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Neurons, Astrocytes, and Oligodendrocytes of the Rat Cerebral Cortex Originate from Separate Progenitor Cells: an Ultrastructural Analysis of Clonally Related Cells

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Neurons, Astrocytes, and Oligodendrocytes of the Rat Cerebral Cortex Originate from Separate Progenitor Cells: an Ultrastructural Analysis of Clonally Related Cells The Journal of Neuroscience, April 1993, 134): 1730-l i’50 Neurons, Astrocytes, and Oligodendrocytes of the Rat Cerebral Cortex Originate from Separate Progenitor Cells: An Ultrastructural Analysis of Clonally Related Cells Marla B. Luskin,ls2 John G. Parnavelas,3 and James A. Barfield’** ‘Department of Anatomy and Cell Biology and *Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia 30322 and 3Department of Anatomy and Developmental Biology, University College London, London WClE 6BT, United Kingdom The diverse array of neurons and glia in the mammalian cytes, or all neurons. Furthermore, each particular cell type cerebral cortex arises from proliferating cells of the ventric- exhibited a different pattern and intensity of staining. The ular zone that surrounds the lateral ventricles of the devel- neuronal clones, with one exception, were composed of ei- oping brain. A fundamental but unresolved question is ther all pyramidal cells (projection neurons), or all nonpy- whether the individual cells of the ventricular zone are com- ramidal cells (interneurons). The size and composition of mitted to producing progeny of only one particular pheno- neuronal clones did not seem related to their position in the type or whether they generate progeny of more than one cerebral cortex. phenotype. We have begun to address this question by ask- Collectively, our observations indicate that separate pro- ing if individual cells of the ventricular zone generate exclu- genitor cells exist for pyramidal neurons, nonpyramidal neu- sively neurons or glia at the onset of cortical neurogenesis rons, astrocytes, and oligodendrocytes. The striking phe- in the rat. notypic homogeneity in the clones arising from individual To assess the phenotypes of cells derived from a common progenitor cells suggests that by the onset of cortical neu- progenitor cell, retroviral-mediated gene transfer was used rogenesis, at least some lineage restrictions have already to introduce the repotter gene, Escherichia co/i B-galacto- occurred among the precursor cell population. Thus, our re- sidase, into ventricular zone cells at embryonic day 15 or sults suggest that lineage may play a pivotal role in deter- 16. We used histochemistry to reveal B-galactosidase-ex- mining some of the functionally important phenotypic attri- pressing cells in the mature rat cerebral cortex. Isolated butes of cells in the cerebral cortex. clusters of B-galactosidase-expressing cells, presumably [Key words: lineage, recombinant retrovirus, glia, pyra- clones, were identified in serial sections. Since the histo- midal cells, nonpyramidal cells, cortical development, inter- chemical reaction product is electron dense, each cell could neurons, phenotype, cell identity] be examined at the ultrastructural level and assigned defin- itively to one of the major classes of cells in the cerebral Many aspectsof the development of the mammalian cerebral cortex on the basis of well-established morphological cri- cortex have been elucidated by analyzing the patterns of gen- teria. This approach overcomes the problems of cell type eration and migration of cells destined for the telencephalon identification encountered with light microscopy, where it is (for review, seeMcConnell, 1988, 1991). A generalobservation not always possible to distinguish between different cell is that the early development of the cerebral cortex is strikingly phenotypes. similar in all speciesof mammals examined. The ventricular We found that virtually all clones contained cells of ex- zone, a layer of seemingly homogeneouscells, surrounds the clusively one type: either all astrocytes, all oligodendro- lateral ventricles and is the sourceof all neuronsand glia in the cerebral cortex. Once generated, neurons migrate toward the pial surface and complete their differentiation in the cortical plate, the forerunner of the mature cerebral cortex. Both the generation and migration of neurons occur over an extended Received May 27, 1992; revised Aug. 3 1, 1992; accepted Nov. 10, 1992. period. Neurons destined for the deep layers of the cortex are We are extremely grateful to Eileen Breding for photographic assistance, to Meean Maultsbv and Eva Franke for technical assistance. and to Dr. W. Scott generated and then migrate away from the ventricular zone Clark for advice on statistical analysis. We also extend our appreciation to Drs. earlier than neurons destined for progressively more superficial Joel Glover, Joshua Sanes, Carla Shatz, and Donald Wigston for helpful comments on the manuscript. M.B.L. was supported by a fellowship from the Alfred P. Sloan layers. In many species,including the rat, neurogenesisoccurs Foundation, a Biomedical Scholars Award from the Pew Charitable Trusts, and prenatally, although migration of cortical neurons may extend a Basil O’Connor Starter Scholar Research Award from the March of Dimes Birth into the postnatal period. On the other hand, in the developing Defects Foundation (5-FY90-798). Additional support was provided by a grant from NIH (NS28380), the University Research Committee of Emory University, telencephalon the greatest production of astrocytes and oligo- and NATO to M.B.L and the MRC to J.G.P (Grant G9006412N). dendrocytes occurs postnatally, after the production of neurons Correspondence should be addressed to Dr. Marla B. Luskin, Department of has ceased,although glial cells are also derived initially from Anatomy and Cell Biology, Emory University School of Medicine, Atlanta, GA 30322. progenitor cells of the ventricular zone. Even though much is Copyright 0 1993 Society for Neuroscience 0270-6474/93/131730-21$05.00/O known about the sequenceofevents underlying the development The Journal of Neuroscience, April 1993, 13(4) 1731 of the mammalian cerebral cortex, less is known about how cells Materials and Methods of the cerebral cortex acquire their identity. Current hypotheses suggest that the determination of cell fate in the cerebral cortex Production of retrovirus. Two different replication-defective recombi- involves a series of decisions restricting the potential of a pro- nant retrovirnses were used to analyze lineage relationships among cells genitor cell or its offspring. The long-term goal of our study is in the cerebral cortex. The construction and production of the retro- viruses have been described in detail elsewhere (Bonnerot et al., 1987; to reveal how, when, and where these decisions are made. Price et al., 1987). Briefly, the recombinant retrovirnses were constrnct- To generate the many cell types that characterize the complex ed by replacing the viral genes gag, pal, and env of the Maloney murine mature cerebral cortex, the developing nervous system must leukemia virus, with the E. coli P-galactosidase gene, abbreviated lacZ. have the ability to regulate cellular production and differenti- We adhere to the convention of abbreviating the encoded gene product ation. A number of mechanisms have been proposed to explain of 1ucZ as 1acZ. Most of the animals used in our studies received an injection of the BAG retrovirns vector (Price et al., 1987; ATTC CRL how cell diversity in the cerebral cortex arises (for review, see 1858). In the progeny of cells infected by the BAG retrovirus, the fl-ga- Levitt et al., 198 1). Some authors have suggested that cells re- lactosidase gene product is localized predominantly in the cytoplasm. main multipotential even after migrating away from the ven- The other retroviral vector used, nls-1acZ (Nuclear Localizing Signal) tricular zone (for review, see Rakic, 198 1). Multipotentiality has (Bonnerot et al., 1987) targets the P-galactosidase gene product to the nucleus. Expression of &galactosidase in the BAG vector construct is been construed as evidence that environmental factors, such as driven by the viral long terminal repeat sequence, and in the nls-1acZ cell-cell interactions, control the fate of cells. However, recent vector, expression is controlled by the SV40 early promotor. reports have suggested that a cortical cell’s identity may be The replication-defective recombinant retrovirns of both BAG and specified even before it leaves the ventricular zone, implying a nls-1acZ was obtained by collecting and filtering the culture medium used to grow the virus-producing cells. The NLS recombinant retrovirns greater influence of inherited factors, such as cytoplasmic de- producer cell line was generously provided by Dr. Gary Nolan (MIT, terminants, in dictating cell fate (Levitt et al., 198 1, 1983; Levine Cambridge, MA). The BAG virus-producing cells purchased from ATTC and Goldman, 1988; Luskin et al., 1988). To further our un- were subcloned to generate a higher-titer-producing cell line. Titers of derstanding about the contribution of inherited and environ- retrovirus, determined according to published procedures (Cepko, 1990), mental factors to the determination of cell fate, we have at- were in the range of lo4 colony-forming units (cfu)/ml for nls-1acZ and lo* to lob cfu/ml for BAG. In addition, a greater than IO-fold increase tempted to elucidate the lineage relationships of the different in viral titers was obtained by concentrating the BAG virus, which cell types in the cerebral cortex. A fundamental question is proved unnecessary. Using a standard test, as described by Luskin et whether the cells of the ventricular zone are as homogeneous al. (1988) each lot of recombinant retrovirus used in our experiments as their morphological similarity suggests (Sauer, 1935; Hinds was proven not to manufacture
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