1 Wolfson Institute for Biomedical Research, The Cruciform Building, University College London, Gower Street, London WC1E 6AE, UK
2 Department of Pediatric Oncology and
3 Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02115, USA
4 Laboratory of Metabolism, National Cancer Institute, NIH, 9000 Rockville Pike, Bethesda, MD 20892, USA
5 Western Regional Research Center, ARS, United States Department of Agriculture, Albany, CA 94710, USA
* Present address: Laboratory of Molecular Neurobiology, Karolinska Institute, Berzeliusväg 3, 17177 Stockholm, Sweden
Author for correspondence (e-mail: w.richardson{at}ucl.ac.uk)
Accepted April 10, 2001
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SUMMARY |
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Key words: Telencephalon, Hedgehog signalling, Oligodendrocyte, Rat, Mouse
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INTRODUCTION |
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The origins of oligodendrocytes at more anterior levels of the neuraxis are less well established. Timsit et al. showed that the myelin proteolipid protein gene Plp/Dm20 is expressed in the ventral neuroepithelium of the embryonic mouse diencephalon from as early as E9 (Timsit et al., 1992); they proposed that this region later goes on to generate oligodendrocytes. Pringle and Richardson (Pringle and Richardson, 1993) described a cluster of Pdgfra-positive presumptive oligodendrocyte progenitors (OLPs) in the ventral forebrain of the rat embryo that appeared to proliferate and migrate throughout the developing forebrain. Spassky et al. and Perez-Villegas et al. also provided histological evidence for a ventral source of oligodendrocytes in the rodent and chick forebrains, respectively (Spassky et al., 1998; Perez-Villegas et al., 1999). In keeping with all these studies, it has been reported that precursor cells from E15 rat striatum (ventral telencephalon) have a greater propensity to generate oligodendrocytes than do precursors from the neocortex (dorsal telencephalon), either when cultured in vitro (Birling and Price, 1998) or when transplanted into the eye (Kalman and Tuba, 1998).
It therefore seems likely that there is a region of the ventral forebrain that is specialized for oligodendrogenesis. The experiments reported here further define the location of this site and explore how and when it is established. Our data support the idea that oligodendrogenesis in the rodent telencephalon depends on a localized source of SHH in the ventral forebrain. Even in cultures derived from embryonic neocortex, which does not appear to express SHH or related molecules in situ, generation of oligodendrocyte progenitors was blocked by cyclopamine and appeared to depend on SHH and/or IHH produced in the cultures. Altogether, the available data suggest that at least some oligodendrocytes in the cortex might be derived from progenitor cells that originate in the basal telencephalon as, for example, the precursors of certain cortical neurons (Parnavelas, 2000).
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MATERIALS AND METHODS |
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Tissue preparation and in situ hybridization
The heads of embryos were fixed by immersion in 4% (w/v) paraformaldehyde (PFA) in phosphate-buffered saline (PBS) pH7.5 for 24 hours at 4°C. To aid fixation, the skin and skull were removed from E14.5 and older embryos. Tissue was cryoprotected in 20% (w/v) sucrose in PBS and embedded in OCT (Raymond Lamb). Cryosections (15-20 µm) were collected on Vectabond-coated slides (Vecta labs). Our in situ hybridization protocol was as described before (Sun et al., 1998), except that we omitted proteinase K digestion prior to hybridization. The mouse Pdgfra probe was transcribed from a approx. 1.6 kb EcoRI cDNA fragment encoding most of the extracellular domain of mouse PDGFRA cloned into pBluescript KS (Mercola et al., 1990); the probe was generated using T7 RNA polymerase (T7pol) from HindIII-cut plasmid. The rat Pdgfra probe was transcribed (T7pol, HindIII) from an approx. 1.5 kb cDNA encoding most of the extracellular domain of rat PDGFRA cloned into PGEM1 (Pringle et al., 1992). The Shh probe was transcribed (T3pol, KpnI) from an approx. 2.6 kb full-length rat cDNA (XhoI fragment) in pBluescript SK (from A. McMahon, Harvard Medical School). The Ptc1 (Ptch) probe was transcribed (T7pol, HindIII) from a 841 bp partial cDNA (EcoRI fragment) from the 5' end of mouse Ptc1 (Ptch) cloned into pBluescript II KS (also from A. McMahon). The Sox10 probe was transcribed (T7pol, SacI) from plasmid pZL1/sox10.7.7.1, which contains an approx. 2.3 kb partial rat cDNA (Kuhlbrodt et al., 1998). The Olig1 probe was transcribed (T7pol, HindIII) from a plasmid containing 986 bp of a rat cDNA (pBSRGUTR; Lu et al., 2000). The Olig2 probe was transcribed (T3pol, EcoRI) from a plasmid containing an approx. 1 kb mouse cDNA (pRBRAH16; Lu et al., 2000). RNA polymerases were from Promega and the DIG labelling mix from Roche. Hybrids were detected with alkaline phosphatase-conjugated anti-DIG antibodies, using NBT and BCIP (both from Roche) as substrates. To increase sensitivity, 5% (w/v) polyvinyl alcohol was included in the final colour reaction.
Brain cell cultures
Dissociated cell cultures were established from embryonic rat or mouse brain (minus brainstem), neocortex or basal ganglia as previously described for spinal cord cultures (Hall et al., 1996). Brains were dissected in Hepes-buffered minimal essential medium (MEM-H) and meningeal membranes removed. The tissue was transferred to Earles balanced salt solution without calcium or magnesium (EBSS; Gibco BRL) containing trypsin (0.0125% w/v) and DNaseI (0.005% w/v) and incubated at 37°C in 5% CO2 for 30 minutes. Cells were dissociated by gentle trituration in the presence of 10% fetal calf serum (FCS), washed by centrifugation and re-suspended in Dulbeccos modified Eagles medium (DMEM) containing 4% FCS. Cells were plated on poly-D-lysine-coated glass coverslips (3x105 cells in 50 µl) and allowed to attach at 37°C. 350 µl of defined medium (Bottenstein and Sato, 1979) was added and incubation continued at 37°C in 5% CO2.
For explant cultures, fragments of E15.5 rat neocortex or basal ganglia were dissected in MEM-H and cultured in collagen gels (Guthrie and Lumsden, 1994) in defined medium (Bottenstein and Sato, 1979) containing 0.5% FCS.
Immunoselection
A single-cell suspension of E19 rat brain (minus brainstem), neocortex or striatum, prepared as described above, was passed over two bacteriological Petri dishes coated with monoclonal antibody Ran-2 (Bartlett et al., 1981) to remove astrocytes, meningeal cells and macrophages (the latter by non-specific adherence to plastic), then over a dish coated with anti-PDGFRA rabbit serum (#3979; Fretto et al., 1993) as described by Hall et al. (Hall et al., 1996). The PDGFRA-positive (PDGFRA+) cells were removed from the panning dish with trypsin and plated (1000 cells in a 3 µl droplet) on 6 mm diameter poly-D-lysine-coated glass coverslips in defined medium (Bottenstein and Sato, 1979) containing 0.5% or 10% FCS.
Immunocytochemistry
Cells on coverslips were lightly fixed in 4% (w/v) PFA in PBS for 5 minutes at room temperature and washed in PBS. The following primary antibodies were used: anti-PDGFRA rabbit serum, (#3709; Fretto et al., 1993) diluted 1:100 in PBS, monoclonal antibody A2B5 (Eisenbarth et al., 1979), anti-NG2 rabbit serum (Chemicon) or monoclonal anti-NG2 (clone N11.4; Stallcup and Beasley, 1987; Levine and Stallcup, 1987), monoclonal antibody O4 (Sommer and Schachner, 1981; Sommer and Schachner, 1982; Bansal et al., 1992), monoclonal anti-galactocerebroside (GC; Ranscht et al., 1982; Bansal and Pfeiffer, 1992) monoclonal anti-glial fibrillary acidic protein (GFAP; clone GA-5, Sigma). For intracellular antigens the cells were made permeable with 0.1% (v/v) Triton X-100 in PBS. Primary antibody treatments were for 1 hour in a humid chamber at room temperature. Fluorescent secondary antibodies (Perbio Science, UK) were applied for 30 minutes at room temperature. Cells were post-fixed for 5 minutes in 4% (w/v) PFA in PBS and mounted under coverslips in Citifluor (City University, UK).
Cultured explants were fixed with 4% PFA in PBS for 10 minutes at room temperature and washed in PBS. Antibody labelling was at 4°C overnight.
RT-PCR
RNA was prepared from freshly dissociated or cultured cells using Trizol® reagent (Gibco BRL). Reverse-transcription was carried out using the SuperscriptTM first-strand synthesis system (Gibco BRL). Primers for polymerase chain amplification (PCR) of mouse or rat cDNAs were as follows: Shh, 5'-GTG ATG AAC CAG TGG CCT GG-3' and 5'-GCC GCC ACG GAG TTC TCT GC-3'; Ihh, 5'-GGC CAT CTC TGT CAT GAA CC-3' and 5'-CAG CCA CCT GTC TTG GCA GC-3'; Dhh, 5'-GTG CGC AAG CAA CTT GTG CC-3' and 5'-GAA TCC TGT GCG TGG TGG CC-3'; Gapdh, 5'-CCC AGA ACA TCA TCC CTG C-3' and 5'-GCC ATG AGG TCC ACC ACC C-3'. Typically, we employed a 38 cycle PCR reaction, except for Gapdh where the number of cycles was reduced to 30 to avoid saturation.
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RESULTS |
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Expression of Sox10, Olig1 and Olig2 in the telencephalon
Recent studies have shown that two novel SHH-inducible bHLH genes, Olig1 and Olig2, as well as the HMG protein Sox10, are expressed in oligodendrocyte lineage cells in the spinal cord, including the oligodendrogenic part of the ventral VZ where Pdgfra+ progenitors originate (Kuhlbrodt et al., 1998; Lu et al., 2000; Zhou et al., 2000; Takebayashi et al., 2000). We therefore compared expression of Sox10, the Olig genes and Pdgfra in the rat telencephalon. At E14.5 all of these were expressed in the anterior hypothalamic neuroepithelium and in scattered cells in the adjacent SVZ and mantle zone (Fig. 3A-F). Olig2 was more widespread in the neuroepithelium than the others, extending through the MGE and LGE.
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PDGFRA+ cells in the embryonic rat brain are oligodendrocyte progenitors
To test directly whether PDGFRA+ cells in the telencephalon are OLPs, we purified them from E19 rat brains (minus brainstem), neocortex or striatum by immunoselection with antibodies raised in rabbits against PDGFRA (Hall et al., 1996; A. C. Hall, PhD thesis, University of London, 1999; see Materials and Methods). After overnight culture in defined medium containing 0.5% FCS and 10 ng/ml PDGF-AA, which stimulates progenitor cell proliferation and inhibits differentiation (Raff et al., 1988; Richardson et al., 1988; Noble et al., 1988), more than 99% of the immunoselected cells had the morphological and antigenic features of early OLPs: i.e. they were small process-bearing cells that labelled with anti-NG2 proteoglycan (Fig. 4A) in addition to anti-PDGFRA and monoclonal A2B5 (not shown). They did not label with antibody O4 or anti-GC, which identify later stages of the oligodendrocyte lineage, nor with anti-glial fibrillary acidic protein (anti-GFAP; not shown). After overnight culture in the presence of PDGF, the medium was replaced with defined medium lacking PDGF and containing either 0.5% or 10% FCS. After a further 36 hours in the presence of 0.5% FCS, the immunoselected cells developed a more highly branched morphology and many were O4+ (not shown), typical of late oligodendrocyte progenitors (Sommer and Schachner, 1982; Bansal and Pfeiffer, 1992). By 48 hours, almost all (>98%) of the immunoselected cells had differentiated into GC+ oligodendrocytes (Fig. 4B). In the presence of 10% FCS, most of the immunoselected cells differentiated into GFAP+ astrocytes (Fig. 4C), many of which also labelled with A2B5 (Fig. 4D). Therefore, the great majority of PDGFRA+ cells in the late embryonic forebrain, including the neocortex, resemble the oligodendrocyte-type-2 astrocyte (O-2A) progenitors previously characterized in cultures of cells from perinatal rat optic nerve (Raff et al., 1983) or spinal cord (Hall et al., 1996). We refer to these as OLPs. These data are in accord with previous observations in situ showing that PDGFRA is co-expressed with markers for early stages of the oligodendrocyte lineage in several regions of the brain including the cerebral cortex (Ellison and de Vellis, 1994; Nishiyama et al., 1996).
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We cultured E10.5 mouse ventral forebrain cells in the presence or absence of the drug cyclopamine, which inhibits HH signalling (Cooper et al., 1998; Incardona et al., 1998; Taipale et al., 2000). The cells were then fixed and OLPs visualized with anti-NG2 antibodies. In the absence of cyclopamine, significant numbers of NG2+ process-bearing progenitor cells appeared between 4 and 6 days in vitro (4-6DIV; Fig. 5). Cyclopamine strongly inhibited the appearance of these cells (Fig. 5), suggesting that in the forebrain, as in more posterior regions of the CNS, oligodendrogenesis is under the control of SHH and/or other related hedgehog molecules.
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Control explant cultures of E15.5 rat MGE (Fig. 8C, D) or E18.5 neocortex (not shown) contained numerous PDGFRA+ NG2+ cells after overnight incubation and they increased in number during the culture period (Fig. 8D; Table 1 and not shown). GC+ oligodendrocytes also developed in these cultures after 8DIV (not shown). In contrast, no PDGFRA+ or NG2+ progenitor cells were generated from E15.5 cortical explants either overnight or after 4DIV (Fig. 8A; Table 1). However, if E15.5 cortical cells were cultured for a longer period of time 6DIV or more many PDGFRA+ NG2+ OLPs did then appear (Fig. 8B and Table 1). Adding cyclopamine to E15.5 cultures strongly inhibited production of OLPs in a dose-dependent manner (Fig. 9A,B), indicating that in long-term cortical cultures SHH or a related molecule (IHH or DHH) was responsible for OLP development. This conclusion was supported by the fact that we could detect mRNA encoding SHH and IHH in cultured cells from E15.5 cortex (Fig. 9C).
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DISCUSSION |
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Oligodendrogenesis in the ventral telencephalon appears to be dependent on locally produced SHH from ventral neuroepithelial cells. The neuroepithelial expression domain of Pdgfra in the anterior hypothalamus is nested within broader expression domains of Shh and its receptor Ptc1, indicating that the forerunners of Pdgfra+ progenitors are normally exposed to and responsive to SHH. Oligodendrogenesis in cultures of rat ventral forebrain cells was blocked by cyclopamine, an inhibitor of HH signalling pathways. Moreover, Pdgfra+ OLPs did not develop in the VZ or SVZ of the ventral forebrain of Nkx2.1 null mutant mice, which lack the telencephalic domain of Shh expression. Further evidence for the involvement of SHH comes from in vivo gain-of-function experiments in which SHH was ectopically expressed in the telencephalic neuroepithelium of mouse embryos using a retrovirus vector (Nery et al., 2001). SHH-expressing cells developed into OLPs and later into oligodendrocytes (Nery et al., 2001). These cells would not normally express SHH persistently but, nevertheless, this experiment provides a striking demonstration of the OLP-inducing activity of SHH in vivo.
Pdgfra+ progenitors did appear belatedly in the telencephalon of Nkx2.1 null mice. However, they first appeared in small numbers scattered outside the germinal zones, not tightly packed within the VZ and SVZ as in wild-type mice, suggesting that Pdgfra+ cells might migrate into the Nkx2.1 null forebrain from more caudal regions where Shh expression is unaffected in the mutant. OLPs also appeared in cultures derived from Nkx2.1 null ventral forebrain, where there was no possibility of immigration from other brain regions. However, their production was suppressed by cyclopamine and furthermore we could detect expression of Shh and Ihh in the cultures, so HH signalling was presumably responsible for OLP induction in vitro. Whether the Shh and Ihh expression we detected reflects aberrant up-regulation of these molecules following cell dissociation, or normal expression that is undetectable in situ, we do not know. In any case, it is not necessary to invoke a SHH-independent pathway of oligodendrogenesis to explain the appearance of OLPs in the Nkx2.1 null telencephalon, as suggested recently by Nery et al. (Nery et al., 2001). Indeed, expression of IHH in culture might explain the observation that some OLPs developed in cultures derived from Shh knockout brain (Nery et al., 2001). This could be tested by culturing Shh null brain cells in the presence of cyclopamine. It remains possible that there is a SHH-independent (or HH-independent) route(s) to oligodendrocyte development but this requires further investigation.
Do cortical oligodendrocytes originate in the ventral telencephalon?
A possibility raised by this work is that some cortical oligodendrocytes might develop, not from endogenous cortical precursors, but rather from OLPs that migrate from the ventral forebrain. At present we have no direct evidence for migration. Nevertheless, Pdgfra+ OLPs in the telencephalon appear similar to those in optic nerve or spinal cord, which are known to migrate relatively long distances during development (Small et al., 1987; Miller et al., 1997; Ono et al., 1997; Pringle et al., 1998) or following transplantation into dysmyelinating or demyelinating hosts (e.g. Warrington et al., 1993; Vignais et al., 1993). Moreover, there is a delay in appearance of Pdgfra+ progenitors in the cortex of Nkx2.1 mutant embryos, which are primarily defective in ventral structures (Sussel et al., 1999), consistent with the idea that at least the early-appearing cortical OLPs are derived from the ventral forebrain. There is ample precedent for migration of progenitor cells into the neocortex from ventral telencephalon. For example, the progenitors of many GABAergic non-pyramidal neurons migrate into the developing cortex from the MGE or LGE (Anderson et al., 1997; Lavdas et al., 1999; reviewed by Parnavelas, 2000).
Latent oligodendrogenic potential of cortical precursors
We found that E15.5 rat cortical cells did not generate oligodendrocyte lineage cells in short term (4DIV) cultures, in contrast to cells from E15.5 ventral forebrain or E18.5 cortex. This confirms the finding of Birling and Price (Birling and Price, 1998) that E15 rat cortical cells have a reduced oligodendrogenic capacity in vitro compared to either E15 striatal cells or E18 cortical cells. It also tallies with the experiments of Kalman and Tuba (Kalman and Tuba, 1998) who showed that fragments of E18 rat cortex, but not E15 cortex, generate oligodendrocytes when transplanted into the eye of a new-born rat. Thus, the oligodendrogenic capacity of rat neocortex increases markedly between around E15 and E18, consistent with and as predicted by the in situ hybridization data, which show Pdgfra+ progenitors apparently migrating into the cortex after E16.5 (Figs 3, 4).
Nevertheless, E15.5 rat cortical cells did generate OLPs when cultured for long periods of time (6DIV), demonstrating that rat cortical precursors have latent oligodendrogenic potential. This is analogous to the recent report that E14 rat dorsal spinal cord cells can generate oligodendrocytes in long-term, though not short-term cultures (Sussman et al., 2000). Several previous studies have demonstrated that early cortical cells from rodents can generate oligodendrocytes in vitro (e.g. Williams et al., 1991; Davis and Temple, 1994).
The appearance of OLPs in long-term cultures of E15.5 rat neocortex was puzzling, since we could not detect expression of Shh, Ihh or Dhh in the embryonic cortex by in situ hybridization. However, production of oligodendrocytes in vitro was inhibited by cyclopamine and, in addition, mRNAs coding for SHH and IHH could be detected in the cultures. Perhaps HH expression is normally repressed in the cortex in vivo but under our culture conditions the inhibitory signals are rendered ineffective by dilution or otherwise. Note that Shh is clearly expressed in certain neurons in the rat cortex from about a week after birth (Traiffort et al., 1999 and our unpublished results). Whether this late-onset expression contributes to postnatal oligodendrogenesis is not known.
Is Olig2 oligodendrocyte lineage-specific?
We found that Olig2 is expressed more widely than Olig1, Sox10 or Pdgfra in both the neuroepithelium and the surrounding mantle zone (not shown). Whether the Olig1- Olig2+ cells are OLPs is an open question, since it is by no means clear that the Olig genes and particularly Olig2 are restricted to the oligodendrocyte lineage(s). It is known, for example, that Olig2 is expressed by olfactory neurons and their precursors in the olfactory epithelium (Takebayashi et al., 2000). It is possible that the Olig1- Olig2+ cells in the telencephalon might be related to the pluripotent neuroglial precursors that Goldman and colleagues identified in the germinal zones surrounding the lateral ventricles after birth (Levison and Goldman, 1993; Levison and Goldman, 1997).
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ACKNOWLEDGMENTS |
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