PKCɛ induces astrogenesis in multipotential neural precursor cells

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

that only a subset of ZII− Purkinje cells are ectopic in cdf and taken together this indicates that there are at least two classes of ZII− Purkinje cells. Interestingly, recent experiments examining the origins of ZII− cells during development also suggest that at least two immunologically distinct populations of Purkinje cells contribute to the ZII− population in the adult cerebellum - one that expresses neurogranin alone (NG+) and a second that expresses a combination of heat shock protein25, calbindin and neurogranin (HSP25+). Our data reveals that both Purkinje cell phenotypes are present in the cdf cerebellum. However, we observed that the overwhelming majority of ectopic Purkinje cells in the perinatal cdf cerebellum are represented by the HSP25+ subset. This observation supports the adult data because a population of cells predicted to contribute to the adult ZII− population is ectopic in the perinatal cerebellum. Finally, despite the ectopia present in the cdf cerebellum, overall, parasagittal patterning is very similar to wild type. Thus, our evidence indicates that mediolateral patterning is established prior to Purkinje cell dispersal and that this dispersal process is differentially regulated across Purkinje cell subclasses. Keywords: Purkinje cell; Parasagittal; Patterning; Zebrin II doi:10.1016/j.ijdevneu.2006.09.217 [P158] The bHLH gene NSCL1 in the vertebrate nervous system: expression and functional studies M. Katidou 1,∗ , K. Theodorakis 2 , E. Renieri 3 , M. Kalantzaki 3 , A. Gavalas 3 , D. Karagogeos 3 1 University

of Crete Medical School, Greece; 2 Institute of Molecular Biology and Biotechnology, Greece; 3 Institute for Biomedical Research of the Academy of Athens, Greece

The basic helix-loop-helix (bHLH) genes encode for a family of transcription factors involved in developmental processes such as neurogenesis, myogenesis, hematopoeisis and sex determination. NSCL1 is a bHLH transcription factor expressed in various populations of postmitotic neurons of the CNS and the PNS. In situ hybridization demonstrated expression of NSCL1 in the subependymal layer of the neuroepithelium throughout the CNS, in the dorsal root ganglia, trigeminal and other cranial ganglia, sensory nasal epithelium, sensory layer of the developing optic cup, in the forebrain, hippocampus, septum, tectum, hypothalamic nuclei, hindbrain and spinal cord both in mouse embryos, postnatal and adult animals. NSCL1 behaves as an initiator factor of cerebellar granule cell growth and differentiation. Expression in the cerebellum, spinal cord and dorsal root ganglia corresponds to the expression of the adhesion molecule TAG-1/axonin-1. NSCL1 is detected in rhombomere boundaries both in mouse and chick. NSCL1 expression in these structures is coincident with their formation and is maintained until these structures are gone at later stages of development. cNSCL1 expression is also observed during

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rhombomere boundary regeneration in chick embryos, after their microsurgical ablation. In addition, a stronger NSCL1 signal is observed in r4, corresponding to facial branchiomotor (fbm) and visceromotor neurons. The expression pattern of mNSCL1 in Hoxb1−/− mice is strongly affected in r4 where migrating fbm neurons are located. To further investigate the role of NSCL-1 as a transcriptional activator or repressor, we performed luciferase assays using different truncated forms of cNSCL-1 fused to GAL-4, and a UAS luciferase reporter. In a complementary system, ME1a, a binding partner of NSCL-1 and a truncated form of mNSCL-1 are being tested for the activation or the repression of a luciferace reporter in which, the DNA binding sequence called E-box is upstream of a basal promoter and the luciferase gene. Acknowledgements Funded by the European Social Fund and Ministry of Education (PYTHAGORAS I). Keywords: bHLH transcription factor; Rhombomeres; Postmitotic neurons; Hoxb1 doi:10.1016/j.ijdevneu.2006.09.218 [P159] PKC␧ induces astrogenesis in multipotential neural precursor cells R. Steinhart 1,∗ , G. Kazimirsky 1 , H. Okhrimenko 1 , T. BenHur 2 , C. Brodie 3 1 Bar-Ilan

Israel;

University, Israel; 2 Hadassah-Hebrew University, Ford Hospital, USA

3 Henry

The PKC family plays important roles in various cellular functions of the central nervous system. In this study, we explored the role of PKC in the differentiation of multipotential neural precursor cells (MNPCs). We found that MNPCs expressed PKC␣, PKC␤2, PKC␦, PKC␧, PKC␨ and low levels of PKC␥. Treatment of the neurospheres with the PKC activator PMA selectively increased the number of astrocytes, whereas it decreased the generation of neurons and oligodendrocytes. To identify the specific PKC isoforms that mediate the astrocytic differentiation of the MNPCs, we overexpressed PKC␣, PKC␦ and PKC␧ in these cells using adenovirus vectors. Overexpression of PKC␣ or PKC␦ did not exert significant effects whereas PKC␧ selectively increased the generation of astrocytes. Moreover, a PKC␧KD mutant abolished the PMA effect. Since PMA induces phosphorylation of PKC␧ on serine 729, we examined the role of this phosphorylation site using a PKC␧S729A mutant. Overexpression of the PKC␧S729A mutant inhibited the effect of PMA on the generation of astrocytes, suggesting an important role of this phosphorylation site in the astrocytic differentiation of MNPCs. To delineate the mechanisms involved in the effect of PKC␧ on the astrocytic generation, we examined the expression of Notch1, which has been associated with this process. We

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

found that PMA and PKC␧ induced a large increase in Notch expression and that the PKC␧S729A mutant abolished the PMA effect. To examine the role of Notch1 in PMA and PKC␧ effects, we employed the ␥-secretase inhibitor L-685,458. Treatment of the cells with L-685,458 decreased the number of GFAP+ cells whereas it increased the number of ␤-tubulinIII+ cells. Moreover, L-685,458 abolished the effect of PMA on the generation of astrocytes. Our data suggest an important role of PKC␧ in astrocytic differentiation and implicate Notch1 as a possible mediator of this effect. Keywords: Neural progenitor cells; Astrogenesis; PKC; Notch 1 doi:10.1016/j.ijdevneu.2006.09.219 [P160] Origin and migration of oligodendrocytes in the zebrafish hindbrain D. Zannino ∗ , B. Appel Vanderbilt University, USA Uniform myelination requires that oligodendrocytes, the myelinating cell type of vertebrates, are uniformly distributed throughout the central nervous system. The origin and migratory pathways of oligodendrocytes have been well studied in the developing spinal cord. For example, spinal cord oligodendrocytes arise from ventral precursors that also produce motor neurons and then migrate dorsally. However, much less is known about oligodendrocyte development in the brain. We are investigating the origins and migratory behaviours of oligodendrocyte progenitor cells (OPCs) using transgenic zebrafish that express green fluorescent protein under control of olig2 regulatory DNA. Consistent with recently published work describing mouse oligodendrocyte development, our time lapse microscopy revealed that zebrafish OPCs originate in both ventral and dorsal hindbrain and cells subsequently intermix through dorsally and ventrally directed migration. Additionally, OPCs migrate long distances along the anteroposterior axis. Our preliminary analysis indicates that, in contrast to spinal cord, only a subset of hindbrain oligodendrocytes share a common precursor origin with motor neurons. Thus, the molecular mechanisms that specify oligodendrocytes might differ between the spinal cord and hindbrain. Keywords: Oligodendrocyte; Specification; Zebrafish; Hindbrain doi:10.1016/j.ijdevneu.2006.09.220

[P161] Role of BDNF in early gustatory neuron development D. Harlow ∗ , L. Barlow University of Colorado Denver Health Sciences Center, USA Cranial ganglion cells provide gustatory and non-gustatory lingual innervation. We have shown using embryos of an aquatic salamander, the axolotl, that the embryonic origin of ganglion cells predicts mature neuron fate; gustatory neurons derive from epibranchial placodes, while non-gustatory neurons derive from neural crest (Harlow and Barlow, in preperation). Previous studies of brain-derived neurotrophic factor (BDNF) knockout mice revealed that BDNF is necessary for gustatory innervation and ganglion cell survival (Nosrat et al., 1997; Liebl et al., 1997), but the embryonic neuronal populations affected are unknown. Are placodal neurons specifically dependent upon BDNF, and if so when, and from what source? We are using axolotls to answer these questions. We have determined that the receptor for BDNF, trkB, is expressed in the fibers that innervate taste buds, as well as within cells of the VIIth, IXth and Xth cranial ganglia, similar to the pattern observed in mice. We are now performing double label experiments to discern if placodal neurons specifically express trkB, and are thus responsive to BDNF in vivo. Cultured placodal ectoderm will generate sensory neurons de novo (Gross et al., 2003), and when exposed to BDNF in vitro, placodal neurons demonstrate increased axon outgrowth, branching and possibly survival. Moreover, the BDNF effect appears restricted to discrete times in vitro, corresponding to specific developmental events in vivo. Placodal neuron outgrowth is most substantial when explants are exposed to neurotrophin either: (1) during the period placodal neurons would coalesce with neural crest neurons into cranial ganglia in vivo or (2) as these sensory neurons reach their hindbrain target. Thus, our data indicate that placodes give rise to gustatory neurons, which are sensitive to BDNF long before these neurons innervate the tongue and resident taste buds. These data are consistent with reports in mice, where ganglionic sensory neurons are lost in BDNF−/− mice prior to axons reaching the tongue. In sum, these results suggest that gustatory neurons receive neurotrophic support from additional sources early on during embryonic development. Acknowledgements Supported by NIDCD DC003947 to LAB & NIDCD DC007796 to DEH. Keywords: BDNF; Gustatory system; Ganglion neurons; Development doi:10.1016/j.ijdevneu.2006.09.221