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Latexin: a molecular marker for regional specification in the neocortex
NEUROSCIENCE RESERRCH ELSEVIER
Neuroscience Research 20 (1994) 131-135
Update article
Latexin: a molecular marker for regional specification in the neocortex Yasuyoshi Arimatsu Mitsubishi Kasei Institute of Life Sciences, 11 Minamiooya, Machida-shi, Tokyo 194, Japan
Received 16 February 1994; revision received 25 May 1994; accepted 25 May 1994
Abstract It largely remains to be elucidated how the mammalian neocortex is regionally specified during development. In an attempt to obtain molecular markers in the neocortex, we have generated a monoclonal antibody PC3.1 which recognizes a subset of neurons located in lateral, but not dorsal, neocortical areas. The antigen is a novel class of protein, named latexin, having a molecular weight of 29 000. Our in vitro studies have revealed that the neocortical regional specification for the production of latexin-positive neurons occurs very early prior to thalamocortical interactions and the completion of neurogenesis, indicating that elements intrinsic to the neocortex play important roles in the neocortical specification. Furthermore, our recent analyses have suggested that this regional specification is attributable, at least in part, to an early restriction of developmental potential in neocortical progenitor cells to become latexin-positive neurons. Keywords: Neocortex; Regional specification; Neurogenesis; Cell differentiation; Migration; Latexin
1. Introduction Although the basic structure of the mammalian neocortex is quite similar, it is divided into various subregions such as visual, somatosensory and motor cortices that contribute to their specialized physiological functions (Gilbert, 1983). The developmental mechanism regulating regional specification within the neocortex is one of the major interests of current neurobiology (Rakic, 1988; O'Leary, 1989; Shatz, 1992; Kennedy and Dehay, 1993). The clarification, however, has been hampered by the extremely complex organization of the neocortex where neurons of diverse phenotypes form a variety of connections with cortical and subcortical structures. The lack of appropriate molecular markers that define specific cell populations of neocortical neurons has contributed to the limited knowledge of the mechanism underlying neocortical development. With the use of morphological characteristics like laminar fate and connectional pattern as an indicator, it is impossible to define neuronal
identity until a neuron of interest has migrated to its final destination and has extended its axons along a pathway unique to its subtype. Cell-type-specific molecular markers in the neocortex would certainly promote studies on cortical development in relatively simple and defined experimental systems. Here I will consider some aspects of regional specification during the early stage of neocortical development in the rat. They have been clarified recently with the use of a novel molecular marker named latexin.
2. Intraneocortical regional specificity represented by latexin-positive neurons In an effort to obtain specific molecular markers in the neocortex, we have generated a monoclonal antibody, designated PC3.1, that detects a neuronal entity confined to infragranular layers within lateral (but not dorsal) neocortical areas in adult rats (Arimatsu et al., 1992). These include the secondary somatosensory, visceral sensory, primary and secondary auditory, and
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Y Arimatsu/Neurosci. Res. 20 (1994) 131-135
Fig. 1. Distribution of latexin-positiveneurons in the cerebral cortex of the rat. Cortical regions containing latexin-positive neurons (dotted) are superimposedon the schematicillustration of a lateral surface view of the forebrain. Boundaries of cortical areas are shown according to Zilles (1985, 1990)and Zilles et al. (1990). The boundaries between latexin-positive and -negative regions do not necessarily correspond strictly to the cytoarchitectonic boundaries. Parl, for example, contains some latexin-positive neurons on the border with Par2. The claustrum (CI) and endopiriform nuclei (En), which also contain latexin-positive neurons, are also shown (shaded). AI, agranular insular cortex; Ent, entorhinal cortex; Frl, frontal cortex, area 1; Fr2, frontal cortex, area 2; Fr3, frontal cortex, area 3; LO, lateral orbital cortex; Ocl, occipital cortex, area 1; Oc2L, occipital cortex, area 2, lateral; Parl, parietal cortex, area 1; Par2, parietal cortex, area 2; Pir, piriformcortex; PRh, perirhinal cortex; Te 1, temporal cortex, area !; Te2, temporal cortex, area 2; Te3, temporal cortex, area 3; Vi, visceral sensory area. Ocl and Oc2L correspond to the primary visual cortex and the lateral portion of the secondary (association) visual cortex, respectively.Parl and Par2 correspond to the primary and secondary somatosensory cortex, respectively. Tel and Te2/3 correspond to the primary and secondary (association) auditory cortex, respectively.
secondary visual cortices (see Fig. 1). Considering the high degree of similarity in the structure of the neocortex across areas (Rockel, 1960; Zilles, t990; Zilles et al., 1990), it is striking to find that latexin-positive neurons are located only in a limited sector of the neocortex. Although systematic characterization of latexin-positive neurons has not yet been performed for either their morphologies, connectional patterns or neurotransmitter phenotypes, these include typical and atypical pyramidal neurons (Arimatsu et al., 1994) and glutamate-like immunoreactive neurons. The antibody PC3.1 specifically binds a polypeptide epitope on a 29 kDa protein, named latexin (Hatanaka et al., 1994). Analysis of c D N A clones for latexin showed that the predicted primary structure of latexin consists of 223 amino-acid residues with no strict homology to any protein sequences so far known. The latexin genelike sequence is well conserved in the genome of the mouse, rabbit, cat, cow, monkey and human, all mammalian species tested. In situ hybridization analysis showed that latexin m R N A is synthesized in a subset of neurons in the lateral neocortex. These results indicate
that latexin is a novel class of protein that represents intraneocortical regional specificity. To understand the mechanisms underlying regional specification in the neocortex, the region-specific molecular marker, latexin, that identifies and defines a neuronal subset is useful. We have already analyzed some aspects of neocortical specification by using latexin as a marker. The major issues have been: (i) when does regional specification occur for the production of latexin-positive neurons; (ii) to what extent do elements intrinsic to the neocortex and extracortical influences contribute to the regional specification; (iii) whether or not thalamic afferents play critical roles in the regional specification, and (iv) what kind of roles, if any, cell lineage, local cell environment, and cell migration play for the specification.
3. Early regional specification for the production of latexin-positive neurons We have shown that some kind of regional specification in the neocortex occurs very early along the dorsalventral axis. We demonstrated the selective appearance of PC3.1-positive neurons in cultures derived from a lateral, but not dorsal, sector of the neocortical anlage at El2 or later (Arimatsu et al., 1992). Since thalamic and other afferents invade the neocortex at El6 or later (Catalano et al., 1991) and corticofugal axons do not exit the cerebral cortex before El3 (Blakemore and Molnar, 1990; De Carlos and O'Leary, 1992), it can be concluded that the regional specification for the production of latexin-positive neurons is established by elements intrinsic to the neocortex prior to interactions between cortical and extracortical cells. To clarify in more detail the tangential heterogeneity of the neocortical anlage, we examined the in vitro capacity for the production of latexin-positive neurons at various rostrocaudal and dorsolateral levels (Arimatsu et al., 1993). In an organotypic slice culture system, a substantial number of neurons became latexin-positive in the tissue fragments derived from the lateral sector of the neocortical anlage. Within the lateral sector, the number of the latexin-positive neurons was highest in the fragments from the middle portion along the rostralcaudal axis, with progressively decreasing numbers at more rostral and caudal levels. Much fewer latexinpositive neurons were found in cultures from the dorsal neocortex at all rostral-caudal levels. The result indicates that, at least along the dorsal-ventral axis, the distribution pattern of the capacity for the production of latexin-positive neurons correlates exactly with that of latexin-positive neurons in the adult cortex. It remains to be clarified whether or not this is also the case for the rostral-caudal axis. The answer will be available after quantitative examination of latexin-positive neurons along the rostral-caudal axis in the adult cortex. If the
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fetal in vitro pattern and the adult in vivo one are closely correlated to each other also along the rostral-caudal axis, then it can be concluded that there is some kind of tangential 'blueprint' or 'protomap' (Rakic, 1988) in the neocortical anlage for the future intracortical regional specificity. Early regional specification has also been shown for the expression of the limbic system-associated membrane protein (LAMP) in the limbic cortex (Barbe and Levitt, 1991, 1992; Ferri and Levitt, 1993). Furthermore, a recent study using a transgenic mouse line has shown an early regional commitment to the expression of a somatosensory cortex marker (Cohen-Tannoudji et al., 1994). It has, however, been suggested by heterotopic transplantation experiments that certain connectional and cytoarchitectonic features in some neocortical regions are not fully specified at the time of neurogenesis, but that they can be modified during later stages by epigenetic interactions with environmental signals (O'Leary and Stanfield, 1989; Schlaggar and O'Leary, 1991). Thus, it seems likely that neocortical regional specification involves multiple steps including both early and later specification events. With respect to latexin-positive neurons, it will be important to investigate whether extrinsic elements like thalamic innervation, in addition to the predicted intrinsic mechanism, play significant roles in establishing the regional specificity observed in the mature cortex. 4. Neurogenesis of latexin-positive neurons To facilitate a better understanding of molecular and cellular mechanisms underlying early regional specification, it is important to know about time as well as site of generation of these neurons. We found in a study combining [3H]thymidine autoradiography and PC3.1immunohistochemistry that latexin-positive neurons in layers V and VI of the lateral neocortical areas were generated concurrently at El5 (Arimatsu et al., 1994). It was unexpected to find a strict correlation between the latexin phenotype of neocortical neurons and the time of their generation irrespective of their final positions along radial and tangential axes, since previous [3H]thymidine birth-dating studies dealing with neocortical neurons as a single population had shown radial and tangential neurogenetic gradients (Bayer and Altman, 1991; for a review, see Jacobson, 1991). In many mammalian species, earlier-generated neurons in a particular area tend to be located in a deeper layer, and later-generated neurons in a more superficial layer (radial neurogenetic gradient). Neurons in a particular lamina of rostral and ventral regions in the neocortex tend to be born earlier than those in caudal and dorsal regions, respectively (tangential neurogenetic gradients). The simultaneous generation of latexin-positive neurons across tangentially widespread cortical regions indicates
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that laminar and tangential locations of neocortical neurons in the adult animal are not established solely by a combination of mechanisms for inside-out migration of newly generated neurons in each cortical area and for broad tangential neurogenetic gradients. The mechanism involved in neocortical histogenesis should be more complicated than has ever been thought (for a detailed discussion, see Arimatsu et al., 1994). 5. Early restriction of developmental potential in neocortical progenitor cells to become latexin-positive neurons Although early regional heterogeneity within the neocortical anlage is predicted from the experimental evidence described above, it is still unclear when individual progenitor cells or young postmitotic neurons are committed to express latexin. Since McConnell and Kaznowski (1991) suggested that neocortical progenitor cells are initially multipotent, but become committed to a laminar fate by environmental signals acting around their final mitosis, it will be of great interest to know whether the commitment to the latexin phenotype occurs at around El5, the birthdate of latexin-positive neurons in the neocortex (Arimatsu et al., 1994). To address this question, we have begun to examine latexin expression in the fetal neocortical cells cultured under various environmental conditions (Arimatsu and Miyamoto, 1993). When E13 cells from a middle portion of the lateral neocortical anlage, which had been labeled with BrdU in vivo, were cocultured in a monolayer together with an excess of unlabeled cells from either the lateral or the dorsal neocortex, a substantial portion of the BrdU-labeled cells expressed latexin. In contrast, a much smaller proportion of dorsal neocortical cells expressed latexin when cocultured with either lateral or dorsal neocortical cells. These results suggest that the composition of El3 neuroepithelium is regionally different along the dorsal-ventral axis. The simplest hypothesis is that the restriction of the developmental potential for the latexin phenotype has occurred in the progenitor cells in the dorsal sector of the neocortical anlage, whereas some cells in the lateral sector are competent for the differentiation. In addition, it was found that a much greater proportion of El3 lateral neocortical cells became to express latexin when maintained in a reaggregateted-cell culture with excess cells from the lateral neocortex than from the dorsal neocortex (Arimatsu and Miyamoto, 1993). This suggests that differentiation of individual progenitor cells to express latexin can be influenced by signal(s) from surrounding neocortical cells. Thus, the latexin phenotype may be established by a certain combination of multiple mechanisms, but the exact nature of epigenetic signals, if they indeed occur, remains to be elucidated. It has been suggested that, in the vertebrate retina, many of the neuronal types arise from multipotent pro-
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genitor cells depending upon extracellular signals at respective developmental stages but not upon cell lineage (Turner et al., 1990; Watanabe and Raft, 1992; Altshuler et al., 1993). In the cerebral cortex, however, an early lineage restriction for some neuronal phenotypes has been suggested to occur in pyramidal vs nonpyramidal cells (Parnavelas et al., 1991; Luskin et al., 1993), and in glutamate- vs GABA-immunoreactive neurons (Mione et al., 1994). In this connection, it will be of interest to clarify in future whether any lineage restriction of cortical progenitor cells is already established for the production of latexin-positive neurons.
6. Cell migration It has been proposed that young cortical neurons migrate radially from the ventricular zone to their final destination (Rakic, 1988; Misson et al., 1991). However, recent lineage-tracing studies using retroviral vectors as a tracer have raised the possibility that some young neurons or their precursor cells migrate across widespread cortical areas (Walsh and Cepko, 1992). In fact, the extent of tangential dispersion of fluorescently labeled cells observed in a slice of developing cerebral cortex differed from one cell to another (O'Rourke et al., 1992; Fishell et al., 1993). A recent transgenic study demonstrated that both radial mosaicism and tangential cell migration contribute to neocortical development (Tan and Breen, 1993). Thus, it is now necessary to examine the migration of neurons not as a whole but with respect to particular neuronal subpopulations. In this regard, our experiments with the use of latexin as a marker may provide some insight into this problem. Since latexin-positive neurons are cogenerated at E 15 irrespective of their eventual laminar and tangential locations, they are likely to represent a developmentally unique population. This led us to consider possible migratory pathways of presumptive iatexin-positive neurons (Arimatsu et al., 1994). In view of the likelihood that cells in the dorsal neocortical anlage at El3 are already incompetent to become latexin-positive, neurons expressing latexin in the adult lateral neocortex should originate in the lateral but not the dorsal sector of the neocortical anlage. Thus, presumptive latexin-positive neurons are not likely to migrate extensively in the tangential plane, at least from the dorsal to the lateral sector, in the developing neocortex.
7. Concluding remarks Having used latexin as a molecular marker, it was possible to clarify some aspects of regional specification and cell-type determination during the early phase of neocortical development. The following conclusions have been drawn: (i) Some kind of regional specification has occurred very early within the neocortical anlage as
to the capacity to produce latexin-positive neurons. (ii) Restriction of developmental potential in progenitor cells to become latexin-positive neurons is likely to contribute to this early regional specification. (iii) An extensive tangential migration of young neurons from the dorsal to the lateral sector of the developing neocortex is likely to play no or, if any, very limited roles in the parcellation of latexin-positive neurons. Now that we know the primary structure of latexin and its mRNA, it will be possible to analyze further the molecular mechanism of the regional specification and the cell-type determination by examining the way in which latexin synthesis is initiated and controlled. The recent evidence supports the idea that transcription factors and secreted proteins contribute to the regional specification of the midbrain, hindbrain and spinal cord in the vertebrate species (McGinnis and Krumlauf, 1992; McMahon, 1992; Basler et al., 1993; Carpenter et al., 1993). Moreover, it has been shown that some transcription factors and regulatory proteins are expressed in the mouse forebrain (Puelles and Rubenstein, 1993). Then, it will be important to investigate whether or not similar mechanisms operate during the early phase of specification events within the neocortex.
Acknowledgements The author wishes to thank Drs. Yumiko Hatanaka, Keiko Takiguchi-Hayashi, Yoshihiko Uratani and Shinobu C. Fujita for their kind comments on the manuscript.
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Neuroscience Research 20 (1994) 131-135
Update article
Latexin: a molecular marker for regional specification in the neocortex Yasuyoshi Arimatsu Mitsubishi Kasei Institute of Life Sciences, 11 Minamiooya, Machida-shi, Tokyo 194, Japan
Received 16 February 1994; revision received 25 May 1994; accepted 25 May 1994
Abstract It largely remains to be elucidated how the mammalian neocortex is regionally specified during development. In an attempt to obtain molecular markers in the neocortex, we have generated a monoclonal antibody PC3.1 which recognizes a subset of neurons located in lateral, but not dorsal, neocortical areas. The antigen is a novel class of protein, named latexin, having a molecular weight of 29 000. Our in vitro studies have revealed that the neocortical regional specification for the production of latexin-positive neurons occurs very early prior to thalamocortical interactions and the completion of neurogenesis, indicating that elements intrinsic to the neocortex play important roles in the neocortical specification. Furthermore, our recent analyses have suggested that this regional specification is attributable, at least in part, to an early restriction of developmental potential in neocortical progenitor cells to become latexin-positive neurons. Keywords: Neocortex; Regional specification; Neurogenesis; Cell differentiation; Migration; Latexin
1. Introduction Although the basic structure of the mammalian neocortex is quite similar, it is divided into various subregions such as visual, somatosensory and motor cortices that contribute to their specialized physiological functions (Gilbert, 1983). The developmental mechanism regulating regional specification within the neocortex is one of the major interests of current neurobiology (Rakic, 1988; O'Leary, 1989; Shatz, 1992; Kennedy and Dehay, 1993). The clarification, however, has been hampered by the extremely complex organization of the neocortex where neurons of diverse phenotypes form a variety of connections with cortical and subcortical structures. The lack of appropriate molecular markers that define specific cell populations of neocortical neurons has contributed to the limited knowledge of the mechanism underlying neocortical development. With the use of morphological characteristics like laminar fate and connectional pattern as an indicator, it is impossible to define neuronal
identity until a neuron of interest has migrated to its final destination and has extended its axons along a pathway unique to its subtype. Cell-type-specific molecular markers in the neocortex would certainly promote studies on cortical development in relatively simple and defined experimental systems. Here I will consider some aspects of regional specification during the early stage of neocortical development in the rat. They have been clarified recently with the use of a novel molecular marker named latexin.
2. Intraneocortical regional specificity represented by latexin-positive neurons In an effort to obtain specific molecular markers in the neocortex, we have generated a monoclonal antibody, designated PC3.1, that detects a neuronal entity confined to infragranular layers within lateral (but not dorsal) neocortical areas in adult rats (Arimatsu et al., 1992). These include the secondary somatosensory, visceral sensory, primary and secondary auditory, and
0168-0102/94/$07.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0168-0102(94)00802-W
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Fig. 1. Distribution of latexin-positiveneurons in the cerebral cortex of the rat. Cortical regions containing latexin-positive neurons (dotted) are superimposedon the schematicillustration of a lateral surface view of the forebrain. Boundaries of cortical areas are shown according to Zilles (1985, 1990)and Zilles et al. (1990). The boundaries between latexin-positive and -negative regions do not necessarily correspond strictly to the cytoarchitectonic boundaries. Parl, for example, contains some latexin-positive neurons on the border with Par2. The claustrum (CI) and endopiriform nuclei (En), which also contain latexin-positive neurons, are also shown (shaded). AI, agranular insular cortex; Ent, entorhinal cortex; Frl, frontal cortex, area 1; Fr2, frontal cortex, area 2; Fr3, frontal cortex, area 3; LO, lateral orbital cortex; Ocl, occipital cortex, area 1; Oc2L, occipital cortex, area 2, lateral; Parl, parietal cortex, area 1; Par2, parietal cortex, area 2; Pir, piriformcortex; PRh, perirhinal cortex; Te 1, temporal cortex, area !; Te2, temporal cortex, area 2; Te3, temporal cortex, area 3; Vi, visceral sensory area. Ocl and Oc2L correspond to the primary visual cortex and the lateral portion of the secondary (association) visual cortex, respectively.Parl and Par2 correspond to the primary and secondary somatosensory cortex, respectively. Tel and Te2/3 correspond to the primary and secondary (association) auditory cortex, respectively.
secondary visual cortices (see Fig. 1). Considering the high degree of similarity in the structure of the neocortex across areas (Rockel, 1960; Zilles, t990; Zilles et al., 1990), it is striking to find that latexin-positive neurons are located only in a limited sector of the neocortex. Although systematic characterization of latexin-positive neurons has not yet been performed for either their morphologies, connectional patterns or neurotransmitter phenotypes, these include typical and atypical pyramidal neurons (Arimatsu et al., 1994) and glutamate-like immunoreactive neurons. The antibody PC3.1 specifically binds a polypeptide epitope on a 29 kDa protein, named latexin (Hatanaka et al., 1994). Analysis of c D N A clones for latexin showed that the predicted primary structure of latexin consists of 223 amino-acid residues with no strict homology to any protein sequences so far known. The latexin genelike sequence is well conserved in the genome of the mouse, rabbit, cat, cow, monkey and human, all mammalian species tested. In situ hybridization analysis showed that latexin m R N A is synthesized in a subset of neurons in the lateral neocortex. These results indicate
that latexin is a novel class of protein that represents intraneocortical regional specificity. To understand the mechanisms underlying regional specification in the neocortex, the region-specific molecular marker, latexin, that identifies and defines a neuronal subset is useful. We have already analyzed some aspects of neocortical specification by using latexin as a marker. The major issues have been: (i) when does regional specification occur for the production of latexin-positive neurons; (ii) to what extent do elements intrinsic to the neocortex and extracortical influences contribute to the regional specification; (iii) whether or not thalamic afferents play critical roles in the regional specification, and (iv) what kind of roles, if any, cell lineage, local cell environment, and cell migration play for the specification.
3. Early regional specification for the production of latexin-positive neurons We have shown that some kind of regional specification in the neocortex occurs very early along the dorsalventral axis. We demonstrated the selective appearance of PC3.1-positive neurons in cultures derived from a lateral, but not dorsal, sector of the neocortical anlage at El2 or later (Arimatsu et al., 1992). Since thalamic and other afferents invade the neocortex at El6 or later (Catalano et al., 1991) and corticofugal axons do not exit the cerebral cortex before El3 (Blakemore and Molnar, 1990; De Carlos and O'Leary, 1992), it can be concluded that the regional specification for the production of latexin-positive neurons is established by elements intrinsic to the neocortex prior to interactions between cortical and extracortical cells. To clarify in more detail the tangential heterogeneity of the neocortical anlage, we examined the in vitro capacity for the production of latexin-positive neurons at various rostrocaudal and dorsolateral levels (Arimatsu et al., 1993). In an organotypic slice culture system, a substantial number of neurons became latexin-positive in the tissue fragments derived from the lateral sector of the neocortical anlage. Within the lateral sector, the number of the latexin-positive neurons was highest in the fragments from the middle portion along the rostralcaudal axis, with progressively decreasing numbers at more rostral and caudal levels. Much fewer latexinpositive neurons were found in cultures from the dorsal neocortex at all rostral-caudal levels. The result indicates that, at least along the dorsal-ventral axis, the distribution pattern of the capacity for the production of latexin-positive neurons correlates exactly with that of latexin-positive neurons in the adult cortex. It remains to be clarified whether or not this is also the case for the rostral-caudal axis. The answer will be available after quantitative examination of latexin-positive neurons along the rostral-caudal axis in the adult cortex. If the
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fetal in vitro pattern and the adult in vivo one are closely correlated to each other also along the rostral-caudal axis, then it can be concluded that there is some kind of tangential 'blueprint' or 'protomap' (Rakic, 1988) in the neocortical anlage for the future intracortical regional specificity. Early regional specification has also been shown for the expression of the limbic system-associated membrane protein (LAMP) in the limbic cortex (Barbe and Levitt, 1991, 1992; Ferri and Levitt, 1993). Furthermore, a recent study using a transgenic mouse line has shown an early regional commitment to the expression of a somatosensory cortex marker (Cohen-Tannoudji et al., 1994). It has, however, been suggested by heterotopic transplantation experiments that certain connectional and cytoarchitectonic features in some neocortical regions are not fully specified at the time of neurogenesis, but that they can be modified during later stages by epigenetic interactions with environmental signals (O'Leary and Stanfield, 1989; Schlaggar and O'Leary, 1991). Thus, it seems likely that neocortical regional specification involves multiple steps including both early and later specification events. With respect to latexin-positive neurons, it will be important to investigate whether extrinsic elements like thalamic innervation, in addition to the predicted intrinsic mechanism, play significant roles in establishing the regional specificity observed in the mature cortex. 4. Neurogenesis of latexin-positive neurons To facilitate a better understanding of molecular and cellular mechanisms underlying early regional specification, it is important to know about time as well as site of generation of these neurons. We found in a study combining [3H]thymidine autoradiography and PC3.1immunohistochemistry that latexin-positive neurons in layers V and VI of the lateral neocortical areas were generated concurrently at El5 (Arimatsu et al., 1994). It was unexpected to find a strict correlation between the latexin phenotype of neocortical neurons and the time of their generation irrespective of their final positions along radial and tangential axes, since previous [3H]thymidine birth-dating studies dealing with neocortical neurons as a single population had shown radial and tangential neurogenetic gradients (Bayer and Altman, 1991; for a review, see Jacobson, 1991). In many mammalian species, earlier-generated neurons in a particular area tend to be located in a deeper layer, and later-generated neurons in a more superficial layer (radial neurogenetic gradient). Neurons in a particular lamina of rostral and ventral regions in the neocortex tend to be born earlier than those in caudal and dorsal regions, respectively (tangential neurogenetic gradients). The simultaneous generation of latexin-positive neurons across tangentially widespread cortical regions indicates
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that laminar and tangential locations of neocortical neurons in the adult animal are not established solely by a combination of mechanisms for inside-out migration of newly generated neurons in each cortical area and for broad tangential neurogenetic gradients. The mechanism involved in neocortical histogenesis should be more complicated than has ever been thought (for a detailed discussion, see Arimatsu et al., 1994). 5. Early restriction of developmental potential in neocortical progenitor cells to become latexin-positive neurons Although early regional heterogeneity within the neocortical anlage is predicted from the experimental evidence described above, it is still unclear when individual progenitor cells or young postmitotic neurons are committed to express latexin. Since McConnell and Kaznowski (1991) suggested that neocortical progenitor cells are initially multipotent, but become committed to a laminar fate by environmental signals acting around their final mitosis, it will be of great interest to know whether the commitment to the latexin phenotype occurs at around El5, the birthdate of latexin-positive neurons in the neocortex (Arimatsu et al., 1994). To address this question, we have begun to examine latexin expression in the fetal neocortical cells cultured under various environmental conditions (Arimatsu and Miyamoto, 1993). When E13 cells from a middle portion of the lateral neocortical anlage, which had been labeled with BrdU in vivo, were cocultured in a monolayer together with an excess of unlabeled cells from either the lateral or the dorsal neocortex, a substantial portion of the BrdU-labeled cells expressed latexin. In contrast, a much smaller proportion of dorsal neocortical cells expressed latexin when cocultured with either lateral or dorsal neocortical cells. These results suggest that the composition of El3 neuroepithelium is regionally different along the dorsal-ventral axis. The simplest hypothesis is that the restriction of the developmental potential for the latexin phenotype has occurred in the progenitor cells in the dorsal sector of the neocortical anlage, whereas some cells in the lateral sector are competent for the differentiation. In addition, it was found that a much greater proportion of El3 lateral neocortical cells became to express latexin when maintained in a reaggregateted-cell culture with excess cells from the lateral neocortex than from the dorsal neocortex (Arimatsu and Miyamoto, 1993). This suggests that differentiation of individual progenitor cells to express latexin can be influenced by signal(s) from surrounding neocortical cells. Thus, the latexin phenotype may be established by a certain combination of multiple mechanisms, but the exact nature of epigenetic signals, if they indeed occur, remains to be elucidated. It has been suggested that, in the vertebrate retina, many of the neuronal types arise from multipotent pro-
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genitor cells depending upon extracellular signals at respective developmental stages but not upon cell lineage (Turner et al., 1990; Watanabe and Raft, 1992; Altshuler et al., 1993). In the cerebral cortex, however, an early lineage restriction for some neuronal phenotypes has been suggested to occur in pyramidal vs nonpyramidal cells (Parnavelas et al., 1991; Luskin et al., 1993), and in glutamate- vs GABA-immunoreactive neurons (Mione et al., 1994). In this connection, it will be of interest to clarify in future whether any lineage restriction of cortical progenitor cells is already established for the production of latexin-positive neurons.
6. Cell migration It has been proposed that young cortical neurons migrate radially from the ventricular zone to their final destination (Rakic, 1988; Misson et al., 1991). However, recent lineage-tracing studies using retroviral vectors as a tracer have raised the possibility that some young neurons or their precursor cells migrate across widespread cortical areas (Walsh and Cepko, 1992). In fact, the extent of tangential dispersion of fluorescently labeled cells observed in a slice of developing cerebral cortex differed from one cell to another (O'Rourke et al., 1992; Fishell et al., 1993). A recent transgenic study demonstrated that both radial mosaicism and tangential cell migration contribute to neocortical development (Tan and Breen, 1993). Thus, it is now necessary to examine the migration of neurons not as a whole but with respect to particular neuronal subpopulations. In this regard, our experiments with the use of latexin as a marker may provide some insight into this problem. Since latexin-positive neurons are cogenerated at E 15 irrespective of their eventual laminar and tangential locations, they are likely to represent a developmentally unique population. This led us to consider possible migratory pathways of presumptive iatexin-positive neurons (Arimatsu et al., 1994). In view of the likelihood that cells in the dorsal neocortical anlage at El3 are already incompetent to become latexin-positive, neurons expressing latexin in the adult lateral neocortex should originate in the lateral but not the dorsal sector of the neocortical anlage. Thus, presumptive latexin-positive neurons are not likely to migrate extensively in the tangential plane, at least from the dorsal to the lateral sector, in the developing neocortex.
7. Concluding remarks Having used latexin as a molecular marker, it was possible to clarify some aspects of regional specification and cell-type determination during the early phase of neocortical development. The following conclusions have been drawn: (i) Some kind of regional specification has occurred very early within the neocortical anlage as
to the capacity to produce latexin-positive neurons. (ii) Restriction of developmental potential in progenitor cells to become latexin-positive neurons is likely to contribute to this early regional specification. (iii) An extensive tangential migration of young neurons from the dorsal to the lateral sector of the developing neocortex is likely to play no or, if any, very limited roles in the parcellation of latexin-positive neurons. Now that we know the primary structure of latexin and its mRNA, it will be possible to analyze further the molecular mechanism of the regional specification and the cell-type determination by examining the way in which latexin synthesis is initiated and controlled. The recent evidence supports the idea that transcription factors and secreted proteins contribute to the regional specification of the midbrain, hindbrain and spinal cord in the vertebrate species (McGinnis and Krumlauf, 1992; McMahon, 1992; Basler et al., 1993; Carpenter et al., 1993). Moreover, it has been shown that some transcription factors and regulatory proteins are expressed in the mouse forebrain (Puelles and Rubenstein, 1993). Then, it will be important to investigate whether or not similar mechanisms operate during the early phase of specification events within the neocortex.
Acknowledgements The author wishes to thank Drs. Yumiko Hatanaka, Keiko Takiguchi-Hayashi, Yoshihiko Uratani and Shinobu C. Fujita for their kind comments on the manuscript.
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