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The Evi1 proto-oncogene maintains the self replicative cell cycle in olfactory neural precursors
Abstracts / Neuroscience Research 71S (2011) e108–e415
tors in the ventricular zone throughout the stages examined. The signals for either GABA or Taurine were also observed with specific laminar patterns in the cortical primordium at each stage. Exposure to either Phenobarbital or Pentobarbital, both of which are GABAA agonists, on E10–11 increased the frequency of Tbr2-positive BPs at E12, which resulted in the increase in the thickness of layers of Doublecortin-positive postmitotic neurons. Exposure to a GABAA antagonist, either Picrotoxin or Pentylenetetrazole, on E10–13 decreased the frequency of BPs and the thickness of Doublecortin-positive layers at E14. These results suggest that GABAA receptors are involved in the regulation of neurogenesis by promoting the transition from APs to BPs. Research fund: KAKENHI #21791035 from the Japan Society for the Promotion of Science. doi:10.1016/j.neures.2011.07.532
P2-d15 Roles of the epithelial structure in the neural progenitor self-renewal in the mammalian developing neocortex Atsunori Shitamukai , Daijiro Konno, Fumio Matsuzaki Lab. for Cell Asymmetry, RIKEN CDB, Kobe, Japan Radial glia cells function as neural stem cells in the developing brain and generate self-renewing and differentiating daughter cells by asymmetric cell divisions. During these cell division, apical process or basal process of the elongated epithelial structure is asymmetrically partitioned into daughter cells, depending on developmental contexts. However, in mammalian neurogenesis, the relationship between these subcellular structures and cell fate decision is largely unknown. We addressed this issue by perturbing the mitotic spindle orientation of progenitors, enabling us to follow the fate of daughters exclusively inheriting the apical or basal process of the dividing progenitor in slice culture. We observed that the more basal daughter cell that inherits the basal process self-renews outside of the ventricular zone (VZ), while the more apical daughter cell differentiates. These self-renewing progenitors, termed outer VZ progenitors (OVZ), retain the basal but not the apical process, as recently reported for the outer subventricular zone (OSVZ) progenitors in primates and ferret. Interestingly, the Delta-Notch signaling between sibling daughter cells is important for their self-renewal. We also found a small endogenous population of OVZ progenitors in the mouse embryonic neocortex, consistent with a low frequency of oblique radial glia divisions. Our results describe the general role of the basal process in the self-renewal of neural progenitors and implicate the loss of the apical junctions during oblique divisions as a possible mechanism for generating OSVZ progenitors. We hypothesize that basal process mediates feedback signals from the basal side of the brain to promote progenitor self-renewal. To test this, we are screening for the candidate basal signals that are involved in the self-renewal of neural progenitors. Our preliminary screen suggests that a FGF signal is one of candidate signals from the basal side. doi:10.1016/j.neures.2011.07.533
P2-d16 Generation and characterization of human ES cells carrying Sox1-reporter gene for neural differentiation Kenji Yoshida 1 , Shin-ichi Ota 1 , Chikako Hara 2 , Yohei Okada 1,2 , Hideyuki Okano 1 1 2
Department of Physiology, Keio University, School of Medicine, Tokyo, Japan Kanrinmaru Project, Keio University, School of Medicine, Tokyo, Japan
Embryonic stem (ES) cells are powerful tools to investigate central nervous system development and to understand its underlying molecular mechanisms. Recently, several neural differentiation methods of human ES cells have been developed including our own neurosphere method. Therefore, to identify key molecules of human neural development, simple method to precisely detect neural differentiation from human ES cells has been required. For the establishment of high throughput screening system, here, we report generation of human ES cells carrying reporter gene for neural differentiation. SOX1 gene, well known transcription factor specifically expressed in neural progenitor cells, was utilized as a marker gene, and a gene encoding a fluorescent protein-fused luciferase (ffLuc), was utilized as a reporter gene to visualize neural progenitors. The ffLuc reporter gene was subcloned into bacterial artificial chromosome (BAC) vector containing more than hundreds Kb of regulatory region of SOX1 gene. This BAC reporter construct was introduced into KhES1 by electroporation and selected by antibiotics and genomic PCR. These clones retain growth capacity equivalent to nontransgenic KhES1 (WT-KhES1) in undifferentiated state. Moreover, neural differentiation, including embryoid body and neurosphere formation, and differentiation of neurospheres into postmitotic neurons were observed
e125
similar to those in WT-KhES1. The expression of reporter gene in each differentiation stage was evaluated by luciferase assay. As expected, embryoid bodies and neurospheres exhibited high luciferase activity on their neural differentiation, whereas undifferentiated ES cells did not. These results indicated that transgenes were appropriately inserted into chromosome and this reporter system properly responds to the intracellular signaling cascade of neural differentiation. This transgenic human ES cells are widely applicable to the screening of varieties of genes, small molecules, and so on. Research fund: Keio Kanrinmaru-Project. doi:10.1016/j.neures.2011.07.534
P2-d17 The Evi1 proto-oncogene maintains the self replicative cell cycle in olfactory neural precursors Elaine K.Y. Chung , Tobias Hohenauer, Hiroaki Taniguchi, Li Foong Yoong, Emi Kinameri, Adrian W. Moore Brain Science Institute, RIKEN, Wako, Japan Development of cell diversity in the nervous system involves precisely regulated events coordinating the proliferation and differentiation of neural progenitor cells. Recently we described members of the evolutionarily conserved Prdm (PRDI-BF1 and RIZ homology domain containing) protooncogene transcription factor as new candidates to control neurogenesis. Evi1 (Prdm3), a founder member of this family, was originally identified as an oncogene causing myeloid leukemia in humans and mice. Evi1 is expressed in bone marrow and plays a crucial role in the maintenance and proliferation of embryonic and adult haemopoietic stem cells. We show that Evi1 is expressed in several domains of neural precursors during embryonic neurogenesis, and we specifically investigate Evi1 function in the development of olfactory neurons. Development of the olfactory neurons from the olfactory neuro-epithelium (OE), the lineage leading from stem cell to neuron is relatively simple and well characterized. Previous studies have shown that the activity of the Notch–Hes pathway is essential to maintain olfactory stem cell self replication. Here we show that Evi1 is expressed in the Notch2/SOX2 positive apical precursor (AP) stem cell population; furthermore in Evi1−/− animals, similar to Notch pathway mutants, AP cells exit the proliferative self replication cycle early leading to an increase in intermediate precursor (transit amplifying cells) positive for the proneural gene Neurogenin 1 (Ngn1). This premature differentiation results in a smaller size and a disrupted cellular organization of the OE. Our findings suggest that Evi1 may control cell cycle progression and regulate the decision of progenitors to exit the proliferative stem cell cycle, and we investigate the interactions between Evi1 action and Notch activity. doi:10.1016/j.neures.2011.07.535
P2-d18 The rule of primary cilia in adult neurogenesis Natsuko Kumamoto 1,2 , Yan Gu 2 , Jia 2 3 Janoschka , Ken-ichi Takemaru , Joel Tohyama 1 , Ge Shaoyu 2
Wang 2 , Stephen Levine 2 , Masaya
1
Dept. of Anat. & Neurosci, Grad. Sch. of Med., Osaka Univ., Suita, Japan 2 Dept. of Neurobiol. & Behav., SUNY at Stony Brook, Stony Brook, NY, USA 3 Dept. of Pharmacol. Science, SUNY at Stony Brook, Stony Brook, NY, USA
The sequential integration of newborn neurons into adult neural circuits has been widely examined in the past few years, yet the mechanisms regulating the precise integration remain largely unknown. Primary cilium, a microtubule-based organelle mediating a variety of signaling, has been recently found to play essential roles in the vertebrate development. To decipher the regulatory mechanisms of sequential integration of adult-born neurons, we examined assembly and function of the primary cilium in neurons born in the adult hippocampus. Strikingly, primary cilia are absent in early developmental stages and assemble right at the stage when adult-born neurons approach their final destination, extend full dendrites and robustly form entorhinal cortex projecting synapses. Specific and conditional depletion of primary cilia in these neurons induced severe dendritic elongation and synapse formation defects. Mechanistically, we found that the timely assembly of primary cilia arrests Wnt/-catenin signaling to regulate dendritic development of newborn neurons. In summary, our study identified primary cilium assembly as a critical regulatory event in the development and synapse integration of adult-born neurons. Furthermore, these results may provide insight into the cause of cilia-associated brain diseases. doi:10.1016/j.neures.2011.07.536
tors in the ventricular zone throughout the stages examined. The signals for either GABA or Taurine were also observed with specific laminar patterns in the cortical primordium at each stage. Exposure to either Phenobarbital or Pentobarbital, both of which are GABAA agonists, on E10–11 increased the frequency of Tbr2-positive BPs at E12, which resulted in the increase in the thickness of layers of Doublecortin-positive postmitotic neurons. Exposure to a GABAA antagonist, either Picrotoxin or Pentylenetetrazole, on E10–13 decreased the frequency of BPs and the thickness of Doublecortin-positive layers at E14. These results suggest that GABAA receptors are involved in the regulation of neurogenesis by promoting the transition from APs to BPs. Research fund: KAKENHI #21791035 from the Japan Society for the Promotion of Science. doi:10.1016/j.neures.2011.07.532
P2-d15 Roles of the epithelial structure in the neural progenitor self-renewal in the mammalian developing neocortex Atsunori Shitamukai , Daijiro Konno, Fumio Matsuzaki Lab. for Cell Asymmetry, RIKEN CDB, Kobe, Japan Radial glia cells function as neural stem cells in the developing brain and generate self-renewing and differentiating daughter cells by asymmetric cell divisions. During these cell division, apical process or basal process of the elongated epithelial structure is asymmetrically partitioned into daughter cells, depending on developmental contexts. However, in mammalian neurogenesis, the relationship between these subcellular structures and cell fate decision is largely unknown. We addressed this issue by perturbing the mitotic spindle orientation of progenitors, enabling us to follow the fate of daughters exclusively inheriting the apical or basal process of the dividing progenitor in slice culture. We observed that the more basal daughter cell that inherits the basal process self-renews outside of the ventricular zone (VZ), while the more apical daughter cell differentiates. These self-renewing progenitors, termed outer VZ progenitors (OVZ), retain the basal but not the apical process, as recently reported for the outer subventricular zone (OSVZ) progenitors in primates and ferret. Interestingly, the Delta-Notch signaling between sibling daughter cells is important for their self-renewal. We also found a small endogenous population of OVZ progenitors in the mouse embryonic neocortex, consistent with a low frequency of oblique radial glia divisions. Our results describe the general role of the basal process in the self-renewal of neural progenitors and implicate the loss of the apical junctions during oblique divisions as a possible mechanism for generating OSVZ progenitors. We hypothesize that basal process mediates feedback signals from the basal side of the brain to promote progenitor self-renewal. To test this, we are screening for the candidate basal signals that are involved in the self-renewal of neural progenitors. Our preliminary screen suggests that a FGF signal is one of candidate signals from the basal side. doi:10.1016/j.neures.2011.07.533
P2-d16 Generation and characterization of human ES cells carrying Sox1-reporter gene for neural differentiation Kenji Yoshida 1 , Shin-ichi Ota 1 , Chikako Hara 2 , Yohei Okada 1,2 , Hideyuki Okano 1 1 2
Department of Physiology, Keio University, School of Medicine, Tokyo, Japan Kanrinmaru Project, Keio University, School of Medicine, Tokyo, Japan
Embryonic stem (ES) cells are powerful tools to investigate central nervous system development and to understand its underlying molecular mechanisms. Recently, several neural differentiation methods of human ES cells have been developed including our own neurosphere method. Therefore, to identify key molecules of human neural development, simple method to precisely detect neural differentiation from human ES cells has been required. For the establishment of high throughput screening system, here, we report generation of human ES cells carrying reporter gene for neural differentiation. SOX1 gene, well known transcription factor specifically expressed in neural progenitor cells, was utilized as a marker gene, and a gene encoding a fluorescent protein-fused luciferase (ffLuc), was utilized as a reporter gene to visualize neural progenitors. The ffLuc reporter gene was subcloned into bacterial artificial chromosome (BAC) vector containing more than hundreds Kb of regulatory region of SOX1 gene. This BAC reporter construct was introduced into KhES1 by electroporation and selected by antibiotics and genomic PCR. These clones retain growth capacity equivalent to nontransgenic KhES1 (WT-KhES1) in undifferentiated state. Moreover, neural differentiation, including embryoid body and neurosphere formation, and differentiation of neurospheres into postmitotic neurons were observed
e125
similar to those in WT-KhES1. The expression of reporter gene in each differentiation stage was evaluated by luciferase assay. As expected, embryoid bodies and neurospheres exhibited high luciferase activity on their neural differentiation, whereas undifferentiated ES cells did not. These results indicated that transgenes were appropriately inserted into chromosome and this reporter system properly responds to the intracellular signaling cascade of neural differentiation. This transgenic human ES cells are widely applicable to the screening of varieties of genes, small molecules, and so on. Research fund: Keio Kanrinmaru-Project. doi:10.1016/j.neures.2011.07.534
P2-d17 The Evi1 proto-oncogene maintains the self replicative cell cycle in olfactory neural precursors Elaine K.Y. Chung , Tobias Hohenauer, Hiroaki Taniguchi, Li Foong Yoong, Emi Kinameri, Adrian W. Moore Brain Science Institute, RIKEN, Wako, Japan Development of cell diversity in the nervous system involves precisely regulated events coordinating the proliferation and differentiation of neural progenitor cells. Recently we described members of the evolutionarily conserved Prdm (PRDI-BF1 and RIZ homology domain containing) protooncogene transcription factor as new candidates to control neurogenesis. Evi1 (Prdm3), a founder member of this family, was originally identified as an oncogene causing myeloid leukemia in humans and mice. Evi1 is expressed in bone marrow and plays a crucial role in the maintenance and proliferation of embryonic and adult haemopoietic stem cells. We show that Evi1 is expressed in several domains of neural precursors during embryonic neurogenesis, and we specifically investigate Evi1 function in the development of olfactory neurons. Development of the olfactory neurons from the olfactory neuro-epithelium (OE), the lineage leading from stem cell to neuron is relatively simple and well characterized. Previous studies have shown that the activity of the Notch–Hes pathway is essential to maintain olfactory stem cell self replication. Here we show that Evi1 is expressed in the Notch2/SOX2 positive apical precursor (AP) stem cell population; furthermore in Evi1−/− animals, similar to Notch pathway mutants, AP cells exit the proliferative self replication cycle early leading to an increase in intermediate precursor (transit amplifying cells) positive for the proneural gene Neurogenin 1 (Ngn1). This premature differentiation results in a smaller size and a disrupted cellular organization of the OE. Our findings suggest that Evi1 may control cell cycle progression and regulate the decision of progenitors to exit the proliferative stem cell cycle, and we investigate the interactions between Evi1 action and Notch activity. doi:10.1016/j.neures.2011.07.535
P2-d18 The rule of primary cilia in adult neurogenesis Natsuko Kumamoto 1,2 , Yan Gu 2 , Jia 2 3 Janoschka , Ken-ichi Takemaru , Joel Tohyama 1 , Ge Shaoyu 2
Wang 2 , Stephen Levine 2 , Masaya
1
Dept. of Anat. & Neurosci, Grad. Sch. of Med., Osaka Univ., Suita, Japan 2 Dept. of Neurobiol. & Behav., SUNY at Stony Brook, Stony Brook, NY, USA 3 Dept. of Pharmacol. Science, SUNY at Stony Brook, Stony Brook, NY, USA
The sequential integration of newborn neurons into adult neural circuits has been widely examined in the past few years, yet the mechanisms regulating the precise integration remain largely unknown. Primary cilium, a microtubule-based organelle mediating a variety of signaling, has been recently found to play essential roles in the vertebrate development. To decipher the regulatory mechanisms of sequential integration of adult-born neurons, we examined assembly and function of the primary cilium in neurons born in the adult hippocampus. Strikingly, primary cilia are absent in early developmental stages and assemble right at the stage when adult-born neurons approach their final destination, extend full dendrites and robustly form entorhinal cortex projecting synapses. Specific and conditional depletion of primary cilia in these neurons induced severe dendritic elongation and synapse formation defects. Mechanistically, we found that the timely assembly of primary cilia arrests Wnt/-catenin signaling to regulate dendritic development of newborn neurons. In summary, our study identified primary cilium assembly as a critical regulatory event in the development and synapse integration of adult-born neurons. Furthermore, these results may provide insight into the cause of cilia-associated brain diseases. doi:10.1016/j.neures.2011.07.536