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Ngn2 acts upstream of Tbr2 for neuron supply in the developing neocortex
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Abstracts / Neuroscience Research 58S (2007) S1–S244
P2-c42 Meis2 activities in the control of three distinct developmental steps during the corticogenesis Yasuto Tanabe 1,2 1 Laboratory of Neuroscience, Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan; 2 MITILS, Tokyo, Japan
P2-c48 Ngn2 acts upstream of Tbr2 for neuron supply in the developing neocortex Wataru Ochiai 1 , Taishi Takahara 1 , Takaki Miyata 1,2 Department of Anatomy & Cell Biology, Nagoya University, Aichi, Japan; 2 CREST, JST, Japan
1
Previous studies on the cerebral cortical development suggested that there are at least five distinct developmental programs operated for the control of cortical projection neuron generation: neuronal differentiation, radial migration, cell cycle regulation, laminar and regional specifications. Meis2 is a homeodomain-containing transcription factor among TALE family proteins. Our analyses on the potential roles of Meis2 in the control of cortical projection neuron generation using electroporationmediated gene transfer approach showed that Meis2 activities are critical for the control of rate of progression from mitosis to differentiation in cortical progenitors, temporal switch between neuronal versus glial differentiation, and proper radial migration of cortical neurons in nearly the entire developmental period of corticogenesis. Our results therefore suggest that TALE family proteins play fundamental roles in the control of cerebral cortical development.
Loss- and gain-of-function studies have shown that Neurogenin 2 (Ngn2) is important for neocortical progenitor cells to supply pyramidal neurons Ngn2 promotes progenitor cells to move to the subventricular zone (SVZ) for non-surface mitosis. These non-surface dividing cells have been shown to express Tbr2, a T-domain transcription factor, although the relationship between Ngn2 and Tbr2 has never been directly examined. Coexpression of these two factors was detected in many cells in the upper ventricular zone (VZ) and SVZ. To determine as clearly as possible whether Ngn2 induces Tbr2 in VZ cells, we then introduced Ngn2 by electroporation into the VZ of the ganglionic eminence that normally lacks both Ngn2 and Tbr2. We found that the ectopically introduced Ngn2 induced Tbr2 expression and observed Ngn2-induced transactivation of Tbr2 promoter containing E-box sequences, bHLH transcription factor binding motif. These results suggest that Ngn2 works upstream of Tbr2 for the commitment of progenitor cells to the neuronal lineage.
Research fund: KAKENHI (17390086)
Research fund: Grant-in-Aid for Young Scientist(B) (18700340)
P2-c45 Region-specific degeneration of NG2+ cells in the cere-
1 Non-uniform expression of Rac1 on the ventricular P2-d0 surface of developing neocortex
bral cortex of aged rats Yasuhisa Tamura 1 , Yilong Cui 1 , Yasuyoshi Watanabe 1,2 , Yosky Kataoka 1,2 1 Molecular Probe Dynamics Laboratory, Molecular Imaging Research Program, RIKEN, Japan; 2 Department of Physiology, Osaka City University, Graduate School of Medicine, Japan It has been known that NG2+ cells are broadly present as progenitor cells in the adult mammalian brain. It is still unclear whether aging affects number and morphological feature of NG2+ cells. In the adult (8–10 weeks) rats, NG2+ cells were abundantly observed throughout the layers I–VI in the cerebral cortex and showed well-developed multiple radial processes. In the aged (96–100 weeks) rats, the number of NG2+ cells decreased and the cells showed the retracted processes in the parietal cortex. In the piriform cortex, however, such changes were not observed. These observations indicate region-specific degeneration of cortical NG2+ cells in aging.
P2-c47
Time-table for transcription factor expression in daughter cells generated at the ventricular surface of the developing neocortex
Takaki Miyata 1,2,3 , Wataru Ochiai 1 , Taishi Takahara 1 , Masaharu Ogawa 2 1 Department of Anatomy & Cell Biology, Nagoya University, Japan; 2 BSI, RIKEN, Japan; 3 CREST, JST, Japan Asymmetric cell divisions contribute to cellular diversification in the developing mammalian brain. Models propose that cell-fate determinants are segregated asymmetrically during mitosis of each progenitor cell at the ventricular surface and daughter cells pair-generated from the progenitor cell can already be distinct from each other at the beginning of their lives. But, such models have not been accompanied by cell-fate tracking at the single cell level. To specify timing of the expression of Neurogenin 2 in a daughter cell in relation to that of other transcription factors including Tbr2 immunostained daughter cells (n > 200) that were time-lapse monitored for various durations from their pair-generation by singly DiI-labeled progenitor cells (n > 100) in three-dimensional slice culture. Research funds: Japanese Ministry of Education, Science, Sports and Culture (16026249), Sumitomo Foundation, Takeda Science Foundation, Toyo-Aki Foundation
Sayaka Minobe 1,2 , Wataru Ochiai 2 , Takaki Miyata 2,3 Department of Anatomy, Mie University School of Medicine, Tsu, Japan; 2 Anatomy and Cell Biology, Nagoya University School of Medicine, Aichi, Japan; 3 JST, CREST, Saitama, Japan
1
Rac1 is known to be important for cortical neuronal migration. We now ask whether Rac1 also helps progenitor cells that play multiple histogenetic roles (i.e. cell production, interkinetic nuclear migration, and forming junctional meshwork). Anti-Rac1 immunostaining of coronal sections of developing cerebral walls showed line-like reactivity at the ventricular surface. Whole-mount anti-Rac1 and anti-␣E-catenin double stain followed by en face observation revealed that Rac1 reactivity is detected in a non-uniform pattern. Only about 20% of the ventricular endfeet were positive for Rac1. This was quite contrast with a diffuse surface staining pattern by anti-Cdc42. Similar non-uniform endfoot staining patterns were obtained when we used antibodies against GEFs for Rac1. Reactions to detect the activation of Rac1 (in situ Rac activity assay) further revealed positive signals in the Rac1-immunoreactive endfeet. These data suggest that this non-uniform Rac1 immunoreactivity is associated with physiological functions of Rac1 in progenitor cells.
2 Axonal regressive events in rat corticospinal slice P2-d0 coculture studied with time-lapse confocal imaging of single axons Noboru Yoshioka, Takae Ohno, Masaki Sakurai Department of Physiology, Teikyo University School of Medicine, Japan Corticospinal projections in the gray matter of the rat spinal cord has the phase of axonal regression during postnatal development. In corticospinal slice coculture, we labeled cortical neurons with pCAGGS-EYFP and made daily confocal imaging from 5 DIV to 12 DIV. We observed, (1) gradual shortening, (2) fragmentation of the axon terminals, (3) amputation at branching points followed by shrinkage of the distal parts, and (4) abrupt disappearance of the total length of axons within an observation field in a day. We then tried to capture the event of abrupt disappearance by making higher time-resolution time-lapse imaging of 30–60 min interval for more than 8 h. This enabled us to detect single axons showing degenerative changes. The event started with formation of beaded structure over considerable length followed by rapid disappearance. The rapid most case showed elimination of 1 mm length within 2.5 h. This may explain the long branch elimination of corticospinal axons during early postnatal period. Research fund: KAKENHI 17023040 (M.S.)
Abstracts / Neuroscience Research 58S (2007) S1–S244
P2-c42 Meis2 activities in the control of three distinct developmental steps during the corticogenesis Yasuto Tanabe 1,2 1 Laboratory of Neuroscience, Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan; 2 MITILS, Tokyo, Japan
P2-c48 Ngn2 acts upstream of Tbr2 for neuron supply in the developing neocortex Wataru Ochiai 1 , Taishi Takahara 1 , Takaki Miyata 1,2 Department of Anatomy & Cell Biology, Nagoya University, Aichi, Japan; 2 CREST, JST, Japan
1
Previous studies on the cerebral cortical development suggested that there are at least five distinct developmental programs operated for the control of cortical projection neuron generation: neuronal differentiation, radial migration, cell cycle regulation, laminar and regional specifications. Meis2 is a homeodomain-containing transcription factor among TALE family proteins. Our analyses on the potential roles of Meis2 in the control of cortical projection neuron generation using electroporationmediated gene transfer approach showed that Meis2 activities are critical for the control of rate of progression from mitosis to differentiation in cortical progenitors, temporal switch between neuronal versus glial differentiation, and proper radial migration of cortical neurons in nearly the entire developmental period of corticogenesis. Our results therefore suggest that TALE family proteins play fundamental roles in the control of cerebral cortical development.
Loss- and gain-of-function studies have shown that Neurogenin 2 (Ngn2) is important for neocortical progenitor cells to supply pyramidal neurons Ngn2 promotes progenitor cells to move to the subventricular zone (SVZ) for non-surface mitosis. These non-surface dividing cells have been shown to express Tbr2, a T-domain transcription factor, although the relationship between Ngn2 and Tbr2 has never been directly examined. Coexpression of these two factors was detected in many cells in the upper ventricular zone (VZ) and SVZ. To determine as clearly as possible whether Ngn2 induces Tbr2 in VZ cells, we then introduced Ngn2 by electroporation into the VZ of the ganglionic eminence that normally lacks both Ngn2 and Tbr2. We found that the ectopically introduced Ngn2 induced Tbr2 expression and observed Ngn2-induced transactivation of Tbr2 promoter containing E-box sequences, bHLH transcription factor binding motif. These results suggest that Ngn2 works upstream of Tbr2 for the commitment of progenitor cells to the neuronal lineage.
Research fund: KAKENHI (17390086)
Research fund: Grant-in-Aid for Young Scientist(B) (18700340)
P2-c45 Region-specific degeneration of NG2+ cells in the cere-
1 Non-uniform expression of Rac1 on the ventricular P2-d0 surface of developing neocortex
bral cortex of aged rats Yasuhisa Tamura 1 , Yilong Cui 1 , Yasuyoshi Watanabe 1,2 , Yosky Kataoka 1,2 1 Molecular Probe Dynamics Laboratory, Molecular Imaging Research Program, RIKEN, Japan; 2 Department of Physiology, Osaka City University, Graduate School of Medicine, Japan It has been known that NG2+ cells are broadly present as progenitor cells in the adult mammalian brain. It is still unclear whether aging affects number and morphological feature of NG2+ cells. In the adult (8–10 weeks) rats, NG2+ cells were abundantly observed throughout the layers I–VI in the cerebral cortex and showed well-developed multiple radial processes. In the aged (96–100 weeks) rats, the number of NG2+ cells decreased and the cells showed the retracted processes in the parietal cortex. In the piriform cortex, however, such changes were not observed. These observations indicate region-specific degeneration of cortical NG2+ cells in aging.
P2-c47
Time-table for transcription factor expression in daughter cells generated at the ventricular surface of the developing neocortex
Takaki Miyata 1,2,3 , Wataru Ochiai 1 , Taishi Takahara 1 , Masaharu Ogawa 2 1 Department of Anatomy & Cell Biology, Nagoya University, Japan; 2 BSI, RIKEN, Japan; 3 CREST, JST, Japan Asymmetric cell divisions contribute to cellular diversification in the developing mammalian brain. Models propose that cell-fate determinants are segregated asymmetrically during mitosis of each progenitor cell at the ventricular surface and daughter cells pair-generated from the progenitor cell can already be distinct from each other at the beginning of their lives. But, such models have not been accompanied by cell-fate tracking at the single cell level. To specify timing of the expression of Neurogenin 2 in a daughter cell in relation to that of other transcription factors including Tbr2 immunostained daughter cells (n > 200) that were time-lapse monitored for various durations from their pair-generation by singly DiI-labeled progenitor cells (n > 100) in three-dimensional slice culture. Research funds: Japanese Ministry of Education, Science, Sports and Culture (16026249), Sumitomo Foundation, Takeda Science Foundation, Toyo-Aki Foundation
Sayaka Minobe 1,2 , Wataru Ochiai 2 , Takaki Miyata 2,3 Department of Anatomy, Mie University School of Medicine, Tsu, Japan; 2 Anatomy and Cell Biology, Nagoya University School of Medicine, Aichi, Japan; 3 JST, CREST, Saitama, Japan
1
Rac1 is known to be important for cortical neuronal migration. We now ask whether Rac1 also helps progenitor cells that play multiple histogenetic roles (i.e. cell production, interkinetic nuclear migration, and forming junctional meshwork). Anti-Rac1 immunostaining of coronal sections of developing cerebral walls showed line-like reactivity at the ventricular surface. Whole-mount anti-Rac1 and anti-␣E-catenin double stain followed by en face observation revealed that Rac1 reactivity is detected in a non-uniform pattern. Only about 20% of the ventricular endfeet were positive for Rac1. This was quite contrast with a diffuse surface staining pattern by anti-Cdc42. Similar non-uniform endfoot staining patterns were obtained when we used antibodies against GEFs for Rac1. Reactions to detect the activation of Rac1 (in situ Rac activity assay) further revealed positive signals in the Rac1-immunoreactive endfeet. These data suggest that this non-uniform Rac1 immunoreactivity is associated with physiological functions of Rac1 in progenitor cells.
2 Axonal regressive events in rat corticospinal slice P2-d0 coculture studied with time-lapse confocal imaging of single axons Noboru Yoshioka, Takae Ohno, Masaki Sakurai Department of Physiology, Teikyo University School of Medicine, Japan Corticospinal projections in the gray matter of the rat spinal cord has the phase of axonal regression during postnatal development. In corticospinal slice coculture, we labeled cortical neurons with pCAGGS-EYFP and made daily confocal imaging from 5 DIV to 12 DIV. We observed, (1) gradual shortening, (2) fragmentation of the axon terminals, (3) amputation at branching points followed by shrinkage of the distal parts, and (4) abrupt disappearance of the total length of axons within an observation field in a day. We then tried to capture the event of abrupt disappearance by making higher time-resolution time-lapse imaging of 30–60 min interval for more than 8 h. This enabled us to detect single axons showing degenerative changes. The event started with formation of beaded structure over considerable length followed by rapid disappearance. The rapid most case showed elimination of 1 mm length within 2.5 h. This may explain the long branch elimination of corticospinal axons during early postnatal period. Research fund: KAKENHI 17023040 (M.S.)