Laboratory of Neurobiology, Department of Neurosurgery, Kumamoto University Medical School, 1-1-1 Honjo, Kumamoto 860-8556, Japan
Satoshi Goto, Department of Neuro-surgery, Kumamoto University Medical School, 1-1-1 Honjo, Kumamoto 860-8556, Japan. Email: sgoto{at}kaiju.medic.kumamoto-u.ac.jp.
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Abstract |
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Introduction |
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Due to anomalous migration and positioning of postmitotic neurons during development, the reeler mouse, an autosomal recessive mouse mutant (Falconer, 1951), manifests abnormal laminar organization of the cerebral cortex (Caviness and Rakic, 1978
; Goffinet, 1984
; Rakic and Caviness, 1995
). This pheno-type is a result of the disruption of the reeler gene (Relnrl, formerly rl) encoding a diffusible protein, reelin, that has several structural characteristics of extracellular matrix proteins (DArcangelo et al., 1995
; Hirotsune et al., 1995
). During corticogenesis in normal mice, reelin is produced by preplate CajalRetzius cells (DArcangelo et al., 1995
; Hirotsune et al., 1995
; Ogawa et al., 1995
), which are located just below the pial surface (Bayer and Altman, 1991
; Jacobson, 1993
). The preplate then splits into two components, the marginal zone and the subplate layer, and the young postmitotic neurons that have migrated along radial glial fibers from the germinal ventricular zone (Rakic, 1995
) form the cortical plate between these components (Marin-Padilla, 1998
). The vertical position of cortical plate neurons is determined by the time of their origination and then clear horizontal layering is formed in the cortex (Rakic, 1995
). In reeler mutant mice, CajalRetzius cells do not produce reelin and the preplate does not split. As a consequence, the cortical plate ectopically locates underneath the subplate neurons and the characteristic inside-out layering is perturbed. This is evidence for the role of reelin in the vertical positioning of cortical plate neurons that is essential for the formation of horizontal layering. In the development of the CNS, reelin has been identified as a crucial molecule that defines architectonic patterns by controlling neuronal migration (DArcangelo et al., 1995
; Ogawa et al., 1995
; Miyata et al., 1997
; Nakajima et al., 1997
; Dulabon et al., 2000
; Yip et al., 2000
; Ohshima et al., 2001
; Magdaleno et al., 2002
) and axon growth and synaptic connectivity (Del Río et al., 1997
; Ghosh, 1997
; Borrell et al., 1999
; Rice et al., 2001
). Recently, reelin receptors and other molecules involved in the reelin signaling cascades also have been identified (Rice and Curran, 2001
).
We now report that during the early postnatal stage, vertical columnar arrays of cortical neurons and their dendritic processes are conspicuous in the mouse presubiculum, a multi-layered and periallocortical region (OMara et al., 2001). They provide a good model for investigating the cellular and molecular cues that direct both the vertical and horizontal positioning of postmitotic cortical neurons. Results from our present study suggest that reelin secreted by CajalRetzius cells may control the developmental formation of the vertical structures in the presubicular cortex by acting as an inhibitory or stop signal for cortical plate neurons and their dendritic extensions.
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Materials and Methods |
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The reeler mouse colony was originally derived from heterozygous B6C3Fe-a/a-rl adults (The Jackson Laboratory). Timed pregnancies were induced by mating homozygous (rl-/-) males to heterozygous (rl+/-) females, or by bleeding heterozygotes of both sexes. The day of birth was recorded as postnatal day 0 (P0). Embryos and neonates of known age were kept in an in-house breeding colony. Care of the animals was in accordance with regulations promulgated by the Center for Animal Resources and Development of Kumamoto University.
Tissue Preparations
Newborn mice (P0) and mice aged P1, P2, P3, P4, P6, P9, P14 and P28, as well as adult mice were used. They received an i.p. injection of a lethal dose of pentobarbital and were perfused transcardially with 0.9% (w/v) saline in 0.01 M phosphate buffer, pH 7.4 (PBS), followed by ice-cold 4% (w/v) paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 (PB). The brains were removed, postfixed with the same fixative at 4°C overnight and then kept overnight at 4°C in 0.1 M PB containing 30% (w/v) sucrose for cryoprotection. The brains were subsequently embedded in OCT compound (Sakura Finetechnical, Japan) and frozen in dry-ice/acetone. Cryostat sections were cut and kept in PBS until use.
Immunofluorescence Stainings
CR-50, a mouse monoclonal antibody that recognizes an epitope in the N-terminal region of reelin (DArcangelo et al., 1997), was a gift from Dr M. Ogawa, The Institute of Physical and Chemical Research (RIKEN), Japan (Ogawa et al., 1995
; Nishikawa et al., 1999
; Hamasaki et al., 2001a
). A mouse monoclonal antibody to microtubule-associated protein 2 (MAP2; Sigma, MO) was also used as primary antibody (Hamasaki et al., 2001a
). The sections were blocked with 3% bovine serum albumin (BSA)PBS for 1 h and then incubated overnight at 4°C in 3% (w/v) BSAPBS containing primary antibody. Immunoreactivity was detected by FITC (Vector, CA) or Texas Red (Vector) conjugated secondary antibodies. Propidium iodide (PI) staining was also used (Hamasaki et al., 2001b
). The fluorescence activities were observed and recorded under a confocal laser-scanning microscope (Fluoview, Olympus, Japan). The images obtained were printed using Pictrography 3000 (Fuji Film, Japan).
Scoring of Apical Dendrite Orientation
Apical dendrites of cortical pyramidal neurons from the presubicular cortex of heterozygous (rl+/-) or homozygous (rl-/-) mice aged P14 were visualized with anti-MAP2 antibody. According to the previous report (Polleux et al., 2000), they were scored as being directed towards the pia (45°135°), the white matter (225°315°), or as horizontal (135°225° or 45°315°).
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Results |
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At age P3 and P4, vertical columnar arrays of neuronal cell bodies running orthogonal to the horizontal laminae were clearly present in the mouse presubicular cortex. Vertical columns were visible even to the untutored eye in PI (Fig. 1A) or Nissl (Fig. 1B,C
) stained preparations. They were conspicuous in the upper half of the cortical plate that corresponds to the supragranular layer (i.e. layers II and III) and had a periodicity of ~55 µm (55.2 ± 12.3 mm) in the horizontal extent. Each vertical column was composed of a cluster of young postmitotic neurons; between columns, a narrow inter-columnar space poor in neurons could be seen (Fig. 1B,C
).
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At birth (P0), neurons in layers IVVI have arrived at their final position, while neurons in layers II and III are still migrating toward their final destinations (Jacobson, 1993). At this stage, the superficial marginal zone appeared to contain CajalRetzius cells stained for reelin with CR-50 (Fig. 2A,B
). Young postmitotic neurons formed the cortical plate beneath the marginal zone enriched in reelin. No definite vertical structures were found in either the cortical plate or the marginal zone at P0.
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Relationship between Reelin and Dendritic Clusters in the Marginal Zone
By P14, all cortical plate neurons have settled in their final positions and are engaged in the formation of fiber connections (Jacobson, 1993). The apical dendrites of cortical pyramidal cells of layers II, III and V form clusters that ascend through the cortical plate and send their terminal arrays to the marginal zone (Fleischhauer et al., 1972
; Peters and Walsh, 1972
) also see Fig. 5A
. At this stage, the columnar arrays of neuronal cell bodies were no longer discernible in the cortical plate (Fig. 3A,C
), although CR-50 labeling continued to show periodic modulation in the marginal zone (Fig. 3B,C
). Interestingly, reelin-poor zones contained columnar tufts of dendritic processes of cortical pyramidal neurons positive for MAP2 (Fig. 3D,E
). Sections cut parallel to the cortical surface revealed a mosaic-like pattern of irregularly spaced spots poor in CR-50 labeling (Fig. 3F
) or enriched in MAP2 labeling (Fig. 3G
) in the marginal zone. Comparison with adjacent sections in the marginal zone showed that the distributions of CR-50 labeling and MAP2-immuno-reactive dendrites were almost complementary: regions with poor CR-50 immunoreactivity exhibited clusters of dendrites of cortical pyramidal neurons (Fig. 3H,I
). Thus, the dendritic branches of cortical pyramidal neurons cluster by avoiding reelin-rich zones in the marginal zone, suggesting that reelin may act as a barrier to their extensions.
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Disruption of the Vertical Structures in reeler Mutant Mice
Compared to wild-type (rl+/+) or heterozygous (rl+/-) mice (see Fig. 1), in homozygous (rl-/-) P3 mice there were no detectable vertical columnar arrays or horizontal laminae of neurons in the cortical plate (Fig. 4
). At P14, clusters of dendritic branches of cortical pyramidal neurons in the marginal zone were clearly evident in wild-type and heterozygous littermates (Fig. 5A
), but not in homozygous mice (Fig. 5B
). Furthermore, the orientation and positioning of cortical plate neurons varied in the homo-zygous mice (Caviness, 1976
; Landrieu and Goffinet, 1981
). Although the apical dendrites of cortical pyramidal neurons were oriented almost orthogonal to the pial surface in wild-type or heterozygous mice (Fig. 5A
), many appeared to be oblique, to run horizontally, or to be inverted in homozygous mice (Fig. 5B
). Our semi-quantitative study (Fig. 5C
) showed that most apical dendrites were directed towards the pial surface in the heterozygous (77.5 ± 3.5%; n = 200), but not in the homozygous (28.5 ± 2.6%; n = 200) mice. These findings indicate that reelin function is required for the developmental formation of both the horizontal layering and the vertically orientated architectures in the presubicular cortex.
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Discussion |
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In this study we showed the presence of two transient vertical structures, identified with simple anatomical techniques in the developing mouse presubicular cortex (for reference, see Fig. 6). One structure consists of the vertical columnar arrays of young cortical plate neurons in the supragranular layer. These occur most conspicuously at P3P4, when the late-born cortical neurons ascend in linear arrays along a scaffold of radial fibers of astroglia or are proceeding to their final positions (Jacobson, 1993
). The other structure is comprised of columnar tufts of dendritic processes of cortical pyramidal neurons in the marginal zone that are most remarkable at P14 when dendritic deployment proceeds and the fiber connections are being established (Jacobson, 1993
): it seems to be a prototype of the dendritic clusters that group cortical neurons into modules of microcolumnar size (Mountcastle, 1997
). In P28 as well as adult mice, these structures can no longer be clearly discerned because they are veiled by tightly packed cell bodies and dendritic branches of cortical neurons. As previously suggested (Vogt Weisenhorn et al., 1994
; Del Río et al., 1995
, 1996
; Spreafico et al., 1995
; Marin-Padilla, 1998
), at this stage there is only a small population of CajalRetzius cells in the marginal zone. Our present results lead us to posit that the structures represent micro-anatomical units that underlie the development of the territorial organization that results in the positioning of cortical neurons and their formation of fiber connections in the horizontal dimension. According to the radial unit hypothesis (Rakic, 1988
), the horizontal coordinates of cortical neurons are determined by the relative positions of their precursor cells in the germinal ventricular zone. It has been suggested that the germinal ventricular zone contains a mosaic of ontogenetic units that are composed of the neuronal precursors for a cortical column; alternatively, the mosaicism of the germinal ventricular zone is reproducible in the cortical plate. It is presently unknown whether or how the cellular microcolumns shown here are anatomically and functionally related to the microcolumns originating from the clonal modality. We are currently investigating the possible role of these microcolumns as functional units in cortical activities related to intracortical fiber connections.
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Finally, we hypothesize that reelin and CajalRetzius cells in the marginal zone may play a role in the formation of not only horizontal laminations, but also vertical columnar structures in the developing cerebral cortex. This is supported by the present finding that the vertical structures shown here are totally disorganized in reeler mutant mice. Our hypothesis coincides with suggestions that CajalRetzius cells may coordinate positional information essential for the early areal and columnar specification of the underlying cortex (Galuske and Singer, 1996; Schmidt et al., 1996; Schwartz et al., 1998
; Soria and Fairén, 2000
; Hevner et al., 2001
; Zecevic and Rakic, 2001
). However, this hypothesis is based on developmental and anatomical evidence found in a specialized region of the mouse cortex, i.e. the presubicular cortex. Further studies are needed to determine whether our hypothesis applies to other cortical areas in rodents or different species as a fundamental mechanism that underlies the developmental formation of the cerebral cortex.
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Acknowledgments |
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References |
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Borrell V, Del Rio JA, Alcántara S, Derer M, Martinez A, DArcangelo G, Nakajima K, Mikoshiba K, Derer P, Curran T, Soriano E (1999) Reelin regulates the development and synaptogenesis of the layer-specific entorhino-hippocampal connections. J Neurosci 15:13451358.
Caviness VS (1976) Reeler mutant mice and laminar distribution of afferents in the neocortex. Exp Brain Res 1:267273.
Caviness VS Jr, Rakic P (1978) Mechanisms of cortical development: a view from mutations in mice. Annu Rev Neurosci 1:297326.[ISI][Medline]
Curran T, DArcangelo G (1998) Role of reelin in the control of brain development. Brain Res Rev 26:285294.[ISI][Medline]
DArcangelo G, Miao GG, Chen SC, Morgan JI, Curran T (1995) A protein related to extracellular matrix proteins deleted in the mouse mutant reeler. Nature 374:719723.[ISI][Medline]
DArcangelo G, Nakajima K, Miyata T, Ogawa M, Mikoshiba K, Curran T (1997) Reelin is a secreted glycoprotein recognized by the CR-50 monoclonal antibody. J Neurosci 17:2331.
Del Río JA, Martinez A, Fonseca M, Auladell C, Soriano E (1995) Glutamate-like immunoreactivity and fate of CajalRetzius cells in the murine cortex as identified with calretinin antibody. Cereb Cortex 1:1321.
Del Río JA, Heimrich B, Supèr H, Borrell V, Frotscher M, Soriano E (1996) Differential survival of CajalRetzius cells in organotypic cultures of hippocampus and neocortex. J Neurosci 16:68966907.
Del Río JA, Heimrich B, Borrell V, Förster E, Drakew A, Alcántara S, Nakajima K, Miyata T, Ogawa M, Mikoshiba K, Derer P, Frotscher M, Soriano E. (1997) A role for CajalRetzius cells and reelin in the development of hippocampal connections. Nature 385:7175.
Dulabon, Olson EC, Taglienti MG, Eisenhuth S, McGrath B, Walsh CA, Kreidberg JA, Anton ES (2000) Reelin binds a3b1 integrin and inhibits neuronal migration. Neuron 27:3344.[ISI][Medline]
Eccles JC (1984) In: Cerebral cortex (Jones EG, Peters A, eds), vol. 2, pp. 136. New York: Plenum Press.
Falconer DS (1951) Two new mutants, trembler and reeler, with neurological actions in the house mouse (Mus musculus L). J Genet 50:192201.[ISI]
Fleischhauer K, Petsche H, Wittkowski W (1972) Vertical bundles of dendrites in the neocortex. Z Anat Entwicklungsgesch 136:213223.[ISI][Medline]
Frotscher M (1998) CajalRetzius cells, reelin, and the formation of layers. Curr Opin Neurobiol 8:570575.[ISI][Medline]
Galuske RA, Singer W (1996) The origin and topography of long-range intrinsic projections in cat visual cortex: a developmental study. Cereb Cortex 6:417430.[Abstract]
Ghosh A (1997) Axons follow reelin routes. Nature 385:2324.[ISI][Medline]
Goffinet AM (1984) Events governing organization of postmigratory neurons: studies on brain development in normal and reeler mice. Brain Res Rev 7:261296.[ISI]
Hamasaki T, Goto S, Nishikawa S, Ushio Y (2001a) Early-generated preplate neurons in the developing telencephalon: inward migration into the developing striatum. Cereb Cortex 11:474484.
Hamasaki T, Goto S, Nishikawa S, Ushio Y (2001b) A role of netrin-1 in the formation of the subcortical structure striatum: repulsive action of the migration of late-born striatal neurons. J Neurosci 21:42724280.
Hevner RF, Shi L, Justice N, Hsueh Y-P, Sheng M, Smiga S, Bulfone A, Goffinet AM, Rubenstein JLR (2001) Tbr1 regulates differentiation of the preplate and layer 6. Neuron 29:353366.[ISI][Medline]
Hirotsune S, Takahara T, Sasaki N, Hirose K, Yoshiki A, Ohashi T, Kusakabe M, Murakami Y, Muramatsu M, Watanabe S, et al. (1995) The reeler gene encodes a protein with an EGF-like motif expressed by pioneer neurons. Nat Genet 10:7783.[ISI][Medline]
Jacobson M (1993) Developmental neurobiology, pp. 401451. New York: Plenum Press.
Jones EG (2000) Microcolumns in the cerebral cortex. Proc Natl Acad Sci USA 97:50195021.
Landrieu P, Goffinet AM (1981) Inverted pyramidal neurons and their axons in the neocortex of reeler mutant mice. Cell Tissue Res 218:293301.[ISI][Medline]
Magdaleno S, Keshvara L, Curran T (2002) Rescue of ataxia and preplate splitting by ectopic expression of reelin in reeler mice. Neuron 33:573586.[ISI][Medline]
Marin-Padilla M (1998) CajalRetzius cells and the development of the neocortex. Trends Neurosci 21:6471.[ISI][Medline]
Miyata T, Nakajima K, Mikoshiba K, Ogawa M (1997) Regulation of Purkinje cell alignment by reelin as revealed with CR-50 antibody. J Neurosci 17:35993609.
Mountcastle VB (1997) The columnar organization of the neocortex. Brain 120:701722.[Abstract]
Nakajima K, Mikoshiba K, Miyata T, Kudo C, Ogawa M (1997) Disruption of hippocampal development in vivo by CR-50 mAb against reelin. Proc Natl Acad Sci USA 94:81968201.
Nishikawa S, Goto S, Hamasaki T, Ogawa M, Ushio Y (1999) Transient and compartmental expression of the reeler gene product reelin in the developing rat striatum. Brain Res 850:244248.[ISI][Medline]
Ogawa M, Miyata T, Nakajima K, Yagyu K, Seike M, Ikenaka K, Yamamoto H, Mikoshiba K (1995) The reeler gene-associated antigen on CajalRetzius neurons is a crucial molecule for laminar organization of cortical neurons. Neuron 14:899912.[ISI][Medline]
Ohshima T, Ogawa M, Hirasawa M, Longenecker G, Ishiguro K, Pant HC, Brady RO, Kulkarni AB, Mikoshiba K (2001) Synergic contributions of cyclin-dependent kinase 5/p35 and reelin/Dab1 to the positioning of cortical neurons in the developing mouse brain. Proc Natl Acad Sci USA 98:27642769.
OMara SM, Commins S, Anderson M, Gigg J (2001) The subiculum: a review of form, physiology and function. Prog Neurobiol 64:129155.[ISI][Medline]
Pearlman AL, Faust PL, Hatten ME, Brunstrom JE (1998) New directions for neuronal migration. Curr Opin Neurobiol 8:4554.[ISI][Medline]
Peters A, Walsh TM (1972) A study of the organization of apical dendrites in the somatic sensory cortex of the rat. J Comp Neurol 144:253268.[ISI][Medline]
Polleux F, Marrow T, Ghosh A (2000) Semaphorin 3A is a chemoattractant for cortical apical dendrites. Nature 404:567573.[ISI][Medline]
Rakic P (1988) Specification of cerebral cortical areas. Science 241:170176.[ISI][Medline]
Rakic P (1995) Radial versus tangential migration of neuronal clones in the developing cerebral cortex. Proc Natl Acad Sci USA 92: 1132311327.
Rakic P, Caviness VS Jr (1995) Cortical development: view from neurological mutants two decades later. Neuron 14:11011104.[ISI][Medline]
Rice DS, Curran T (2001) Role of the reelin signaling pathway in central nervous system development. Annu Rev Neurosci 24:10051039.[ISI][Medline]
Rice DS, Nusinowitz S, Azimi AM, Martinez A, Soriano E, Curran T (2001) The reelin pathway modulates the structure and function of retinal synaptic circuitry. Neuron 31:929941.[ISI][Medline]
Schmidt KE, Galuske RAW, Singer W (1999) Matching the modules: cortical maps and long range intrinsic connections in visual cortex during development. J Neurobiol 41:1017.[ISI][Medline]
Schwartz TH, Rabinowitz D, Unni V, Kumar VS, Smetters DK, Tsiola A, Yuste R (1998) Networks of coactive neurons in developing layer I. Neuron 20:541552.[ISI][Medline]
Soria JM, Fairén A (2000) Cellular mosaics in the rat marginal zone define an early neocortical territorialization. Cereb Cortex 10:400412.
Spreafico R, Frassoni C, Arcelli P, Selvaggio M, DeBiasi S (1995) In situ labeling of apoptotic cell death in the cerebral cortex and thalamus of rats during development. J Comp Neurol 363:281295.[ISI][Medline]
Vogt Weisenhorn DM, Weruaga-Prrieto E, Celio MR (1994) Localization of calretinin in cells of layer I (CajalRetzius cells) of the developing cortex of the rat. Dev Brain Res 82:293297.[ISI][Medline]
Walsh CA, Goffinet AM (2000) Potential mechanisms of mutations that affect neuronal migration in man and mouse. Curr Opin Genet Dev 10:270274.[ISI][Medline]
Yip JW, Yip YPL, Nakajima K, Capriotti C (2000) Reelin controls position of autonomic neurons in the spinal cord. Proc Natl Acad Sci USA 97:86128616.
Zecevic N, Rakic P (2001) Development of layer I neurons in the primate cerebral cortex. J Neurosci 21:56075619.