Shinji Sasaki

Complete Loss of Ndel1 Results in Neuronal Migration Defects and Early Embryonic Lethality

ABSTRACT Regulation of cytoplasmic dynein and microtubule dynamics is crucial for both mitotic cell division and neuronal migration. NDEL1 was identified as a protein interacting with LIS1, the protein product of a gene mutated in the lissencephaly. To elucidate NDEL1 function in vivo, we generated null and hypomorphic alleles of Ndel1 in mice by targeted gene disruption. Ndel1 −/− mice were embryonic lethal at the peri-implantation stage like null mutants of Lis1 and cytoplasmic dynein heavy chain. In addition, Ndel1 −/− blastocysts failed to grow in culture and exhibited a cell proliferation defect in inner cell mass. Although Ndel1 +/− mice displayed no obvious phenotypes, further reduction of NDEL1 by making null/hypomorph compound heterozygotes (Ndel1 cko/− ) resulted in histological defects consistent with mild neuronal migration defects. Double Lis1 cko/+ -Ndel1 +/− mice or Lis1 +/− -Ndel1 +/− mice displayed more severe neuronal migration defects than Lis1 cko/+ -Ndel1 +/ + mice or Lis1 +/− -Ndel1 +/+ mice, respectively. We demonstrated distinct abnormalities in microtubule organization and similar defects in the distribution of β-COP-positive vesicles (to assess dynein function) between Ndel1 or Lis1-null MEFs, as well as similar neuronal migration defects in Ndel1- or Lis1-null granule cells. Rescue of these defects in mouse embryonic fibroblasts and granule cells by overexpressing LIS1, NDEL1, or NDE1 suggest that NDEL1, LIS1, and NDE1 act in a common pathway to regulate dynein but each has distinct roles in the regulation of microtubule organization and neuronal migration.

The cortical subventricular zone-specific molecule Svet1 is part of the nuclear RNA coded by the putative Netrin receptor gene Unc5d and is expressed in multipolar migrating cells

Although Svet1 RNA is a widely used marker for the subventricular zone (SVZ) of the embryonic cerebral cortex, its function remains completely unknown. We report finding that Svet1 contains a high proportion of repetitive sequences and maps in the first intron of the putative Netrin receptor gene Unc5d. The direction of transcription of Svet1 is the same as that of Unc5d. The Svet1 RNA was detected in the nucleus but not in the cytoplasm. Both Svet1/Unc5d RNAs and UNC5D protein were localized in the multipolar cells in the SVZ throughout cortical development. These results suggest that Svet1 RNA is part of the sequence of the primary transcript of Unc5d in the nucleus that is spliced out before the mRNA is transported to the cytoplasm. Thus, the previously reported SVZ-specific expression of the Svet1 RNA in fact indicates putative involvement of UNC5D signaling in the multipolar migrating cells.

Laminar and Areal Expression of Unc5d and Its Role in Cortical Cell Survival

The UNC-5 family of netrin receptors is known to regulate axon guidance, cell migration, and cell survival. We have previously demonstrated that unc5d, one of the UNC-5 family member genes, is specifically expressed in layer 4 of the developing rat neocortex (Zhong Y, Takemoto M, Fukuda T, Hattori Y, Murakami F, Nakajima D, Nakayama M, Yamamoto N. 2004. Identification of the genes that are expressed in the upper layers of the neocortex. Cereb Cortex. 14:1144-1152). However, the role of UNC5D in cortical development is still unknown. In this study, we revealed that unc5d was highly expressed in the primary sensory areas of the mouse neocortex at around postnatal day 7. Netrin-4 was also found to be predominantly expressed in layer 4 of the sensory cortex and sensory thalamic nuclei. Cell surface binding assay showed that netrin-4 protein bound to UNC5D-expressing cells. An in vitro study further demonstrated that cell death of unc5d-expressing layer 4 cells was reduced by exogenous application of netrin-4 protein, whereas UNC5D is not sufficient to mediate the effect of netrin-4 in deep layer cells. Taken together, these results suggest that UNC5D is primarily expressed by layer 4 cells in the primary sensory areas of the developing neocortex and may mediate the effect of netrin-4 on cortical cell survival in a lamina-specific manner.

A Phosphatidylinositol Lipids System, Lamellipodin, and Ena/VASP Regulate Dynamic Morphology of Multipolar Migrating Cells in the Developing Cerebral Cortex

In the developing mammalian cerebral cortex, excitatory neurons are generated in the ventricular zone (VZ) and subventricular zone; these neurons migrate toward the pial surface. The neurons generated in the VZ assume a multipolar morphology and remain in a narrow region called the multipolar cell accumulation zone (MAZ) for ∼24 h, in which they extend and retract multiple processes dynamically. They eventually extend an axon tangentially and begin radial migration using a migratory mode called locomotion. Despite the potential biological importance of the process movement of multipolar cells, the molecular mechanisms remain to be elucidated. Here, we observed that the processes of mouse multipolar cells were actin rich and morphologically resembled the filopodia and lamellipodia in growth cones; thus, we focused on the actin-remodeling proteins Lamellipodin (Lpd) and Ena/vasodilator-stimulated phosphoprotein (VASP). Lpd binds to phosphatidylinositol (3,4)-bisphosphate [PI(3,4)P2] and recruits Ena/VASP, which promotes the assembly of actin filaments, to the plasma membranes. In situ hybridization and immunohistochemistry revealed that Lpd is expressed in multipolar cells in the MAZ. The functional silencing of either Lpd or Ena/VASP decreased the number of primary processes. Immunostaining and a Förster resonance energy transfer analysis revealed the subcellular localization of PI(3,4)P2 at the tips of the processes. A knockdown experiment and treatment with an inhibitor for Src homology 2-containing inositol phosphatase-2, a 5-phosphatase that produces PI(3,4)P2 from phosphatidylinositol (3,4,5)-triphosphate, decreased the number of primary processes. Our observations suggest that PI(3,4)P2, Lpd, and Ena/VASP are involved in the process movement of multipolar migrating cells.

Identity of neocortical layer 4 neurons is specified through correct positioning into the cortex

Many cell-intrinsic mechanisms have been shown to regulate neuronal subtype specification in the mammalian neocortex. However, how much cell environment is crucial for subtype determination still remained unclear. Here, we show that knockdown of Protocadherin20 (Pcdh20), which is expressed in post-migratory neurons of layer 4 (L4) lineage, caused the cells to localize in L2/3. The ectopically positioned “future L4 neurons” lost their L4 characteristics but acquired L2/3 characteristics. Knockdown of a cytoskeletal protein in the future L4 neurons, which caused random disruption of positioning, also showed that those accidentally located in L4 acquired the L4 characteristics. Moreover, restoration of positioning of the Pcdh20-knockdown neurons into L4 rescued the specification failure. We further suggest that the thalamocortical axons provide a positional cue to specify L4 identity. These results suggest that the L4 identity is not completely determined at the time of birth but ensured by the surrounding environment after appropriate positioning. DOI: http://dx.doi.org/10.7554/eLife.10907.001

Specification of neocortical layer IV fate by Protocadherin20 through regulation of neuronal positioning

The mammalian cerebral cortex consists of well-organized six layers of neurons, each of which contains one or more distinct subtypes of neurons. The neurons are born in the ventricular and subventricular zones and migrate to beneath the marginal zone, where they form the cortical plate. The laterborn neurons migrate past the early-born neurons and align in the superficial area, resulting in the “inside-out” progression of cortical plate development. Among many subtypes in the cerebral cortex, layer IV neurons integrate inputs from outside the cortex into cortical neuronal networks, although molecular determinants of this type of neurons remain largely unknown. Here we show that Protocadherin20 (Pcdh20) plays an essential role in the development of layer IV neurons. Expression of Pcdh20 was detected preferentially in a lineage of layer IV neurons, but started only after neurons reach to the cortical plate. Knockdown of Pcdh20 inhibited not only the fate specification of future layer IV neurons but also their laminar fate. Moreover, malpositioning of neurons by another method also disrupted the fate specification of future layer IV neurons when they were located in other layers. These results may suggest a previously-unrecognized role of the cadherin superfamily in the regulation of the laminar fates of cortical neurons.

Screening and functional analyses of the molecules that are preferentially expressed in the upper cortical plate of the developing cerebral cortex

s / Neuroscience Research 58S (2007) S1–S244 S203 P3-d13 Screening and functional analyses of the molecules that are preferentially expressed in the upper cortical plate of the developing cerebral cortex Kashiko Tachikawa1, Shinji Sasaki1, Takuya Maeda1, Kazunori Nakajima1,2 1 Department of Anatomy, Keio University School of Medicine, Japan; 2 Department of Molecular Neurobiology, Jikei University School of Medicine, Japan During the cerebral cortical development, major population of excitatory neurons originate in the ventricular zone and migrate to beneath the marginal zone (MZ) to form the cortical plate. To clarify the events that occur beneath the MZ, we screened for the molecules that were preferentially expressed beneath the MZ using GeneChip analysis and in situ hybridization. Consequently, we identified 40 molecules that were preferentially expressed in the upper cortical plate. Especially, Ndrg1 mRNA was strongly expressed in the neurons that were located just beneath the MZ. Interestingly, the Ndrg1 protein was detected as punctates mainly in the MZ, suggesting that it was located on the primitive dendrites of neurons that had just reached beneath the MZ. Thus, to examine the functions of Ndrg1 in cortical development, we applied in utero electroporation system. Here we report the results of loss-of-function experiments of Ndrg1 and discuss its functions during cortical development. P3-d14 Observation on developing cerebellar cortex using acoustic impedance microscope and characterization of L-type calcium channel distribution Shiho Masaki1, Erina Fukushi1, Toshitaka Morishima1, Atsunori Matsuda1, Kazuto Kobayashi2, Naohiro Hozumi3, Sachiko Yoshida1 1 Department of Material Science, Toyohashi University of Technology, Toyohashi, Japan; 2 Honda Electronics Co., Ltd., Toyohashi, Japan; 3 Aichi Institute of Technology, Toyota, Japan Two-dimensional acoustic impedance imaging is useful for observation on living organs with no invasion. We have reported that three-layer structure in cerebellar cortex was observed as the contrast in acoustic impedance. In order to observe more clearly or specifically, we examined substrate conditions and specimen treatments. Either SiO2 or TiO2 coated substrate, which had a contact angle between 25◦ and 55◦, gave us good discrimination. In addition, the application of 100 M CdCl2 induced increment of molecular layer impedance at P21. Neither 50 M NiCl2 nor 2 mM CaCl2 treatment showed that increment. Cd2+ has high affinity for L-type Ca channel, so it suggested that selective binding of Cd2+ to L-type Ca channel could show the distribution of functional L-type Ca channel in living cerebellar cortex. P3-d15 Neural zinc finger (NZF) transcription factors regulate development and survival of dorsal root ganglia neurons Toshiki Kameyama, Fumio Matsushita, Tohru Marunouchi, Yuzo Kadokawa Division of Cell Biology, Fujita Health University, Toyoake, Japan NZF-2b and NZF-3 transcription factors, which has C2HC-type zinc finger motifs, are expressed in subsets of DRG sensory neuron, suggesting that they may regulate development of specific sensory neurons. To elucidate in vivo functions of NZF family, we generated knockout mice of NZF-2 and NZF-3 gene, and double knockout mice both of NZF-2 and NZF-3. Double knockout mice exhibited a forelimb posture abnormality. To find the cause of this defect, we examined the neuronal differentiation and the trajectories of spinal nerves. Double mutant mice embryos showed spinal nerve hypoplasia especially projecting into limb buds. In the cervical DRG of the double mutants, the number of sensory neurons was fewer than that of wild type embryos. Furthermore, TrkCand parvalbumin-positive proprioceptive neurons were markedly decreased in the mutant DRG. Our data suggest that NZF-2 and NZF-3 are important for regulating the development of proprioceptive DRG sensory neurons and may help to elucidate the molecular mechanisms underlying somatosensory-related ataxia. P3-d16 Phenotype analysis of crmp5-deficient mice Mari Miyazaki1, Kozue Ugajin1, Naoya Yamashita1, Fumio Nakamura1, Pappachan Kolattukudy2, Yoshio Goshima1 1 Graduate School of Medicine, Yokohama City University, Yokohama, Japan; 2 Biomol. Sci., University of Central Florida, Florida, USA Collapsin response mediator proteins (CRMPs) are involved in neuronal cell migration and axonal guidance. The expression of CRMP5, one of the CRMP members, is abundantly expressed in fetal brain. CRMP5 is also thought to play a critical role in neuronal degeneration associated with paraneoplastic syndrome. To elucidate the function of CRMP5 in vivo, we examined phenotype of crmp5−/− mice. CRMP5 protein was detected at P21 in all layers of cerebellum by anti CRMP5 antibody staining. In crmp5−/− mice, decreased staining of MAP2 and aberrant dendritic morphogenesis were observed in the cerebellar Purkinje cells. Furthermore, tail hanging test revealed that crmp5−/− mice, but not crmp5+/− mice and crmp5+/+ mice, exhibited abnormal limb-clasping reflexes. These findings suggest that CRMP5 plays an important role in cerebellar development and function. P3-d17 Phosphorylation of CRMP2 at S522 is required for proper dendritic patterning of layer V cortical neurons in vivo Yutaka Uchida1, Toshio Ohshima2, Naoya Yamashita1, Fumio Nakamura1, Papachan Kolattukudy3, Jerome Honnorat4, Yoshio Goshima1,5 1 Department of Molecular Pharmacology & Neurobiology, Yokohama City University Graduate School of Medicine, Yokohama, Japan; 2 RIKEN, BSI, Wako, Japan; 3 University of Central Florida, Orlando, USA; 4 INSERM U433, Lyon, France; 5 CREST, Kawaguchi, Japan We report that CRMP2 is phosphorylated by Cdk5 at S522. This phosphorylation is essential for the sequential phosphorylation by GSK3 at T509, and caused reduction in its affinity to tubulin. To determine the role of the phosphorylation in vivo, we generated CRMP2 knock-in (KI) mice in which S522 was replaced with A. Since the phosphorylated CRMP2 at S522 was abundant at dendrites in cultured cortical neurons, we examined dendritic morphology using YFP-H transgenic mice. CRMP2 KI mice did not show any defects in dendritic patterning. In CRMP2S522A KI;crmp1 double mutant mice, however, we observed disorientation of basal dendrites of Layer V cortical neurons. These findings suggest that phosphorylation of CRMP2 at S522 is essential for proper development of basal dendritic patterning in vivo. Research funds: Grant-in-Aid for Scientific Research on Priority Areas, CREST P3-d18 The role of calmodulin 1 in the migration of precerebellar neurons Hiroaki Kobayashi, Shunsuke Saragai, Atsushi Naito, Daisuke Kawauchi Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan Migration of neurons from their birthplace to the appropriate positions is a fundamental event, and calcium signaling is implicated in regulating this process. Calmodulin (CaM) is an upstream regulator of calcium signaling cascade, and is encoded by three non-allelic genes (calm-1,2,3) in vertebrates. Since all three calm genes encode an identical protein with no amino acid substitutions, they are thought to play differential roles by providing distinct subcellular CaM pools through the regulation of mRNA sorting and translation. Here we investigated the role of CaM signaling in migration of precerebellar neurons (PCNs). We examined the expression profiles of three calm genes in PCNs by in situ hybridization with gene-specific cRNA probes. We found that calm1 is expressed during migration, followed by the expression of all three genes around the period of nucleus formation. RNA-interference-mediated knockdown of calm1 resulted in delayed migration of PCNs. These results suggest that the calm1 signaling is required for the proper tangential migration of PCNs.

Regulation of cortical laminar formation by cell–cell contacts

s / Neuroscience Research 58S (2007) S1–S244 S145 P2-d0 4 Heparanase upregulation in astrocytes and macrophages recruited to the injured spinal cord Yi Zhang1, C.H. Chau1, Y.S. Chan2, D.K.Y. Shum1 1 Department of Biochemistry, The University of Hong Kong, China; 2 Departments of Physiology, The University of Hong Kong, China Mammalian heparanase (Hpa1) is a heparan sulfate-cleaving endoglucuronidase exploited by inflammatory and cancer cells to invade connective tissues. We hypothesize Hpa1 involvement also in the recruitment of reactive astrocytes and macrophages to the injured spinal cord. To test this, spinal cords of adult rats were hemisected at T8. Q-PCR for Hpa1 mRNA in the cord at T7-9 indicated initial (3 dpi) decrease followed by progressive increase to levels in excess of that in the normal cord by 14 dpi. Combined in situ hybridization for Hpa1 mRNA and immunocytochemistry for cell markers indicated increased Hpa1 expression in GFAP+ astrocytes and ED-1+ macrophages in the lesion by 7–14 dpi. Upregulated Hpa1 expression was also observed in astrocytes that invaded the scratched area in an in vitro model. Taken together, our results indicated upregulated expression of Hpa1 in astrocytes and macrophages that were recruited to the injury. Research funds: CRCG10204997 & 10205634 to DKYS P2-d0 5 Wnt5 signaling for glomerular patterning in the Drosophila olfactory system Masao Sakurai1,2, Tomoko Aoki1, Shingo Yoshikawa2, Kyoko Ishikawa1, John B. Thomas2, Chihiro Hama1 1 CDB, Riken, Japan; 2 Salk Institute for Biological Studies, United States In Drosophila, odor specificity is represented as a spatial map of activated glomeruli in the first olfactory relay center called antennal lobe (AL). About 50 glomeruli, each formed by specific synaptic connections between olfactory receptor neurons (ORNs) and projection neurons (PNs), are stereotypically organized by multiple regulatory processes during olfactory development. Here we reveal that glomerular patterning is controlled by Wnt5 signaling transmitted in part from ORNs to PNs. In the Wnt5 mutant, several glomeruli shifted to ectopic positions, and this phenotype was rescued by Wnt5 expression in ORNs. Previous studies in Drosophila embryos have shown that Wnt5 repels the axons expressing Derailed (Drl). In the AL, however, our analyses indicate that Drl, localized in subsets of PN dendrites, has an antagonistic function in Wnt5 signaling. These data suggest that Wnt5 secreted from ORN axons differentially repels pre-patterned PN dendrites expressing varying amounts of Drl and a second receptor to establish the stereotyped glomerular pattern. P2-d0 6 Change in the microtubule-anchoring proteins at the centrosome during the differentiation of mouse neurons Yusaku Ohama, Kensuke Hayashi Life Science Institute, Sophia University, Tokyo, Japan Neurons are rich in non-centrosomal microtubules. These microtubules are thought to be important for axon elongation. In general, microtubules are nucleated at centrosome with -tubulin and they are anchored there by microtubule-anchoring protein, ninein. In this study, we generated antibodies against ninein and studied the distribution of ninein, as well as those of -tubulin and pericentrin (centrosome marker) during the development of the mouse cerebral cortex. In radial cells at the ventricular zone, -tubulin and ninein are localized at the centrosome. In migrating neurons in E11 cortical plate, -tubulin was still detected at the centrosome, but ninein was not detected there. In differentiated cortical neurons of young adult mice, -tubulin and ninein was not detected at the centrosome. Pericentrin-antibody clearly stained centrosomes of all neurons. These observations suggest the change in the microtuble-anchoring function of centrosomes during the differentiation of neurons that might be responsible for the generation of non-centrosomal microtubles. Research funds: KAKENHI (16027207, 16500193) P2-d0 7 Function of draxin in the migration of neural crest cells Su Yuhong, Iftekhar Bin Naser, M.D. Shahidul Islam, Yohei Shinmyo, Sanbing Zhang, Giasudin Ahmed, Sandy Chen, Hideaki Tanaka Department of Developmental Neurobiology, University of Kumamoto, Kumamoto, Japan Neural crests cells originate from dorsal neural tube and migrate out through anterior half of the somite to form diverse derivatives. Several repulsive axon guidance molecules, such as ephrin-B, semaphorin and slit have been shown to play important roles in neural crest migration. Here we show a novel molecule named draxin, which is expressed in roof plate and functions as a repulsive guidance molecule for spinal cord commissural axons. Neural crest cell migration from the neural tube explants was inhibited by draxin. After over expression of draxin in early chick embryos by electroporation at stage 12, many neural crest cells migrated ectopically at stage 18 along the dorsal-lateral pathway which is a normal pathway for later born cells start at stage 20. These studies suggest that draxin, a new repulsive axon guidance molecule, can also repel neural crest cells in migration out from chick neural tube. P2-d0 8 Analysis of a novel guidance molecule, draxin, in chick embryonic tectobulbar axon projection Iftekhar Bin Naser, Yuhong Su, M.D. Shahidul Islam, Yohei Shinmyo, Sanbing Zhang, Giasuddin Ahmed, Sandy Chen, Hideaki Tanaka Department of Developmental Neurobiology, University of Kumamoto, Kumamoto, Japan Tectobulbar tract in chick embryo is the first long distance projecting fiber pathway to appear during the development of the avian optic tectum. It starts from E3 where fibers proceeding downward from dorsal to ventral side on the lateral wall of the optic tectum and join the medial longitudinal fasciculus of the same side or cross the floor of the optic tectum and descend on the opposite side and this tract does not cross the dorsal midline of the tectum. Here we report a molecule named draxin, which plays a role in spinal cord commissural axon guidance. Draxin is expressed in dorsal high to ventral low gradient in chick optic tectum. Draxin protein is deposited in the most dorsal part of the tectum. In vitro experiments show that this molecule inhibits neurite outgrowth from tectum explants and in vivo over expression induced misrouted or stopped axon outgrowth in the tectum. So this molecule may be an important member of the tectobulbar tract formation. P2-d0 9 Regulation of cortical laminar formation by cell–cell contacts Koji Oishi1, Kashiko Tachikawa1, Shinji Sasaki1, Kazunori Nakajima1,2 1 Department of Anatomy, Keio University School of Medicine, Tokyo, Japan; 2 Department of Molecular Neurobiology, Jikei University School of Medicine, Japan Cerebral cortical neurons are sequentially generated in the ventricular/subventricular zones of the dorsal telencephalon, migrate to their destinations within the cortical plate, in which later-born neurons migrate over earlier-born deeper-layer neurons and occupy more superficial layers, and form a six-layered structure. Recently, behaviors of migrating neurons have been revealed from time-lapse imaging analyses and cell labeling experiments. They migrate along radial fibers separately and stay beneath the marginal zone (MZ) for about one day forming a relatively densely structure. These observations prompted us to hypothesize that birth-date-dependent cell adhesive specificity might regulate the birth-date-dependent cortical layer structure. To test this hypothesis, we screened for the adhesion-related molecules that showed a stagedependent expression pattern beneath the MZ. Here we report the results of expression pattern and function of the identified molecule(s).

Functional analyses of the molecules that control the behavior of multipolar migration neurons in the developing cerebral neocortex

In the developing cerebral neocortex, postmitotic neurons in the ventricular zone (VZ) do not directly migrate into the cortical plate (CP) but stay for some time in the subventricular zone (SVZ) with multipolar morphology. In this zone, they dynamically extend and retract multiple processes, moving as if they are seeking for some extracellular signals. However, what molecules control this ‘multipolar migration’ is still unknown. In this study, we identified receptor molecules that are preferentially expressed in the multipolar migration neurons in the SVZ. Although some of the ligand molecules for these receptors were not produced in the CP, these ligand proteins were detected in the intermediate zone or the VZ, facing to the SVZ. These coordinated distribution patterns of receptors and their ligands suggest that the behavior of multipolar migration neurons may be regulated by the adjacent structures through the ligand-receptor interactions.