- Email: [email protected]
In vitro clonal analysis of rat cerebral cortical neurons expressing latexin, a subtype-specific molecular marker of glutamatergic neurons
Developmental Brain Research 132 (2001) 87–90 www.elsevier.com / locate / bres
Short communication
In vitro clonal analysis of rat cerebral cortical neurons expressing latexin, a subtype-specific molecular marker of glutamatergic neurons Keiko Takiguchi-Hayashi* Mitsubishi Kasei Institute of Life Sciences, Minamiooya 11, Machida-shi, Tokyo 194 -8511, Japan Accepted 28 August 2001
Abstract Latexin is expressed in a subset of glutamatergic projection neurons located in the lateral but not the dorsomedial neocortex in rat. In the present study, we performed clonal analysis of embryonic cortical neurons in vitro using latexin as a subtype-specific molecular marker of glutamatergic neurons to address whether certain precursors in early cortical anlage are already committed to a single molecular phenotype. Dissociated cells from lateral cortex at embryonic day 12 were labelled with a replication-deficient retroviral vector containing the bacterial lacZ gene, and cultured in a monolayer for 3 weeks. Double-immunofluorescence staining was used to simultaneously visualize latexin- and b-galactosidase-immunopositive neurons. Out of 572 b-galactosidase-immunopositive clones examined, 27 clones contained latexin-expressing neuron(s). Of these 27 clones, 25 clones that contained three or more neurons were mixed clones containing both latexin-immunopositive and -immunonegative neurons. These results suggest that committed precursors to producing solely latexin-expressing neurons are not exist in the early cerebral cortical anlage. 2001 Elsevier Science B.V. All rights reserved. Theme: Development and regeneration Topic: Cell lineage and determination Keywords: Cell lineage; Cell-type specification; Development; Cerebral cortex; Progenitor; Precursor; Latexin
The mammalian cerebral cortex is comprised of functionally distinct areas, having characteristic histological features and wiring patterns. There have been two complementary hypotheses for the mechanism of neocortical areal specification: the protomap model [16] and the protocortex model [14]. The protomap model postulates that cortical regionalization is primarily controlled by molecular determinants that are intrinsic to the proliferative zone of the neocortex [1,4,6,12,13]. The protocortex model postulates that neocortical regional specification is controlled largely by extrinsic influences, such as thalamocortical axons [15,18]. Most probably, cortical regional specification depends on interactions between intrinsic properties of cortical cells and extrinsic influences of subcortical structures [17]. Previously, we have shown that latexin-immunopositive glutamatergic neurons are localized in the lateral but not *Tel.: 181-42-724-6244; fax: 181-42-724-6312. E-mail address: [email protected] (K. TakiguchiHayashi).
dorsal cortical areas in the rat [1,3]. Using latexin as a molecular marker, we demonstrated that the composition of E13 dorsal and lateral cortical progenitors is distinct in terms of developmental potential [2]. It remains to be elucidated, however, when and how a portion of precursors or young neurons is specified to generate latexin-immunopositive neurons in the lateral cortex. It may be hypothesized that a population of committed progenitors for the latexin phenotype is generated early and localized predominantly in the lateral cortical analage. To address whether this actually occurs, we analyzed the clonal relationship among cerebral cortical neurons expressing latexin in vitro. Dissociated cells from E12 cerebral cortices of timedpregnant Wistar rats (SPF grade; Sankyo, Shizuoka) were prepared as described previously [1]. The replication-deficient BAG retroviral vector [20], which transduces the bacterial lacZ gene, was infected to the cortical precursors. Prior to plating, 2310 7 colony-forming units (cfu) / ml of viral supernatant was added to the suspension of dissociated cells at a final concentration of 2310 3 cfu / 3310 4
0165-3806 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0165-3806( 01 )00262-0
88
K. Takiguchi-Hayashi / Developmental Brain Research 132 (2001) 87 – 90
Fig. 1. Proportion of latexin-immunopositive neurons in the culture derived from the dorsal and lateral cortical anlage. Shaded column, dorsal cortical anlage (DCX); diagonal column, lateral cortical anlage (LCX). Each value represents the mean number (6S.E.M.) of latexin-immunopositive neurons per 10 3 microtubule-associated protein 2 (MAP2)-positive neurons. *, Significantly greater (P,0.001, Student’s t test) than dorsal cortical culture.
cells / well. The cells were plated at a density of 4–5310 4 cells / cm 2 and cultured described previously [1] in a medium supplemented with FGF2 (Sigma, 5 ng / ml). At 6 days in vitro, 2.5 mM cytosine arabinoside (Sigma) was added to the FGF2-nonsupplemented medium for 2 days to eliminate dividing non-neuronal cells. Without this treatment, the culture would become nonviable. The cultures were maintained for an additional 13–15 days in the FGF2-nonsupplemented medium, which was replaced twice a week. A mouse monoclonal antibody PC3.1 against latexin ([1], 13 mg / ml) and a rabbit polyclonal antibody against b-galactosidase (1:500, Cappel, West Chester, PA, USA) were used to simultaneously visualize latexin and b-galactosidase immunoreactivity. Individual b-galactosidaseimmunopositive clones were identified under a 103 objective, and then the number of cells per clone and the percentage of latexin-immunopositive cells were determined under a 203 objective. Neurons were identified based on characteristic phase-contrast morphologies (Figs. 2B, 3A and B), and were found to be MAP2-immunopositive (data not shown). We performed clonal analysis of cortical cells cultured in a medium supplemented with exogenous FGF2 which is known to facilitate mitotic activity. Though it has been reported that FGF2 affects certain aspects of neuronal differentiation [5,19], a substantial proportion of the lateral neurons (5.7960.42 per 10 3 neurons, n524) began to express latexin, while far fewer latexin-expressing neurons appeared in the cultures of dorsal cells (0.9860.16 per 10 3 neurons, n519) (Fig. 1). These results indicate that the region-specific capacity of E12 cerebral cortical cells to
differentiate to latexin-expressing neurons is maintained even under the culture condition with exogenous FGF2. To follow the fate of individual E12 lateral cortical precursors, cells were infected with the BAG retrovirus vector (Fig. 2A and B). A group of b-galactosidaseimmunopositive cells was operationally defined as a clone when the cells were located within a 5003500 mm square. The identified clones were separated from each other by more than 500 mm. Out of 572 clones analyzed, 501 clones were purely neuronal clones. Of the neuronal clones, 40% (197 clones) were single-cell clones, 20% (96 clones) were two-cell clones, and 40% (208 clones) were clones containing three or more neurons (Fig. 2C). Latexin-immunopositive neurons were observed in 27 out of 501 neuronal clones: two were two-cell clones, and 25 were three-or-more-cell clones. Notably, all of the 25 multicell clones, were mixed clones which contained both latexin-immunopositive and -immunonegative neurons (Figs. 3 and 4). These results indicate that none of the E12 cortical precursors were committed to produce solely latexin-expressing neurons in vitro. Neurons in the cerebral cortex are composed of two major types: excitatory (glutamatergic) projection neurons and inhibitory (GABAergic) interneurons. It is generally considered that precursors for the glutamatergic neurons generate various subtypes of neurons in both superficial and deep layers. However, the existence of separate sets of layer-restricted precursors for the superficial and deep cortical layers is also suggested [7–11]. Latexin-immunopositive neurons are glutamate-immunoreactive neurons [3] and are restricted to the deep layers of lateral cortical
Fig. 4. Summary of the number of latexin-expressing neuron(s) in clusters of clonally related neurons. Horizontal axis shows 27 individual clones containing latexin-immunopositive neuron(s), and vertical axis shows the number of cells per individual clone. Black column, the number of latexin-immunopositive neuron(s). White column, the number of latexin-immunonegative neuron(s).
K. Takiguchi-Hayashi / Developmental Brain Research 132 (2001) 87 – 90
89
Fig. 2. Size distribution of clones grown in dissociated cell culture at 22–24 days in vitro. (A and B) An example of cells assumed to be clonally related. Arrowheads in (A) and (B) indicate b-galactosidase-immunopositive cells. (B) Phase-contrast photomicrograph of the same field as (A). Bar5100 mm in (A) and (B). (C) Histogram of the clone size of neuronal clones.
areas [1,3]. It has been shown that the regional specification for these neurons occurs before their neurogenesis at the precursor level [1,2]. To explain this early regional specification, it may be attractive to hypothesize that some committed progenitors for a deep layer-restricted latexin phenotype are generated early in the ventricular zone of the lateral cortical anlage. We performed an in vitro clonal analysis of latexinexpressing neurons derived from the E12 lateral cortex.
Although single cell clones represent 40% of the neuronal clones (Fig. 2C), none expressed latexin. This is consistent with our previous observations that birthdate of cortical latexin-immunopositive neurons is around E15 [1]. Latexin-immunopositive neurons were always found together with latexin-immunonegative neurons within single multicell clones (Figs. 3 and 4). The result indicates that extremely few, if any, E12 cortical precursors are committed to produce a progeny composed of solely latexin-
Fig. 3. Clonal analysis of cortical precursors containing latexin-immunoreactive neurons at 22–24 days in vitro. b-Galactosidase and latexin are visualized by double-immunofluorescence staining with anti-b-galactosidase antiserum and anti-latexin antibody; (A,C,E) and (B,D,F) have the same microscopic field. (A,B) Phase-contrast photomicrographs of two clones, both of which contain only one latexin-expressing neuron. (C,D) Cells infected by the retrovirus or descended from an infected precursor are readily identified by the presence of b-galactosidase immunoreactivity. (E,F) Differentiated neurons exhibiting latexin-immunoreactivity distributed in the cytoplasm of cell bodies. Bar5100 mm in (A–F).
90
K. Takiguchi-Hayashi / Developmental Brain Research 132 (2001) 87 – 90
immunopositive neurons in vitro. Thus, it is highly unlikely that early regional specification of the latexin phenotype is a consequence of the region-specific production of committed progenitors by E12. As the next step, an in vivo study is necessary to address whether radial clone precursors actually give rise to latexin-immunopositive neurons or not.
Acknowledgements The author thanks Dr. Yasuyoshi Arimatsu (Mitsubishi Kasei Inst Life Sci, Japan) for his advice and encouragement during the course of this work. The author also thanks Drs. Christopher Walsh (Harvard Inst. Med., USA) and Constance L. Cepko (Harvard Med Sch / HHMI, USA) for providing a BAG library.
References [1] Y. Arimatsu, M. Miyamoto, I. Nihonmatsu, K. Hirata, Y. Uratani, Y. Hatanaka, K. Takiguchi-Hayashi, Early regional specification for a molecular neuronal phenotype in the rat neocortex, Proc. Natl. Acad. Sci. USA 89 (1992) 8879–8883. [2] Y. Arimatsu, M. Ishida, K. Takiguchi-Hayashi, Y. Uratani, Cerebral cortical specification by early potential restriction of progenitor cells and later phenotype control of postmitotic neurons, Development 126 (1999) 629–638. [3] Y. Arimatsu, M. Ishida, M. Sato, M. Kojima, Corticocortical association neurons expressing latexin: specific cortical connectivity formed in vivo and in vitro, Cereb. Cortex 9 (1999) 569–576. [4] M.F. Barbe, P. Levitt, The early commitment of fetal neurons to the limbic cortex, J. Neurosci. 11 (1991) 519–533. [5] J.F.R. Cavanagh, M.C. Mione, I.S. Pappas, J.G. Parnavelas, Basic fibroblast growth factor prolongs the proliferation of rat cortical progenitor cells in vitro without altering their cell cycle parameters, Cereb. Cortex 7 (1997) 293–302. [6] M. Cohen-Tannoudji, C. Babinet, M. Wassef, Early determination of a mouse somatosensory cortex marker, Nature 31 (1994) 460–463.
[7] J.E. Crandall, K. Herrup, Patterns of cell lineage in the cerebral cortex reveal evidence for developmental boundaries, Exp. Neurol. 109 (1990) 131–139. [8] G. Fishell, J. Rossant, D. Van der Kooy, Neuronal lineages in chimeric mouse forebrain are segregated between compartments and in the rostrocaudal and radial planes, Develop. Biol. 141 (1990) 70–83. [9] L.A. Krushel, J.G. Johnston, G. Fishell, R. Tibshirani, D. Van der Kooy, Spatially localized neuronal cell lineages in the developing mammalian forebrain, Neuroscience 53 (1993) 1035–1047. [10] C.Y. Kuan, E. Elliott, R.A. Flavell, P. Rakic, Restrictive clonal allocation in the chimeric mouse brain, Proc. Natl. Acad. Sci. USA 94 (1997) 3374–3379. [11] M.C. Mione, J.F.R. Cavanagh, B. Harris, J.G. Parnavelas, Cell fate specification and symmetrical / asymmetrical divisions in the developing cerebral cortex, J. Neurosci. 17 (1997) 2018–2029. [12] E.M. Miyashita-Lin, R. Hevner, K.M. Wassarman, S. Martinez, J.L.R. Rubenstein, Early neocortical regionalization in the absence of thalamic innervation, Science 285 (1999) 906–909. [13] Y. Nakagawa, J.E. Johnson, D.D.M. O’Leary, Graded and areal expression patterns of regulatory genes and cadherins in embryonic neocortex independent of thalamocortical input, J. Neurosci. 19 (1999) 10877–10885. [14] D.D.M. O’Leary, Do cortical areas emerge from a protocortex?, Trends Neurosci. 12 (1989) 400–406. [15] D.D.M. O’Leary, B.B. Stanfield, Selective elimination of axons extended by developing cortical neurons is dependent on regional locale: experiments utilizing fetal cortical transplants, J. Neurosci. 9 (1989) 2230–2246. [16] P. Rakic, Specification of cerebral cortical areas, Science 241 (1988) 170–176. [17] J.L.R. Rubenstein, P. Rakic, Genetic control of cortical development, Cereb. Cortex 9 (1999) 521–523. [18] B.L. Schlagger, D.D.M. O’Leary, Potential of visual cortex to develop an array of functional units unique to somatosensory cortex, Science 252 (1991) 1556–1560. [19] F.M. Vaccarino, M.L. Schwartz, D. Hartigan, J.F. Leckman, Basic fibroblast growth factor increases the number of excitatory neurons containing glutamate in the cerebral cortex, Cereb. Cortex 1 (1995) 64–78. [20] C. Walsh, C.L. Cepko, Widespread dispersion of neuronal clones across functional regions of the cerebral cortex, Science 255 (1992) 434–440.
Short communication
In vitro clonal analysis of rat cerebral cortical neurons expressing latexin, a subtype-specific molecular marker of glutamatergic neurons Keiko Takiguchi-Hayashi* Mitsubishi Kasei Institute of Life Sciences, Minamiooya 11, Machida-shi, Tokyo 194 -8511, Japan Accepted 28 August 2001
Abstract Latexin is expressed in a subset of glutamatergic projection neurons located in the lateral but not the dorsomedial neocortex in rat. In the present study, we performed clonal analysis of embryonic cortical neurons in vitro using latexin as a subtype-specific molecular marker of glutamatergic neurons to address whether certain precursors in early cortical anlage are already committed to a single molecular phenotype. Dissociated cells from lateral cortex at embryonic day 12 were labelled with a replication-deficient retroviral vector containing the bacterial lacZ gene, and cultured in a monolayer for 3 weeks. Double-immunofluorescence staining was used to simultaneously visualize latexin- and b-galactosidase-immunopositive neurons. Out of 572 b-galactosidase-immunopositive clones examined, 27 clones contained latexin-expressing neuron(s). Of these 27 clones, 25 clones that contained three or more neurons were mixed clones containing both latexin-immunopositive and -immunonegative neurons. These results suggest that committed precursors to producing solely latexin-expressing neurons are not exist in the early cerebral cortical anlage. 2001 Elsevier Science B.V. All rights reserved. Theme: Development and regeneration Topic: Cell lineage and determination Keywords: Cell lineage; Cell-type specification; Development; Cerebral cortex; Progenitor; Precursor; Latexin
The mammalian cerebral cortex is comprised of functionally distinct areas, having characteristic histological features and wiring patterns. There have been two complementary hypotheses for the mechanism of neocortical areal specification: the protomap model [16] and the protocortex model [14]. The protomap model postulates that cortical regionalization is primarily controlled by molecular determinants that are intrinsic to the proliferative zone of the neocortex [1,4,6,12,13]. The protocortex model postulates that neocortical regional specification is controlled largely by extrinsic influences, such as thalamocortical axons [15,18]. Most probably, cortical regional specification depends on interactions between intrinsic properties of cortical cells and extrinsic influences of subcortical structures [17]. Previously, we have shown that latexin-immunopositive glutamatergic neurons are localized in the lateral but not *Tel.: 181-42-724-6244; fax: 181-42-724-6312. E-mail address: [email protected] (K. TakiguchiHayashi).
dorsal cortical areas in the rat [1,3]. Using latexin as a molecular marker, we demonstrated that the composition of E13 dorsal and lateral cortical progenitors is distinct in terms of developmental potential [2]. It remains to be elucidated, however, when and how a portion of precursors or young neurons is specified to generate latexin-immunopositive neurons in the lateral cortex. It may be hypothesized that a population of committed progenitors for the latexin phenotype is generated early and localized predominantly in the lateral cortical analage. To address whether this actually occurs, we analyzed the clonal relationship among cerebral cortical neurons expressing latexin in vitro. Dissociated cells from E12 cerebral cortices of timedpregnant Wistar rats (SPF grade; Sankyo, Shizuoka) were prepared as described previously [1]. The replication-deficient BAG retroviral vector [20], which transduces the bacterial lacZ gene, was infected to the cortical precursors. Prior to plating, 2310 7 colony-forming units (cfu) / ml of viral supernatant was added to the suspension of dissociated cells at a final concentration of 2310 3 cfu / 3310 4
0165-3806 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0165-3806( 01 )00262-0
88
K. Takiguchi-Hayashi / Developmental Brain Research 132 (2001) 87 – 90
Fig. 1. Proportion of latexin-immunopositive neurons in the culture derived from the dorsal and lateral cortical anlage. Shaded column, dorsal cortical anlage (DCX); diagonal column, lateral cortical anlage (LCX). Each value represents the mean number (6S.E.M.) of latexin-immunopositive neurons per 10 3 microtubule-associated protein 2 (MAP2)-positive neurons. *, Significantly greater (P,0.001, Student’s t test) than dorsal cortical culture.
cells / well. The cells were plated at a density of 4–5310 4 cells / cm 2 and cultured described previously [1] in a medium supplemented with FGF2 (Sigma, 5 ng / ml). At 6 days in vitro, 2.5 mM cytosine arabinoside (Sigma) was added to the FGF2-nonsupplemented medium for 2 days to eliminate dividing non-neuronal cells. Without this treatment, the culture would become nonviable. The cultures were maintained for an additional 13–15 days in the FGF2-nonsupplemented medium, which was replaced twice a week. A mouse monoclonal antibody PC3.1 against latexin ([1], 13 mg / ml) and a rabbit polyclonal antibody against b-galactosidase (1:500, Cappel, West Chester, PA, USA) were used to simultaneously visualize latexin and b-galactosidase immunoreactivity. Individual b-galactosidaseimmunopositive clones were identified under a 103 objective, and then the number of cells per clone and the percentage of latexin-immunopositive cells were determined under a 203 objective. Neurons were identified based on characteristic phase-contrast morphologies (Figs. 2B, 3A and B), and were found to be MAP2-immunopositive (data not shown). We performed clonal analysis of cortical cells cultured in a medium supplemented with exogenous FGF2 which is known to facilitate mitotic activity. Though it has been reported that FGF2 affects certain aspects of neuronal differentiation [5,19], a substantial proportion of the lateral neurons (5.7960.42 per 10 3 neurons, n524) began to express latexin, while far fewer latexin-expressing neurons appeared in the cultures of dorsal cells (0.9860.16 per 10 3 neurons, n519) (Fig. 1). These results indicate that the region-specific capacity of E12 cerebral cortical cells to
differentiate to latexin-expressing neurons is maintained even under the culture condition with exogenous FGF2. To follow the fate of individual E12 lateral cortical precursors, cells were infected with the BAG retrovirus vector (Fig. 2A and B). A group of b-galactosidaseimmunopositive cells was operationally defined as a clone when the cells were located within a 5003500 mm square. The identified clones were separated from each other by more than 500 mm. Out of 572 clones analyzed, 501 clones were purely neuronal clones. Of the neuronal clones, 40% (197 clones) were single-cell clones, 20% (96 clones) were two-cell clones, and 40% (208 clones) were clones containing three or more neurons (Fig. 2C). Latexin-immunopositive neurons were observed in 27 out of 501 neuronal clones: two were two-cell clones, and 25 were three-or-more-cell clones. Notably, all of the 25 multicell clones, were mixed clones which contained both latexin-immunopositive and -immunonegative neurons (Figs. 3 and 4). These results indicate that none of the E12 cortical precursors were committed to produce solely latexin-expressing neurons in vitro. Neurons in the cerebral cortex are composed of two major types: excitatory (glutamatergic) projection neurons and inhibitory (GABAergic) interneurons. It is generally considered that precursors for the glutamatergic neurons generate various subtypes of neurons in both superficial and deep layers. However, the existence of separate sets of layer-restricted precursors for the superficial and deep cortical layers is also suggested [7–11]. Latexin-immunopositive neurons are glutamate-immunoreactive neurons [3] and are restricted to the deep layers of lateral cortical
Fig. 4. Summary of the number of latexin-expressing neuron(s) in clusters of clonally related neurons. Horizontal axis shows 27 individual clones containing latexin-immunopositive neuron(s), and vertical axis shows the number of cells per individual clone. Black column, the number of latexin-immunopositive neuron(s). White column, the number of latexin-immunonegative neuron(s).
K. Takiguchi-Hayashi / Developmental Brain Research 132 (2001) 87 – 90
89
Fig. 2. Size distribution of clones grown in dissociated cell culture at 22–24 days in vitro. (A and B) An example of cells assumed to be clonally related. Arrowheads in (A) and (B) indicate b-galactosidase-immunopositive cells. (B) Phase-contrast photomicrograph of the same field as (A). Bar5100 mm in (A) and (B). (C) Histogram of the clone size of neuronal clones.
areas [1,3]. It has been shown that the regional specification for these neurons occurs before their neurogenesis at the precursor level [1,2]. To explain this early regional specification, it may be attractive to hypothesize that some committed progenitors for a deep layer-restricted latexin phenotype are generated early in the ventricular zone of the lateral cortical anlage. We performed an in vitro clonal analysis of latexinexpressing neurons derived from the E12 lateral cortex.
Although single cell clones represent 40% of the neuronal clones (Fig. 2C), none expressed latexin. This is consistent with our previous observations that birthdate of cortical latexin-immunopositive neurons is around E15 [1]. Latexin-immunopositive neurons were always found together with latexin-immunonegative neurons within single multicell clones (Figs. 3 and 4). The result indicates that extremely few, if any, E12 cortical precursors are committed to produce a progeny composed of solely latexin-
Fig. 3. Clonal analysis of cortical precursors containing latexin-immunoreactive neurons at 22–24 days in vitro. b-Galactosidase and latexin are visualized by double-immunofluorescence staining with anti-b-galactosidase antiserum and anti-latexin antibody; (A,C,E) and (B,D,F) have the same microscopic field. (A,B) Phase-contrast photomicrographs of two clones, both of which contain only one latexin-expressing neuron. (C,D) Cells infected by the retrovirus or descended from an infected precursor are readily identified by the presence of b-galactosidase immunoreactivity. (E,F) Differentiated neurons exhibiting latexin-immunoreactivity distributed in the cytoplasm of cell bodies. Bar5100 mm in (A–F).
90
K. Takiguchi-Hayashi / Developmental Brain Research 132 (2001) 87 – 90
immunopositive neurons in vitro. Thus, it is highly unlikely that early regional specification of the latexin phenotype is a consequence of the region-specific production of committed progenitors by E12. As the next step, an in vivo study is necessary to address whether radial clone precursors actually give rise to latexin-immunopositive neurons or not.
Acknowledgements The author thanks Dr. Yasuyoshi Arimatsu (Mitsubishi Kasei Inst Life Sci, Japan) for his advice and encouragement during the course of this work. The author also thanks Drs. Christopher Walsh (Harvard Inst. Med., USA) and Constance L. Cepko (Harvard Med Sch / HHMI, USA) for providing a BAG library.
References [1] Y. Arimatsu, M. Miyamoto, I. Nihonmatsu, K. Hirata, Y. Uratani, Y. Hatanaka, K. Takiguchi-Hayashi, Early regional specification for a molecular neuronal phenotype in the rat neocortex, Proc. Natl. Acad. Sci. USA 89 (1992) 8879–8883. [2] Y. Arimatsu, M. Ishida, K. Takiguchi-Hayashi, Y. Uratani, Cerebral cortical specification by early potential restriction of progenitor cells and later phenotype control of postmitotic neurons, Development 126 (1999) 629–638. [3] Y. Arimatsu, M. Ishida, M. Sato, M. Kojima, Corticocortical association neurons expressing latexin: specific cortical connectivity formed in vivo and in vitro, Cereb. Cortex 9 (1999) 569–576. [4] M.F. Barbe, P. Levitt, The early commitment of fetal neurons to the limbic cortex, J. Neurosci. 11 (1991) 519–533. [5] J.F.R. Cavanagh, M.C. Mione, I.S. Pappas, J.G. Parnavelas, Basic fibroblast growth factor prolongs the proliferation of rat cortical progenitor cells in vitro without altering their cell cycle parameters, Cereb. Cortex 7 (1997) 293–302. [6] M. Cohen-Tannoudji, C. Babinet, M. Wassef, Early determination of a mouse somatosensory cortex marker, Nature 31 (1994) 460–463.
[7] J.E. Crandall, K. Herrup, Patterns of cell lineage in the cerebral cortex reveal evidence for developmental boundaries, Exp. Neurol. 109 (1990) 131–139. [8] G. Fishell, J. Rossant, D. Van der Kooy, Neuronal lineages in chimeric mouse forebrain are segregated between compartments and in the rostrocaudal and radial planes, Develop. Biol. 141 (1990) 70–83. [9] L.A. Krushel, J.G. Johnston, G. Fishell, R. Tibshirani, D. Van der Kooy, Spatially localized neuronal cell lineages in the developing mammalian forebrain, Neuroscience 53 (1993) 1035–1047. [10] C.Y. Kuan, E. Elliott, R.A. Flavell, P. Rakic, Restrictive clonal allocation in the chimeric mouse brain, Proc. Natl. Acad. Sci. USA 94 (1997) 3374–3379. [11] M.C. Mione, J.F.R. Cavanagh, B. Harris, J.G. Parnavelas, Cell fate specification and symmetrical / asymmetrical divisions in the developing cerebral cortex, J. Neurosci. 17 (1997) 2018–2029. [12] E.M. Miyashita-Lin, R. Hevner, K.M. Wassarman, S. Martinez, J.L.R. Rubenstein, Early neocortical regionalization in the absence of thalamic innervation, Science 285 (1999) 906–909. [13] Y. Nakagawa, J.E. Johnson, D.D.M. O’Leary, Graded and areal expression patterns of regulatory genes and cadherins in embryonic neocortex independent of thalamocortical input, J. Neurosci. 19 (1999) 10877–10885. [14] D.D.M. O’Leary, Do cortical areas emerge from a protocortex?, Trends Neurosci. 12 (1989) 400–406. [15] D.D.M. O’Leary, B.B. Stanfield, Selective elimination of axons extended by developing cortical neurons is dependent on regional locale: experiments utilizing fetal cortical transplants, J. Neurosci. 9 (1989) 2230–2246. [16] P. Rakic, Specification of cerebral cortical areas, Science 241 (1988) 170–176. [17] J.L.R. Rubenstein, P. Rakic, Genetic control of cortical development, Cereb. Cortex 9 (1999) 521–523. [18] B.L. Schlagger, D.D.M. O’Leary, Potential of visual cortex to develop an array of functional units unique to somatosensory cortex, Science 252 (1991) 1556–1560. [19] F.M. Vaccarino, M.L. Schwartz, D. Hartigan, J.F. Leckman, Basic fibroblast growth factor increases the number of excitatory neurons containing glutamate in the cerebral cortex, Cereb. Cortex 1 (1995) 64–78. [20] C. Walsh, C.L. Cepko, Widespread dispersion of neuronal clones across functional regions of the cerebral cortex, Science 255 (1992) 434–440.