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Roles of mammalian Fringe in the developing cerebral cortex
Abstracts P2-e05 Role of structural asymmetry of divided neural progenitor cells in the fate decision during mammalian brain neurogenesis Atsunori Shitamukai, Fumio Matsuzaki RIKEN, CDB, Japan In the mammalian brain neurogenesis, the radial glial cells called apical progenitors act as neural progenitors. These cells have apico-basal polarity and entend an apical process to the ventricular suface and a basal process to the pial surface. However, the roles of the apico-basal structure in cell fate decisions at apical progenitor divisions are largely unknown. To clarify these, we analyzed the relationship between inheritance apico-basal structure at apical divisions and their daughter cell fates by using the time-lapse imaging. As the results, the apical junction was partitioned into both daughter cells at most apical divisions and there is no correlation between the apical domain size inherited by a daughter and its cell fate. Whereas, the most of basal process was partitioned asymmetrically during apical divisions. We found a strong correlation between the apical progenitor cell fate and the inheritance of the basal process. These data suggest that the existence of the basal process an important factor for self-renewal. We currently investigate what signal is involved in this regulation. doi:10.1016/j.neures.2009.09.801
P2-e06 Introducing neurocristopathy-associated PHOX2B mutations in mouse reveals conserved pathogenic effects of mutant PHOX2B on mammalian neural crest cell development Mayumi Nagashimada 1,2 , Hideki Enomoto 1 1
NDR, Riken, CDB, Kobe, Japan;
2
Univ of Osaka, Osaka, Japan
Phox2b is essential for the development of the autonomic nervous system (ANS). Heterozygous mutations of the PHOX2B gene, 931del5 and 693del8, have been identified in a neurocristopathy syndrome where Congenital Central Hypoventilation Syndrome (CCHS), Hirschsprung’s disease (intestinal aganglionosis) and multifocal neuroblastoma are found in the same patients. To understand how these PHOX2B mutations influence ANS development, we introduced those human mutations into the phox2b locus in mice (Phox2bdel5/+ and Phox2bdel8/+ mice, respectively) by gene targeting. Heterozygous mice of both mutants died soon after birth due to impaired spontaneous breathing, and the phenotype was reminiscent of CCHS. Moreover, both mutants displayed abnormal development of the enteric nervous system and sympathetic ganglia. Thus mutant PHOX2B affect normal development of an identical set of neuronal populations between human and mice, suggesting the conserved pathogenic effects of mutant PHOX2B in mammals. doi:10.1016/j.neures.2009.09.802
P2-e07 Characterization of temporal identity of neuronal progenitor cells in the neocortex Ken-ichi Mizutani, Carina Hanashima RIKEN CDB, Japan Developmentally, neocortical excitatory neurons are generated from common progenitors within the dorsal telencephalon, which are thought to produce distinct layer neurons in an inside-out manner. We have utilized Ngn2, a bHLH proneural, Cre-recombinase mouse line to fate-map transiently Ngn2 expressing cells. It has been shown that Ngn2 expression peaks in progenitors that are committed to leave the cell cycle and thus is an ideal candidate to label the cortical lineage, as well as temporal cohorts of differentiating neurons. We crossed the Ngn2-CreER line with ROSA-stop-YFP mice with tamoxifen between E11.5 and E16.5, and confirmed that we could specifically target distinct layer neurons upon tamoxifen administration. Also, we characterized the molecular specificity of isolating distinct cortical progenitors after cell sorting. Our Ngn2 temporal fate-mapping analysis will provide the molecular information by which each neuronal fate is fundamentally distinct from other excitatory neurons. doi:10.1016/j.neures.2009.09.803
P2-e08 Neural excitation generates new astrocytes in the cerebral cortex of adult rats Yasuhisa Tamura 1 , Guanghua Jin 1 , Yilong Cui 1 , Yasuyoshi Watanabe 2 , Yosky Kataoka 1 1
Cellular Function Imaging Lab., RIKEN CMIS, Kobe, Japan; Probe Dynamics Lab., RIKEN CMIS, Kobe, Japan
2
Molecular
Spreading depression (SD) is characterized by slow propagation of neuronal/glial membrane depolarization throughout the unilateral cerebral cortex, and is involved
S157
in several neurological disorders, including migraine aura. We previously reported that many newlygenerated cells appeared in the cortex following SD stimulus even in adult rats. Almost all the cells were immunopositive for NG2, a marker of glial progenitor cells, at 3 days after SD generation. However, fate of the cells following such a neural excitation has remained unclear. In the present study, we observed that such newlygenerated cells differentiated into GFAP-immunopositive astrocytes, although almost all the NG2-immunopositive cells differentiated into mature oligodendrocytes in the absence of such a neural excitation. These findings suggest that neural activity regulates gliogenesis even in the adult brain. doi:10.1016/j.neures.2009.09.804
P2-e09 Roles of mammalian Fringe in the developing cerebral cortex Tomoaki Kato 1,2 , Ayano Kawaguchi 1 , Yoichi Kosodo 1 , Fumio Matsuzaki 1 1
Lab for Cell Asymmetry, CDB RIKEN, Japan; ative Med, Kobe Univ Grad Sch Med, Japan
2
Dept Develop and Regener-
Notch signal has pivotal roles in cell fate determination during organogenesis. Fringe (Fng) was originally identified as a Notch signal modulator in Drosophila, which potentiates Notch activation by Delta and suppresses it by Serrate. In mammals, there are three Fng genes (L-, M-, and R-Fng) and they are expressed in differential patterns in the developing cerebral cortex. Because mammalian Fngs also show distinct responses to different ligands in vitro, effects of Fngs on Notch activation may depend on tissue-environments. Previous studies indicate that L-fng suppresses Notch signaling in somitogenesis as M-fng does in pancreas development. We investigated roles of Fngs in the mouse developing cortex. All Fng genes, when overexpressed in the ventricular zone, augment Notch signaling, and increase the progenitor cell population. On the contrary, knockdown of L-fng, the major isoform in the ventricular zone, suppresses Notch signaling and promotes neurogenesis. Our results thus indicate that Fng cell-autonomously potentiates Notch signaling in neural progenitor cells. doi:10.1016/j.neures.2009.09.805
P2-e10 Temporal influence of neural activity on thalamocortical axon branching Akiko Hayashi, Yasufumi Hayano, Nobuhiko Yamamoto Graduate Sch of Frontier Biosci, Osaka Univ, Suita, Japan Neural activity is known to modify axonal branching. To study the temporal influence of neural activity on branch formation, firing activity and axon morphology were observed simultaneously by a time-lapse study in cocultures of the thalamus and cortex. We found that branch addition of thalamocortical axons was highly correlated with firing frequency on the preceding day. Moreover, the activity blockade suppressed branch addition with the predictable time delay. On the other hand, branches were retracted depending on branch addition on the previous day rather than electrical activity. These results suggest that neural activity acts as a promoter for axon branching and is converted into the morphological change with a long latency. doi:10.1016/j.neures.2009.09.806
P2-e11 Aberrant synapse formation of the phrenic nerves in DINEdeficient mice Kenichi Nagata 1 , Sumiko Kiryu-Seo 1 , Kayo Yoshida 2 , Takashi Morita 2 , Hiroshi Kiyama 1 1 Department of Anat. and Neurobiol., Grad. Sch. Med., Osaka City University, Osaka, Japan; 2 Department of Mol. Genetics, Grad. Sch. Med., Osaka City University, Osaka, Japan
Damage-induced neuronal endopeptidase (DINE) is a metalloprotease which belongs to the neprilysin (NEP) family, and remarkably induced in response to various types of neuronal injuries. DINE mRNA expression is strikingly restricted to neurons from early developmental stage, suggesting important role in developing nervous system. To clarify the role of DINE, we generated DINE-deficient (DINE −/−) mice. These mice died of respiratory failure shortly after birth. Considering neuron-specific expression of DINE, we found on neurological phenotype of the mice. Detailed histochemical analysis showed that muscle innervation of their phrenic nerves, which is responsible for respiratory control, is dramatically reduced. Furthermore, DINE −/− embryos had little or no neuromuscular junction in the diaphragm. However, the number of phrenic motor neuron in spinal cord is normal.
P2-e06 Introducing neurocristopathy-associated PHOX2B mutations in mouse reveals conserved pathogenic effects of mutant PHOX2B on mammalian neural crest cell development Mayumi Nagashimada 1,2 , Hideki Enomoto 1 1
NDR, Riken, CDB, Kobe, Japan;
2
Univ of Osaka, Osaka, Japan
Phox2b is essential for the development of the autonomic nervous system (ANS). Heterozygous mutations of the PHOX2B gene, 931del5 and 693del8, have been identified in a neurocristopathy syndrome where Congenital Central Hypoventilation Syndrome (CCHS), Hirschsprung’s disease (intestinal aganglionosis) and multifocal neuroblastoma are found in the same patients. To understand how these PHOX2B mutations influence ANS development, we introduced those human mutations into the phox2b locus in mice (Phox2bdel5/+ and Phox2bdel8/+ mice, respectively) by gene targeting. Heterozygous mice of both mutants died soon after birth due to impaired spontaneous breathing, and the phenotype was reminiscent of CCHS. Moreover, both mutants displayed abnormal development of the enteric nervous system and sympathetic ganglia. Thus mutant PHOX2B affect normal development of an identical set of neuronal populations between human and mice, suggesting the conserved pathogenic effects of mutant PHOX2B in mammals. doi:10.1016/j.neures.2009.09.802
P2-e07 Characterization of temporal identity of neuronal progenitor cells in the neocortex Ken-ichi Mizutani, Carina Hanashima RIKEN CDB, Japan Developmentally, neocortical excitatory neurons are generated from common progenitors within the dorsal telencephalon, which are thought to produce distinct layer neurons in an inside-out manner. We have utilized Ngn2, a bHLH proneural, Cre-recombinase mouse line to fate-map transiently Ngn2 expressing cells. It has been shown that Ngn2 expression peaks in progenitors that are committed to leave the cell cycle and thus is an ideal candidate to label the cortical lineage, as well as temporal cohorts of differentiating neurons. We crossed the Ngn2-CreER line with ROSA-stop-YFP mice with tamoxifen between E11.5 and E16.5, and confirmed that we could specifically target distinct layer neurons upon tamoxifen administration. Also, we characterized the molecular specificity of isolating distinct cortical progenitors after cell sorting. Our Ngn2 temporal fate-mapping analysis will provide the molecular information by which each neuronal fate is fundamentally distinct from other excitatory neurons. doi:10.1016/j.neures.2009.09.803
P2-e08 Neural excitation generates new astrocytes in the cerebral cortex of adult rats Yasuhisa Tamura 1 , Guanghua Jin 1 , Yilong Cui 1 , Yasuyoshi Watanabe 2 , Yosky Kataoka 1 1
Cellular Function Imaging Lab., RIKEN CMIS, Kobe, Japan; Probe Dynamics Lab., RIKEN CMIS, Kobe, Japan
2
Molecular
Spreading depression (SD) is characterized by slow propagation of neuronal/glial membrane depolarization throughout the unilateral cerebral cortex, and is involved
S157
in several neurological disorders, including migraine aura. We previously reported that many newlygenerated cells appeared in the cortex following SD stimulus even in adult rats. Almost all the cells were immunopositive for NG2, a marker of glial progenitor cells, at 3 days after SD generation. However, fate of the cells following such a neural excitation has remained unclear. In the present study, we observed that such newlygenerated cells differentiated into GFAP-immunopositive astrocytes, although almost all the NG2-immunopositive cells differentiated into mature oligodendrocytes in the absence of such a neural excitation. These findings suggest that neural activity regulates gliogenesis even in the adult brain. doi:10.1016/j.neures.2009.09.804
P2-e09 Roles of mammalian Fringe in the developing cerebral cortex Tomoaki Kato 1,2 , Ayano Kawaguchi 1 , Yoichi Kosodo 1 , Fumio Matsuzaki 1 1
Lab for Cell Asymmetry, CDB RIKEN, Japan; ative Med, Kobe Univ Grad Sch Med, Japan
2
Dept Develop and Regener-
Notch signal has pivotal roles in cell fate determination during organogenesis. Fringe (Fng) was originally identified as a Notch signal modulator in Drosophila, which potentiates Notch activation by Delta and suppresses it by Serrate. In mammals, there are three Fng genes (L-, M-, and R-Fng) and they are expressed in differential patterns in the developing cerebral cortex. Because mammalian Fngs also show distinct responses to different ligands in vitro, effects of Fngs on Notch activation may depend on tissue-environments. Previous studies indicate that L-fng suppresses Notch signaling in somitogenesis as M-fng does in pancreas development. We investigated roles of Fngs in the mouse developing cortex. All Fng genes, when overexpressed in the ventricular zone, augment Notch signaling, and increase the progenitor cell population. On the contrary, knockdown of L-fng, the major isoform in the ventricular zone, suppresses Notch signaling and promotes neurogenesis. Our results thus indicate that Fng cell-autonomously potentiates Notch signaling in neural progenitor cells. doi:10.1016/j.neures.2009.09.805
P2-e10 Temporal influence of neural activity on thalamocortical axon branching Akiko Hayashi, Yasufumi Hayano, Nobuhiko Yamamoto Graduate Sch of Frontier Biosci, Osaka Univ, Suita, Japan Neural activity is known to modify axonal branching. To study the temporal influence of neural activity on branch formation, firing activity and axon morphology were observed simultaneously by a time-lapse study in cocultures of the thalamus and cortex. We found that branch addition of thalamocortical axons was highly correlated with firing frequency on the preceding day. Moreover, the activity blockade suppressed branch addition with the predictable time delay. On the other hand, branches were retracted depending on branch addition on the previous day rather than electrical activity. These results suggest that neural activity acts as a promoter for axon branching and is converted into the morphological change with a long latency. doi:10.1016/j.neures.2009.09.806
P2-e11 Aberrant synapse formation of the phrenic nerves in DINEdeficient mice Kenichi Nagata 1 , Sumiko Kiryu-Seo 1 , Kayo Yoshida 2 , Takashi Morita 2 , Hiroshi Kiyama 1 1 Department of Anat. and Neurobiol., Grad. Sch. Med., Osaka City University, Osaka, Japan; 2 Department of Mol. Genetics, Grad. Sch. Med., Osaka City University, Osaka, Japan
Damage-induced neuronal endopeptidase (DINE) is a metalloprotease which belongs to the neprilysin (NEP) family, and remarkably induced in response to various types of neuronal injuries. DINE mRNA expression is strikingly restricted to neurons from early developmental stage, suggesting important role in developing nervous system. To clarify the role of DINE, we generated DINE-deficient (DINE −/−) mice. These mice died of respiratory failure shortly after birth. Considering neuron-specific expression of DINE, we found on neurological phenotype of the mice. Detailed histochemical analysis showed that muscle innervation of their phrenic nerves, which is responsible for respiratory control, is dramatically reduced. Furthermore, DINE −/− embryos had little or no neuromuscular junction in the diaphragm. However, the number of phrenic motor neuron in spinal cord is normal.