligand, Sema3D, produces a highly similar phenotype, while perturbation of other Sema family members does not appear to affect HB-IPN innervation. Over expression of Sema3D throughout

[P250] Functional interaction between matrix metalloproteinase-3 and semaphorin-3C during cortical axonal growth and guidance B. Gonthier ∗ , C. Nasarre, L. Roth, M. Perraut, N. Thomasset, G. Roussel, D. Aunis, D. Bagnard INSERM U575, France In the developing cortex, axons and dendrites extend progressively in response to environmental cues attracting or repelling growing processes. Recent evidence suggests the existence of a functional link between guidance molecules and metalloproteinases. This has been shown so far for Netrin (Galko and Tessier-Lavigne, 2000) and Ephrin (Hattori and Flanagan, 2000). Here, we analyzed the putative functional interaction of matrix metalloproteinases (MMPs) with guidance cues of the Semaphorin family during growth and guidance of cortical axons. Using cellular and molecular approaches, we demonstrated that the expression pattern and the proteolytic activity of MMP-3 are consistent with a role of this particular MMP during cortical axon outgrowth. We found that MMP-3 is specifically required for an optimal axon extension. Moreover, our investigation revealed that MMP-3 is also specifically involved in the Sema3C-dependent chemoattraction of cortical axons by modulating both the growth capacity and the orientation of cortical axon outgrowth. Interestingly, the inhibitory Sema3A decreased both the expression and activity of MMP-3. Taken together, our results demonstrate a molecular interaction between MMPs and semaphorins providing new insight into the molecular mechanism allowing axonal growth cone to respond to environmental guidance cues in the context of cortical development. Keywords: Semaphorins; Matrix metalloproteinases; Chemoattraction; Growth promotion doi:10.1016/j.ijdevneu.2006.09.308