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Generation of a Radial-Like Glial Cell Line

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Generation of a Radial-Like Glial Cell Line Generation of a Radial-Like Glial Cell Line David R. Friedlander,1 Perry A. Brittis,* Takeshi Sakurai,1 Bronya Shif,1 William Wirchansky,1 Gord Fishell,2,3 Martin Grumet1 1Department of Pharmacology, NYU Medical Center, 550 First Avenue, New York, New York 10016 2Department of Cell Biology, NYU Medical Center, 550 First Avenue, New York, New York 10016 3Skirball Developmental Genetics Program, NYU Medical Center, 550 First Avenue, New York, New York 10016 Received 9 April 1998; accepted 9 June 1998 ABSTRACT: Rat C6 glioma is a cell line that has fraction (;60%) of the C6-R cells in the cortex assumed been used extensively as a model of astroglia. Although a radial orientation and about half of these (;30%) this cell line retains many of the properties of developing made contact with the pial surface. In contrast, the glia, it does not resemble morphologically the specialized parental C6 cells generally formed aggregates and only form of glia found embryonically, the radial glia. In displayed a radial alignment when associated with blood experiments designed to study a mutant form of recep- vessels. These results suggest that we have generated a tor protein tyrosine phosphatase b, we isolated a sub- stable cell line from C6 glioma which has adopted cer- clone of C6 called C6-R which, like radial glia, assumes tain key features of radial glia, including the ability to a highly polarized radial-like morphology in culture. promote neuronal migration in culture and integrate C6-R cells and, to a somewhat lesser extent, C6 cells, radially in vivo in response to local cues. This cell line express cytoskeletal proteins found in developing astro- may be particularly useful for studying receptors on glia including glial fibrillary acidic protein and RC1. As radial glia that mediate neuronal migration. © 1998 John seen with radial glia, cerebellar granule cell bodies and Wiley & Sons, Inc. J Neurobiol 37: 291–304, 1998 neurites migrated along radial processes of C6-R cells in Keywords: radial glia; C6 glioma; cerebellar granule culture. Morphological analysis of dye-labeled cells in- cells; neuronal migration; RPTPb jected into the developing forebrain revealed that a large Glial cells play important roles during development as during development and are thought to give rise to well as in the mature nervous system. The first glia to astroglia (Culican et al., 1990; Levitt et al., 1983; develop are the radial glial cells which serve as guides Alvarez-Buylla et al., 1990; Misson et al., 1988; Gray for radial migration of neural cells from proliferative and Sanes, 1992; Voigt, 1989). Most astroglia are zones to their postmitotic destinations (Rakic, 1990). born after the wave of neurogenesis has subsided, and In contrast to the adult cerebellum and retina where in mammals they increase to constitute the major cell radial glia are found as the Bergmann glia and Mu¨ller type in the mature mammalian nervous system (Ja- fibers, respectively, in most regions of the central cobson, 1991). nervous system radial glia are found only transiently Radial glia are found in regions of the nervous system where laminated structures develop, including *Present address: Department of Neuroscience, Brown Univer- the cortex, cerebellum, spinal, cord and retina (Ramo´n sity, Providence, RI 02912 y Cajal, 1995). They appear very early when the Correspondence to: M. Grumet Contract grant sponsor: NIH; contract grant number: NS33921 neuroepithelium is relatively thin and persist into later © 1998 John Wiley & Sons, Inc. CCC 0022-3034/98/020291-14 developmental stages where they span considerable 291 292 Friedlander et al. distances from the ventricle to the pia. Studies of modified Eagle’s medium (DMEM; Biowhittaker, Walkers- radial glia in vivo indicate that they serve as guides for ville, MD)/10% fetal calf serum (FCS; Life Technologies, radial migration of neurons from proliferative zones Gaithersburg, MD). DNA encoding the human receptor pro- b b (Rakic, 1990; Hatten, 1993). Cell culture experiments tein tyrosine phosphatase- (RPTP ) short form (nucleotides 9 have confirmed that neurons can migrate along radial 1–2655, ClaI site at the 3 end) in Bluescript SKII was kindly provided by Dr. G. Barnea and Dr. J. Schlessinger at the NYU glia (Edmondson and Hatten, 1987). Moreover, anti- Medical Center. The ClaI and ApaI (from the multicloning body perturbation studies indicate that the migration site) fragment of this clone was replaced with a poly- involves astrotactin (Fishell and Hatten, 1991; Zheng merasechain reaction (PCR) fragment encoding an hemagglu- et al., 1996) from the neurons and a neuron–glia tinin (HA) tag followed by a stop codon in frame. Primers junctional protein (Anton et al., 1996). To obtain for PCR were 59-GCAATCGATACGGCCGCATCTTTTA- primary cells as radial glia in culture, however, it is CCC-39 (ClaI site is underlined) and 59-CGTGGGCCCTAG- necessary to culture them with neurons or extracts CACTGAGCAGCGTAATCTG-39 (ApaI site is underlined from brain (Hatten, 1985; Hunter and Hatten, 1995). and stop codon is in bold); the PCR template was DNA This need for extrinsic factors to maintain radial glia encoding HA tag sequences in the NotI site of Bluescript SK, in culture makes it difficult to study these cells in kindly provided by Dr. Schlessinger (Hu et al., 1993). The isolation. Much remains to be learned about these construct was cut by SacI (multicloning site), treated with Klenow fragment in the presence of four deoxynucleotide cells, and the availability of radial glial cell lines triphosphates, digested again with ApaI, and inserted into would make this specialized cell type amenable for pCDNA3 (Invitrogen, San Diego, CA) using EcoRV and ApaI. the first time to molecular and biochemical analysis in Sequences for both strands were confirmed by sequencing. vitro. Twenty micrograms of DNA was mixed with 50 mL of lipo- Studies of astroglia have benefited from investiga- fectin (Life Technologies) and transfected into C6 glioma cells tions of brain tissue as well as the use of primary cells using opti-MEM media (Life Technologies). Stable transfec- and cell lines in culture. Most glial cell lines have tants were selected with G418 (1.5 mg/mL effective concen- been derived from gliomas, and some have been es- tration) and more than 50 colonies were picked from three tablished by immortalizing cells (McKay et al., 1990) independent transfections. Each cell line was screened by or by clonal selection (Alliot and Pessac, 1984). Tu- Western blotting of cell extracts using monoclonal antibody mor cell lines retain many of the hallmarks of astro- 12CA5 against the anti-HA tag (Boehringer Mannheim, Indi- anapolis, IN). glia and have been useful models for molecular and cellular studies of many of their characteristics. While cell lines have been useful for studying various prop- Cell Culture and Interactions erties of astroglia, radial glial cell lines have not yet with Neurons been established. In the course of selecting C6 rat glioma cells that C6 and its transfectants including C6-R were maintained in were transfected with a plasmid encoding a truncated DMEM/10% FCS. For immunofluorescence studies, cells receptor phosphatase (Levy et al., 1993), we obtained were plated onto 2.5-mm wells of 26-well slides (Cel-Line a clone called C6-R that exhibited remarkable radial Associates, Newfield, NJ). Interactions with neurons were morphology in culture. Interestingly, the majority of observed using either dissociated cerebellar granule cells on sparse glia or aggregates of granule cells on confluent glial the C6-R cells had highly polarized morphologies monolayers. Confluent cell monolayers of C6 or C6-R cells following injection of labeled cells into the develop- were prepared by growing 3 3 104 cells per 4-mm well of ing forebrain. In this article, we characterize this 24-well glass slides (Cel-Line Associates) for 2–4 days. radial-like cell line by comparing its properties with Cerebellar granule cells from P4 rats were prepared by parental C6 glioma cells. The results of this analysis trypsinization, fractionation on a two-step (35% and 60%) suggest that we have identified an immortalized cell Percoll gradient (Sigma, St. Louis, MO), and preplating on line with phenotypic properties of radial glia. As such, tissue culture dishes coated with poly-L-polylysine (Sigma) this line provides a valuable model for this elusive cell as described (Hatten, 1985). A total of 105 dissociated type. granule cells were seeded in wells with sparse glia. Aggre- gates of granule cells were formed during overnight incu- bation with shaking (Friedlander et al., 1996), and approx- imately 10 aggregates of labeled cerebellar granule cells MATERIALS AND METHODS were added to each confluent monolayer. The migratory behavior of 1,19-dioctadecyl-3,3,39,39-tetramethyl-indocar- Establishment of C6-R Cell Line bocyanine perchlorate (DiI)-labeled granule cell bodies and neurites was observed under phase and fluorescence micros- C6 glioma cells were obtained from the American Type Cul- copy. Static and time-lapse recordings were made following ture Collection (Bethesda, MD) and cultured in Dulbecco’s overnight incubation. The patterns of the neuronal aggre- Generation of Radial-Like Glial Cell Line 293 gates on confluent glial monolayers were obtained at 1 and 1995; Fishell and van der Kooy, 1987). In brief, following 27 h after introduction of the aggregates. Images were laparotomy, the uterine horns were exposed and embryos digitized using a Cohu CCD camera and a Scion image visualized through transillumination. Using the calvarian grabber, and analyzed using NIH Image software. To quan- sutures as a landmark, 1 mL of cell suspension containing tify the extent and polarity of cell body migration, the area 1.5 3 107 cells/mL in Ham’s F12 medium was injected defined by the margins of the region occupied by labeled through the uterine wall into the cerebral ventricles of host cells, as well as the length of the major and minor axes of embryo forebrains; the use of Fast green confirmed injection the associated fitted elipses, were obtained.
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