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Cxcr4 chemokine signaling is required for placode assembly and sensory axon pathfinding in the zebrafish olfactory system
Abstracts / Neuroscience Research 58S (2007) S1–S244
P3-c48 A drastic change in gene expression profile of human neural progenitor cells under serum-free and serum-containing culture conditions Jun-Ichi Satoh, Tamako Misawa, Nobuya Obayashi, Kenta Sumiyoshi, Kimiko Oosumi, Hiroko Tabunoki Department of Bioinformatics, Meiji Pharmaceutical University, Tokyo, Japan Neural progenitor cells (NPCs) show a self-renewing and proliferating capacity. NPCs expanding in culture provide an unlimited source for transplantation therapy of neurodegenerative diseases. To obtain a desirable source, the interaction of environmental and genetic factors that regulates stem cell fate should be elucidated in the human NPCs. We cultured human NPCs in the DMEM/F-12 medium containing EGF, bFGF and LIF. After several months, nestin+ GFAP+ floating spheres were incubated for 72 hours in the medium with or without 10% FBS. They were processed for analyzing on a microarray (Agilent, 41,000 genes), validated by real-time PCR. The 46 genes (ID1, ID2, ID3, CTGF, TGFA, METRN and GFAP) were upregulated, while 23 genes (NPTX1, DLL1, DLL3, SOX4 and HES5) were downregulated in the culture with the serum. Thus, the serum upregulated the genes that enhance astrocytic differentiation but repressed those involved in maintenance of the stem cell capacity. Research funds: KAKENHI(B18300118) and the Nakatomi Foundation
2 Systematic immunostaining and RNAi experiments for P3-d0 finding the novel molecular marker candidates of the growth cone
Motohiro Nozumi 1,2 , Kazuko Takahashi-Niki 1 , Michihiro Igarashi 1,2 1 Div. Mol. Cell. Biol., Grad. Sch. Med. Dent. Sci., Japan; 2 Trans-Disciplinary Research Program, Niigata University, Niigata, Japan
The growth cone (GC) is the tip of the extending neuronal processes for the accurate synaptogenesis. There were no methods discovering the molecules involved in the GC functions systematically, thus, the molecular basis of the GC behavior is poorly understood and its molecular markers were not known except GAP-43. Here we take the strategy to identify the GC proteins that are important for axonal growth for combining purification of the GCs from rat brain, proteomic analysis by multidimensional protein identification technology of the GC; immunostaining using antibodies against the proteins identified by proteomics; and RNAi that inhibits the axonal growth. Using this approach, we found 90 proteins which are more concentrated in the GC area than GAP-43. Among more than siRNAs against 50 proteins, those against 15 proteins were shown to inhibit the axonal growth significantly. In conclusion, our approach succeeded in effectively finding the proteins that operates the GC behavior. Research funds: KAKENHI(#17023019)
3 Cxcl12/Cxcr4 chemokine signaling is required for plaP3-d0 code assembly and sensory axon pathfinding in the zebrafish olfactory system Nobuhiko Miyasaka 1,2 , Holger Knaut 3 , Yoshihiro Yoshihara 1,2 Laboratory for Neurobiology of Synapse, RIKEN BSI, Japan; 2 CREST, JST, Japan; 3 Department of Molecular and Cellular Biology, Harvard University, USA
1
In the zebrafish olfactory system, precursors of olfactory sensory neurons (OSNs) assemble into a compact cluster to form the olfactory placode. Subsequently, OSNs differentiate and extend their axons to the presumptive olfactory bulb with high precision. The chemokine receptor cxcr4b is expressed in the migrating olfactory neuron precursors, and its ligand cxcl12a is expressed in the abutting anterior neural plate. The expression of cxcr4b persists in the olfactory placode at the initial phase of OSN axon pathfinding. Interfering with Cxcl12a/Cxcr4b signaling perturbs the assembly of the olfactory placode, resulting in the appearance of ventrally displaced olfactory neurons. Moreover, OSN axons frequently fail to exit the olfactory placode and accumulate near the placode-telencephalon border in the absence of Cxcr4b signaling. These data indicate that chemokine signaling contributes to both the olfactory placode assembly and the OSN axon pathfinding in zebrafish. Research funds: KAKENHI 16300105, CREST
S201
4 Key role of Rho/Rho-kinase signaling in hindbrain P3-d0 motor axon patterning Kenta Kobayashi 1 , Tomoyuki Masuda 2 , Masanori Takahashi 3 , Jun-Ichi Miyazaki 4 , Masahiro Nakagawa 5 , Hiroyuki Yaginuma 2 , Noriko Osumi 3 , Kozo Kaibuchi 6 , Kazuto Kobayashi 1 1 Department of Molecular Genetics, Fukushima Medical University, Fukushima, Japan; 2 Department of Anatomy, Fukushima Medical University, Fukushima, Japan; 3 Department of Developmental Neurobiology, Tohoku University Graduate School of Medicine, Sendai, Japan; 4 Div. Stem Cell Reg. Res., Osaka University Graduate School of Medicine, Suita, Japan; 5 Department of Electrical Engineering, Faculty of Engineering, Nagaoka University of Technology, Nagaoka, Japan; 6 Department of Cell Pharmacology, Nagoya University Graduate School of Medicine, Nagoya, Japan In this study, we performed conditional expression of a dominant-negative form for Rho-kinase in transgenic mice by use of Cre-loxP system to suppress the activity of Rho/Rho-kinase signaling in developing hindbrain motor neurons. The patterning of somatic motor (SM) axons, but not of visceral motor axons, was specifically impaired in transgenic embryos. In addition, chemoattractivity for SM axons in response to hepatocyte growth factor (HGF) was inhibited in the explant culture derived from transgenics. These findings suggest that Rho/Rho-kinase pathway dependent on HGF signaling plays a critical role in the patterning of SM axons during hindbrain development.
5 Localization of functional Reelin receptors as revealed P3-d0 by alkaline phosphatase–Reelin fusion protein Takayuki Uchida 1 , Baba Atsushi 1 , Miyata Takaki 2 , Nakajima Kazunori 3 , Mitsuharu Hattori 1 1 Nagoya City University, Graduate School of Pharmaceutical Sciences, Department of Biomedical Sciences, Nagoya, Japan; 2 Nagoya University, School of Medicine, Department of Anatomy, Japan; 3 Keio University, School of Medicine, Department of Anatomy, Japan
Reelin is essential for proper layer formation of mammalian brain. Reelin is received by cells that express Reelin receptors (ApoER2 and VLDLR), but when and where such interaction occurs remains obscure. Here we tried to know the localization of functional Reelin receptors. In situ staining of developing mouse forebrain, both from wild-type and reeler mice, with alkaline phosphatase fusion protein of the receptor-binding domain of Reelin revealed that functional Reelin receptors were mainly localized in the subventricular-intermediate zones, suggesting that Reelin is received by migrating neurons before they path through the subplate and that the receptors are down-regulated thereafter. In developing cerebellum Reelin receptors are expressed in not only purkinje cells but also various kinds of neurons. These observations will help understand how Reelin and its receptors organize neuronal layer formation. Research funds: KAKENHI 17082007
6 Roles of 5-HT1A and 5-HT2A receptors in the dendrite P3-d0
formation and synaptogenesis of cerebral cortex pyramidal neurons
Chisako Kanamaru, Kouji Senzaki, Takashi Shiga University of Tsukuba, Graduate School of Comprehensive Human Sciences, Tsukuba, Japan Recent studies have suggested 5-hydroxytryptoamine (5-HT) affects neural development, but roles of receptor subtypes in neuronal development are largely unknown. We have previously examined roles of 5-HT1A and 5-HT2A receptors in the formation of dendrites and synapses by dissociation culture (Shiga et al., 2006). Embryonic day 16 or 18 rat cerebral cortex was cultured in the presence of 5-HT1A or 5-HT2A receptor agonists. In the chronic presence of the agonists, dendritic outgrowth was promoted by 5-HT1A receptor, while inhibited by 5-HT2A receptor. In addition, synaptogenesis was promoted by 5-HT2A receptor, whereas inhibited by 5-HT1A receptor. In order to clarify the mechanisms of 5-HT1A and 5HT2A receptors underlying the dendrite formation and synaptogenesis, we examined acute effects of 5-HT1A and 5-HT2A receptor agonists. The agonists were added at 7–14 days of culture. We fixed the culture within 24 h, and analyzed synaptogenesis and spine formation. Research funds: The Long-range Research Initiative by Japan Chemical Industry Association
P3-c48 A drastic change in gene expression profile of human neural progenitor cells under serum-free and serum-containing culture conditions Jun-Ichi Satoh, Tamako Misawa, Nobuya Obayashi, Kenta Sumiyoshi, Kimiko Oosumi, Hiroko Tabunoki Department of Bioinformatics, Meiji Pharmaceutical University, Tokyo, Japan Neural progenitor cells (NPCs) show a self-renewing and proliferating capacity. NPCs expanding in culture provide an unlimited source for transplantation therapy of neurodegenerative diseases. To obtain a desirable source, the interaction of environmental and genetic factors that regulates stem cell fate should be elucidated in the human NPCs. We cultured human NPCs in the DMEM/F-12 medium containing EGF, bFGF and LIF. After several months, nestin+ GFAP+ floating spheres were incubated for 72 hours in the medium with or without 10% FBS. They were processed for analyzing on a microarray (Agilent, 41,000 genes), validated by real-time PCR. The 46 genes (ID1, ID2, ID3, CTGF, TGFA, METRN and GFAP) were upregulated, while 23 genes (NPTX1, DLL1, DLL3, SOX4 and HES5) were downregulated in the culture with the serum. Thus, the serum upregulated the genes that enhance astrocytic differentiation but repressed those involved in maintenance of the stem cell capacity. Research funds: KAKENHI(B18300118) and the Nakatomi Foundation
2 Systematic immunostaining and RNAi experiments for P3-d0 finding the novel molecular marker candidates of the growth cone
Motohiro Nozumi 1,2 , Kazuko Takahashi-Niki 1 , Michihiro Igarashi 1,2 1 Div. Mol. Cell. Biol., Grad. Sch. Med. Dent. Sci., Japan; 2 Trans-Disciplinary Research Program, Niigata University, Niigata, Japan
The growth cone (GC) is the tip of the extending neuronal processes for the accurate synaptogenesis. There were no methods discovering the molecules involved in the GC functions systematically, thus, the molecular basis of the GC behavior is poorly understood and its molecular markers were not known except GAP-43. Here we take the strategy to identify the GC proteins that are important for axonal growth for combining purification of the GCs from rat brain, proteomic analysis by multidimensional protein identification technology of the GC; immunostaining using antibodies against the proteins identified by proteomics; and RNAi that inhibits the axonal growth. Using this approach, we found 90 proteins which are more concentrated in the GC area than GAP-43. Among more than siRNAs against 50 proteins, those against 15 proteins were shown to inhibit the axonal growth significantly. In conclusion, our approach succeeded in effectively finding the proteins that operates the GC behavior. Research funds: KAKENHI(#17023019)
3 Cxcl12/Cxcr4 chemokine signaling is required for plaP3-d0 code assembly and sensory axon pathfinding in the zebrafish olfactory system Nobuhiko Miyasaka 1,2 , Holger Knaut 3 , Yoshihiro Yoshihara 1,2 Laboratory for Neurobiology of Synapse, RIKEN BSI, Japan; 2 CREST, JST, Japan; 3 Department of Molecular and Cellular Biology, Harvard University, USA
1
In the zebrafish olfactory system, precursors of olfactory sensory neurons (OSNs) assemble into a compact cluster to form the olfactory placode. Subsequently, OSNs differentiate and extend their axons to the presumptive olfactory bulb with high precision. The chemokine receptor cxcr4b is expressed in the migrating olfactory neuron precursors, and its ligand cxcl12a is expressed in the abutting anterior neural plate. The expression of cxcr4b persists in the olfactory placode at the initial phase of OSN axon pathfinding. Interfering with Cxcl12a/Cxcr4b signaling perturbs the assembly of the olfactory placode, resulting in the appearance of ventrally displaced olfactory neurons. Moreover, OSN axons frequently fail to exit the olfactory placode and accumulate near the placode-telencephalon border in the absence of Cxcr4b signaling. These data indicate that chemokine signaling contributes to both the olfactory placode assembly and the OSN axon pathfinding in zebrafish. Research funds: KAKENHI 16300105, CREST
S201
4 Key role of Rho/Rho-kinase signaling in hindbrain P3-d0 motor axon patterning Kenta Kobayashi 1 , Tomoyuki Masuda 2 , Masanori Takahashi 3 , Jun-Ichi Miyazaki 4 , Masahiro Nakagawa 5 , Hiroyuki Yaginuma 2 , Noriko Osumi 3 , Kozo Kaibuchi 6 , Kazuto Kobayashi 1 1 Department of Molecular Genetics, Fukushima Medical University, Fukushima, Japan; 2 Department of Anatomy, Fukushima Medical University, Fukushima, Japan; 3 Department of Developmental Neurobiology, Tohoku University Graduate School of Medicine, Sendai, Japan; 4 Div. Stem Cell Reg. Res., Osaka University Graduate School of Medicine, Suita, Japan; 5 Department of Electrical Engineering, Faculty of Engineering, Nagaoka University of Technology, Nagaoka, Japan; 6 Department of Cell Pharmacology, Nagoya University Graduate School of Medicine, Nagoya, Japan In this study, we performed conditional expression of a dominant-negative form for Rho-kinase in transgenic mice by use of Cre-loxP system to suppress the activity of Rho/Rho-kinase signaling in developing hindbrain motor neurons. The patterning of somatic motor (SM) axons, but not of visceral motor axons, was specifically impaired in transgenic embryos. In addition, chemoattractivity for SM axons in response to hepatocyte growth factor (HGF) was inhibited in the explant culture derived from transgenics. These findings suggest that Rho/Rho-kinase pathway dependent on HGF signaling plays a critical role in the patterning of SM axons during hindbrain development.
5 Localization of functional Reelin receptors as revealed P3-d0 by alkaline phosphatase–Reelin fusion protein Takayuki Uchida 1 , Baba Atsushi 1 , Miyata Takaki 2 , Nakajima Kazunori 3 , Mitsuharu Hattori 1 1 Nagoya City University, Graduate School of Pharmaceutical Sciences, Department of Biomedical Sciences, Nagoya, Japan; 2 Nagoya University, School of Medicine, Department of Anatomy, Japan; 3 Keio University, School of Medicine, Department of Anatomy, Japan
Reelin is essential for proper layer formation of mammalian brain. Reelin is received by cells that express Reelin receptors (ApoER2 and VLDLR), but when and where such interaction occurs remains obscure. Here we tried to know the localization of functional Reelin receptors. In situ staining of developing mouse forebrain, both from wild-type and reeler mice, with alkaline phosphatase fusion protein of the receptor-binding domain of Reelin revealed that functional Reelin receptors were mainly localized in the subventricular-intermediate zones, suggesting that Reelin is received by migrating neurons before they path through the subplate and that the receptors are down-regulated thereafter. In developing cerebellum Reelin receptors are expressed in not only purkinje cells but also various kinds of neurons. These observations will help understand how Reelin and its receptors organize neuronal layer formation. Research funds: KAKENHI 17082007
6 Roles of 5-HT1A and 5-HT2A receptors in the dendrite P3-d0
formation and synaptogenesis of cerebral cortex pyramidal neurons
Chisako Kanamaru, Kouji Senzaki, Takashi Shiga University of Tsukuba, Graduate School of Comprehensive Human Sciences, Tsukuba, Japan Recent studies have suggested 5-hydroxytryptoamine (5-HT) affects neural development, but roles of receptor subtypes in neuronal development are largely unknown. We have previously examined roles of 5-HT1A and 5-HT2A receptors in the formation of dendrites and synapses by dissociation culture (Shiga et al., 2006). Embryonic day 16 or 18 rat cerebral cortex was cultured in the presence of 5-HT1A or 5-HT2A receptor agonists. In the chronic presence of the agonists, dendritic outgrowth was promoted by 5-HT1A receptor, while inhibited by 5-HT2A receptor. In addition, synaptogenesis was promoted by 5-HT2A receptor, whereas inhibited by 5-HT1A receptor. In order to clarify the mechanisms of 5-HT1A and 5HT2A receptors underlying the dendrite formation and synaptogenesis, we examined acute effects of 5-HT1A and 5-HT2A receptor agonists. The agonists were added at 7–14 days of culture. We fixed the culture within 24 h, and analyzed synaptogenesis and spine formation. Research funds: The Long-range Research Initiative by Japan Chemical Industry Association