Article |
Address correspondence to Ned Mantei, Institute of Cell Biology, Dept. of Biology, Swiss Federal Institute of Technology, ETH-Hönggerberg, CH-8093 Zürich, Switzerland. Tel.: 41-1-633-3685. Fax: 41-1-633-1190. E-mail: mantei{at}cell.biol.ethz.ch
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Abstract |
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Key Words: ectopic expression; brain; Cre-lox P; newborn; apoptosis
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Introduction |
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In vertebrate glial cell development, the Notch pathway has been implicated in a number of crucial events. Activated Notch promotes the formation of radial glia in the fetal forebrain (Gaiano et al., 2000; Chambers et al., 2001), Schwann cells in dorsal root ganglia (Wakamatsu et al., 2000), and Müller glia in the retina (Furukawa et al., 2000). In cell culture, even transient activation of Notch strongly promotes the differentiation of adult hippocampus-derived multipotent progenitors into astroglia (Tanigaki et al., 2001), and of neural crest stem cells into the Schwann cell lineage (Morrison et al., 2000).
However, the oligodendrocyte (OL) lineage appears to respond somewhat differently. Activation of Notch signaling suppresses rather than promotes the differentiation of OLs from multipotent progenitor cells (Gaiano et al., 2000; Tanigaki et al., 2001). Furthermore, a potential regulatory role of the Notch pathway in mammalian oligodendrocyte precursor cell (OPC) differentiation has been suggested by cell culture studies showing that OPCs derived from the postnatal rat optic nerve could be inhibited in their differentiation by incubation with Notch ligands (Wang et al., 1998).
How proliferation and differentiation are regulated in the OL lineage is only partially understood (Barres et al., 1992; Barres and Raff, 1994; Raff et al., 1998; Kondo and Raff, 2000a,b; Qi et al., 2001; Wang et al., 2001; Zhou et al., 2001). On the basis of morphology and molecular phenotype, cells of the OL lineage can be grouped into OPCs, immature OLs (premyelinating), and mature OLs (myelinating cells; Gard and Pfeiffer, 1990; Hardy and Reynolds, 1991; Armstrong et al., 1992; Miller, 1996). OPCs of the developing spinal cord are generated along the length of the neural tube from a narrow zone in the ventral region of the neuroepithelium (Noll and Miller, 1993; Pringle and Richardson, 1993; Yu et al., 1994). Determination of the OL lineage is promoted by the transcription factors Olig1, Olig2, and Nkx2.2 (Lu et al., 2001; Zhou et al., 2001; Fu et al., 2002), but the exact nature of the interplay among them is not clear. OPCs arising in the ventral spinal cord subsequently proliferate and disperse throughout the developing CNS (for reviews see Miller, 1996; Richardson et al., 1997; Spassky et al., 2000).
Postmitotic OLs appear first in the ventral funiculus and later in the dorsal and lateral funiculi, the large axonal bundles that become the white matter tracts of the spinal cord (Jordan et al., 1989; Yu et al., 1994). However, it remains an open question how OPCs are able to migrate through an already established neuronal system around, over, and along the axons they may ultimately myelinate, before differentiating at the appropriate time point (Blaschuk and ffrench-Constant, 1998). Progression past the OPC stage is strongly delayed and reduced in Nkx2.2-/- mice, suggesting a possible role for this transcription factor in later development of OLs (Qi et al., 2001). Ectopic OPCs arising on overexpression of Notch1 together with Olig2 in chick spinal cord do not mature, which is consistent with a negative role for Notch1 in late stages of differentiation (Zhou et al., 2001). In contrast, Givogri et al. (2002) have reported a transient increase of myelination in some parts of the CNS during the early postnatal weeks in heterozygous-null Notch1 mutants. In vitro studies and correlative evidence based on the regulation of Notch1 and its ligand Jagged1 in the developing optic nerve have also implicated the Notch pathway in the control of the timing of OPC differentiation (Wang et al., 1998). This is a tantalizing idea supporting a model in which loss of responsiveness to growth factors may permit OPC differentiation, but local cues (e.g., the Notch pathway) regulate the timing of final maturation in different myelinated tracts (Blaschuk and ffrench-Constant, 1998). Unfortunately, further support for this model has not been particularly forthcoming over the last years and several crucial questions remain open: is the observed function of Notch signaling in the control of rat OPC differentiation restricted to the optic nerve or is it a general phenomenon throughout the rodent CNS? Is the observed effect specifically Notch1 dependent or may other members of the Notch family be involved (Notch 24; Lindsell et al., 1996; Irvin et al., 2001)? Are the findings with isolated OPCs in vitro directly transferable to the in vivo situation, in particular given the recent reappraisal that OL development is under stringent axonal control and the proposed interactions of OPCs with mature OLs via the Notch pathway in tissue (Wang et al., 1998; Barres and Raff, 1999; Casaccia-Bonnefil, 2000)? The "acid test" to answer these questions requires an analysis in a physiologically accurate system in which specifically Notch1 has been reduced or eliminated. Transgenic mice provide such a proper setting. Notch1 knockout mice were not informative, however, due to early embryonic lethality (Swiatek et al., 1994; Conlon et al., 1995). Thus, in this study we have selectively inhibited Notch1 signaling using a conditional Notch1 knockout mouse strain (Radtke et al., 1999). Our data demonstrate a crucial function of Notch1 in late steps of OL differentiation in the spinal cord and suggest a similar function in the brain.
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Results |
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Ectopic appearance of immature OLs in the gray matter at birth
Dramatic changes in the number and spatial distribution of OLs were also evident in spinal cords of newborn (P0) Cnp-Cre /
compared with control (lox/lox) mice (Fig. 3
, compare c and e with d and f). Many immature OLs were abnormally located in the gray matter. This altered distribution was seen along the entire length of the spinal cord in mutant mice (unpublished data). We quantified the MAG-positive cells showing perinuclear staining (immature OLs; Fig. 3 i), using their perinuclear staining to distinguish them from myelinating mature OLs (Trapp et al., 1997). After excluding the mature OLs at the ventral margin of both mutant and control spinal cords (Fig. 3, e and f; see also MBP staining for mature OLs in Fig. 3, g and h for comparison), there were approximately sixfold more immature OLs present in mutants as compared with controls (Fig. 3 l). Similar observations were made in Plp-Cre
/
mice (53 ± 19 vs. 5 ± 4 in mutant and control, respectively). At E17.5, we found no difference between the numbers of cells positive for PDGFR-
mRNA in Cnp-Cre
/
and control mice (Fig. 3, a, b, and k).
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Efficiency of Notch1 ablation
Because our multiple efforts to directly show elimination of the Notch1 protein failed due to a lack of reliable reagents, we demonstrated that Cre recombinase had been active in the differentiating OLs. We prepared short-term cultures of cells isolated from the spinal cord of single Cnp-Cre R26R mice homozygous for the floxed Notch1 allele (/
). Heterozygous littermates (
/wt) were used as controls. The cells were first stained with X-gal (Fig. 4
, a and b) and then for O4 (Fig. 4, c and d), a marker for both immature and mature OLs (Hardy and Reynolds, 1991). 89 out of 100 and 71 out of 100 O4-positive cells derived from homozygous and heterozygous animals, respectively, were also positive for ß-gal, demonstrating expression of Cre recombinase and efficient recombination of the R26R allele within the O4-positive cell population. To directly demonstrate Notch1 recombination, DNA was isolated from spinal cords of newborn mice and analyzed by Southern blotting (Fig. 4 e). Quantification by PhosphorImager analysis revealed
2025% recombination in Cnp-Cre
/
whole spinal cord, after normalization to DNA isolated from control lox/0 mice.
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Discussion |
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An important issue is whether the observed effects of Notch1 deletion on OPC differentiation depend directly on deletion within the OPCs themselves or are an indirect consequence of deletion in some other cell type. We used Cre-mediated recombination driven by Cre recombinase inserted into the Cnp locus. In agreement with published reports on CNP regulation and according to our own data, in the developing spinal cord Cre recombinase was expressed exclusively by early oligodendrocyte progenitors and motoneurons. Given the pattern of X-gal and immunostaining at E17.5, Cre is likely not to be expressed in astrocytes (Fig. 2, compare b with f). Development of neurons appears normal in the spinal cords of mutant mice (Fig. 2, c, d, and g). However, we cannot exclude subtle effects such as altered trophic support or abnormal electrical signaling from the periphery. Electrical activity of neurons has been shown to influence proliferation of OPCs (Barres and Raff, 1993) and the process of myelination (Demerens et al., 1996), but we are not aware of evidence that such electrical activity influences differentiation of OPCs into immature OLs (for review see Barres and Raff, 1999). OLs develop normally in explant cultures from spinal cord of Isl1-/- mice (Sun et al., 1998), even though no motoneurons or V1 interneurons are produced (Pfaff et al., 1996). OLs also develop in hindbrain of Olig2-/- mutants (Lu et al., 2002), which lack somatic motoneurons. The most likely interpretation of our results is therefore that they reflect a direct effect of Notch1 deletion in OPCs.
Although ß-gal expression was seen as early as E11.5, the first immature OLs were not found until E17.5. Several possibilities may be envisaged to explain this delay. Persistence of Notch1 protein might render the cells effectively Notch1-positive for some time after recombination has occurred. This appears unlikely because, although we were not able to directly assess the lifetime of the Notch1 protein, the presence of a PEST sequence near the COOH terminus suggests that Notch1 turns over rapidly (Rechsteiner and Rogers, 1996). Alternatively, ablation of Notch1 might have an immediate effect, but progress through the developmental program might require several days (Durand and Raff, 2000). This would be consistent with the clonal analyses of purified precursor cells isolated from P78 (Barres et al., 1994) and E18 rat optic nerve (Gao et al., 1998) suggesting that a cell intrinsic program plays an important role in determining when OPCs stop dividing and differentiate. Finally, Notch1 may inhibit the transition from OPCs to immature OLs only relatively late in development, shortly before E17.5 in the mouse spinal cord.
Why do many of the prematurely differentiated OLs in Notch1 mutants die? Immunostaining with late differentiation markers and assessment of apoptosis suggest that precociously differentiated OLs cannot terminally differentiate and are unable to survive in the long term if they arise at the wrong place and time, most likely due to missing appropriate survival cues. Similar conclusions were reached by Calver et al. (1998). Overexpression of PDGF led to the appearance of an excess of immature OLs at ectopic sites in the gray matter of transgenic spinal cords, due to an excess production of OPCs, but the OLs survived in large numbers only in white matter. In our study, the number of precursors was normal, and it was precocious and uncontrolled differentiation that led to a high number of immature OLs in the gray matter. However, in both cases, these cells were eliminated by apoptosis, and mature OLs accumulated only in the white matter. It has been suggested that OPCs differentiate preferentially in fiber tracts (Hardy and Friedrich, 1996). However, our results and those of Calver et al. (1998) indicate that progenitor cells can differentiate both in gray and white matter, but survive in large numbers only in white matter.
The increase in immature OLs was not accompanied by a decrease in the number of OPCs. It has been proposed that expansion of OPCs is regulated by a density-dependent mechanism (Zhang and Miller, 1996), which could have served to prevent depletion of the OPC pool in our experiments. We did not observe a significant increase in the number of proliferating cells. However, if the OLs live considerably longer than the cell cycle time of the OPCs (which ranges from 30 h at E13 to
100 h at E17; van Heyningen et al., 2001), the expected increase in proliferation rate might have escaped detection. We also considered the possibility that Notch ablation might have produced MAG-positive OLs that were also still positive for early markers. We tested this by coimmunostaining for MAG and the early marker NG2, but found that the MAG-positive cells in the gray matter were NG2 negative (unpublished data).
Our analysis demonstrates that Notch1 is required for the regulation of the differentiation of OPCs to OLs, and thus contributes to the timing of oligodendrocyte differentiation in vivo. It has been suggested that this timing might be controlled by an intrinsic mechanism that allows a single OPC to undergo controlled cell cycle arrest and to differentiate in vitro without neurons (Temple and Raff, 1986). Our data and the observations by Wang et al. (1998) show that additional regulatory mechanisms that are mediated by direct cellcell interactions via the Notch1 receptor and its ligands throughout the CNS are functional in this process. This most probably involves interactions between the axon and OPCs but there might also be a contribution by interactions between mature OLs and OPCs (Wang et al., 1998). The intracellular mechanisms that underlie precocious OL differentiation in the Notch conditional knockout animals remain unknown at this stage. The finding that maturation of OLs is strongly inhibited in the absence of the transcription factor Nkx2.2 (Qi et al., 2001) suggests the possibility that this transcription factor or one of its regulatory targets is antagonized by the Notch signaling pathway.
Very recently, examination of mutants heterozygous for a Notch1-null allele has provided evidence for an effect of Notch1 on myelination (Givogri et al., 2002). Several changes in the pattern of myelination were observed, including premature myelination in the cortex, increased myelination of small caliber axons in the optic nerve, and ectopic myelination in the molecular layer of the cerebellum. Some of these changes may reflect premature differentiation of OPCs, such as we describe here but in the heterozygous-null setting, there also seem to be effects on survival of OLs and on the choice of axons to be myelinated.
Cellcell interactions mediated by Notch signaling are likely to play a pivotal role in development as part of a local regulatory circuit that forms the basis for the asynchronous myelination of the CNS and its different fiber tracts. The final trigger for OL differentiation is likely to be a down-regulation of the axonal component of the Notch1ligand interaction, possibly linked to electrical activity (Rogister et al., 1999). Likewise, OPCs in the adult nervous system might be held in the undifferentiated state via this regulatory system. If correct, inhibition of Notch1 might be an effective way of activating these quiescent cells for repair in demyelinating diseases like multiple sclerosis. This is particularly warranted by the fact that lesions in multiple sclerosis are not starved of OPCs as previously thought, but rather that other factors, possibly including activated Notch signaling, are responsible for the failure of remyelination (Solanky et al., 2001).
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Materials and methods |
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For most experiments, we compared Cnp-Cre /
or Plp-Cre
/
animals with lox/lox littermates as a control. Findings were reproduced from at least three animals of each genotype and from at least two different litters. Spinal cord sections were cut at the forelimb level in embryos and at the thoracic level in newborns, unless otherwise indicated. The lateral ventricles were used as landmarks for the brain sections.
X-gal histochemistry, in situ hybridization, immunofluorescence, and TUNEL staining
Timed pregnant mothers were killed, and embryos were isolated, fixed for 12 h in 4% paraformaldehyde, incubated for a few hours to overnight in 30% sucrose, embedded in OCT (TissueTech), and immediately frozen on dry ice. Newborns were anesthetized by intraperitoneal injection of a pentobarbital solution, and then perfused with a 0.9% saline solution followed by 4% ice-cold paraformaldehyde. Brains and spinal cords were isolated, fixed for 2472 h in the same fixative at 4°C, incubated in 30% sucrose overnight, and embedded in OCT. 20-µm (embryos) or 5-µm (newborn)frozen sections were thaw-mounted onto Superfrost slides (Mettler) and air dried.
For the X-gal staining, the sections were incubated overnight in a PBS solution containing 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 2 mM magnesium chloride, and 2 mM X-gal (Calbiochem). In situ hybridizations were performed with digoxigenin-labeled RNA probes overnight at 72°C in buffer containing 50% formamide and detected using an antiDIG-AP antibody according to the manufacturer's instructions (Roche Diagnostics). The PDGFR- and PLP/DM20 plasmids were gifts from Drs. W.D. Richardson and B. Zalc, respectively. Immunohistochemistry was performed overnight at 4°C with antibodies against NeuN (1:100; Chemicon Inc.), GFAP (1:500; Accurate), ß-tubulin III (1:300; Sigma-Aldrich), neurofilament 160 (1:20; Sigma-Aldrich) and phosphorylated histone H3 (1:100; Upstate Biotechnology). Antibodies against MBP (1:500), PLP/DM20 (1:500), Isl1/2 (1:500), and MAG (1:500) were provided by Drs. N. Baumann, I.R. Griffiths, T.M. Jessell, and J.L. Salzer, respectively. Secondary antibody incubations (1:300; Jackson Immunochemicals) were performed for 1 h at room temperature.
Apoptotic cell death was analyzed by TUNEL staining using biotin-labeled UTP and an FITC-conjugated streptavidin complex according to the manufacturer's instructions (Roche Diagnostics).
Images were collected using an Axiophot microscope (Carl Zeiss MicroImaging, Inc.) in conjunction with a ProgRes 3008 (Jenoptik) or Hamamatsu CCD camera. Image processing was performed with Adobe Photoshop® 5.0 software. Staining intensity was measured with NIH Image 1.62 software. The number of cells expressing PDGFR- mRNA or showing perinuclear MAG staining was determined by counting all cells in at least 10 sections.
Primary cell dissociation
Spinal cords were individually isolated from newborn animals by incubation for 20 min at 37°C in 200 µl DME medium (Life Technologies) containing 2 mg/ml collagenase type IV (Worthington Biochemical Corp.), 1.2 mg/ml hyaluronidase type IV-S (Sigma-Aldrich) and 0.3 mg/ml trypsin inhibitor (Sigma-Aldrich). After trituration, dissociated cells were plated onto poly-D-lysine (Sigma-Aldrich)coated dishes (35 mm; Corning) in Eagle's medium with 10% FCS (Sera-Tech) and maintained at 37°C in 5% CO2 overnight. These cells were used for X-gal and O4 staining (Fig. 4, ad). The carcasses were genotyped by PCR.
Southern blot and quantification
Spinal cord was isolated from newborn animals and cells were mechanically dissociated as previously described (Milner and Ffrench-Constant, 1994). Southern blotting (5 µg EcoRI-digested genomic DNA) to a HybondTM-N+ membrane (Amersham Biosciences) was performed essentially as described by Radtke et al. (1999), using as probe a 750-bp BamHI-EcoRI fragment derived from the 5' upstream region of the Notch1 locus. The probe was hybridized at 42°C, and the signals quantified using a PhosphorImager (Storm 820; Molecular Dynamics).
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Footnotes |
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Acknowledgments |
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This work was supported by the Swiss National Science Foundation and the Fifth European Community Framework Programme (Control of specification and migration of oligodendrocytes).
Submitted: 1 February 2002
Revised: 15 July 2002
Accepted: 15 July 2002
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