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DOI: 10.1093/brain/awh217 Brain (2004), 127, 1928–1941
Notch1 and Jagged1 are expressed after CNS
demyelination, but are not a major rate-
determining factor during remyelination
Mark F. Stidworthy,
1
Stephane Genoud,
2
Wen-Wu Li,
1
Dino P. Leone,
2
Ned Mantei,
2
Ueli Suter
2
and Robin J. M. Franklin
1
Correspondence to: Dr Robin Franklin, The Veterinary
School, University of Cambridge, Madingley Road,
Cambridge CB3 0ES, UK
E-mail: rjf1000@cam.ac.uk
1
Cambridge Centre for Brain Repair and Centre for
Veterinary Sciences, University of Cambridge, UK and
2
Institute of Cell Biology, ETH Zu
¨
rich, Switzerland
Summary
The reasons for the eventual failure of repair mechanisms
in multiple sclerosis are unkno wn. The presence of precur-
sor and immature oligodendrocytes in some non-repairing
lesions suggests a mechanism in which these cells either
receive insufficient differentiation signals or are exposed
to differentiation inhibitors. Jagged signalling via Notch
receptors on oligodendrocyte precursor cells (OPCs) inhi-
bits their differentiation during development and the find-
ing that both notch and jagged are expressed in multiple
sclerosis lesions has fostered the view that this signalling
pathway may explain remyelination failure. In this study,
we show that Notch1 is expressed on adult OPCs and that
there are multiple cellular sources of its ligand Jagged1 in a
rodent model of remyelination. However, despite their
expression, the lesions undergo complete remyelination.
To establish whether Notch-jagged signalling regulates
the rate of remyelination we compa red their expression
profiles in young animals with those in older animals,
where remyelination occurs more slowly, but could find
no correlation between expression and remyelination
rate. Finally we found that OPC-targeted Notch1 ablation
in cuprizone-treated Plp-creER Notch1
lox/lox
transgenic
mice yielded no significant differences in remyelination
parameters between knock-out and control mice. Thus,
in contrast to developmental myelination, adult expression
of Notch1 and Jagged1 neither prevents nor plays a major
rate-determining role in remyelination. More generally,
the re-expression of developmentally expressed genes fol-
lowing injury in the adult does not per se imply similar
function.
Keywords: remyelination; notch; jagged; multiple sclerosis; oligodendrocyte
Abbreviations: EB = ethidium bromide; ER = estrogen receptor; GFAP = glial fibrillary acidic protein; Hh3 = histone h3; OPC =
oligodendrocyte progenitor cell; Plp = proteolipid protein; PDGFaR = platelet-derived growth factor a receptor; RT–PCR =
reverse transcription–polymerase chain reaction
Received February 25, 2004. Revised April 9, 2004. Accepted April 12, 2004. Advanced Access publication August 2, 2004
Introduction
Remyelination in the adult CNS involves a complex interplay
of multiple signalling events, which, if inappropriately coor-
dinated, lead to impairment in demyelinating disease such as
multiple sclerosis (Franklin, 2002). Some lesions in multiple
sclerosis patients contain oligodendrocyte progenitor cells
(OPCs) (Scolding et al., 1998; Wolswijk, 1998; Chang et al.,
2000; Maeda et al., 2001) or premyelinating oligodendrocytes
(Chang et al., 2002), but remain chronically demyelinated
without these cells differentiating into stable myelinating oligo-
dendrocytes. Identifying factors that regulate differentiation is
crucial to developing strategies for enhancing remyelination in
these lesions. Notch signalling has emerged as an inhibitor of
OPC differentiation that may contribute to remyelination
failure (Wang et al., 1998; Genoud et al., 2002; Givogri
et al., 2002; John et al., 2002).
The Notch family comprises conserved transmembrane re-
ceptors interacting with membrane-bound ligands in the Delta/
Serrate/Jagged families (Lardelli et al., 1995; Weinmaster,
1997; Lewis, 1998; Artavanis-Tsakonas et al., 1999). Signal-
ling is involved in developmental (de la Pompa et al., 1997;
Vargesson et al., 1998; Favier et al., 2000; Singh et al., 2000;
Zine et al., 2000; Gridley, 2001; Pear and Radtke, 2003), and
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adult regenerative processes (Lindner et al., 2001; Conboy
et al., 2003; Mitsiadis et al., 2003). During CNS myelination,
axonally expressed Jagged1 inhibits differentiation of OPCs
via Notch1 receptors and its downstream effector Hes5 (Wang
et al., 1998; Kondo and Raff, 2000), and plays a role in con-
trolling the timing of OPC differentiation and myelination
(Genoud et al., 2002; Givogri et al., 2002). It is proposed
that this mechanism accounts for the failure of differentiation
of OPCs in chronic multiple sclerosis lesions (John et al.,
2002).
To understand the functional role of the Notch1 pathway in
remyelination we have taken advantage of the contribution that
delayed OPC differentiation makes to age-associated delay in
remyelination (Sim et al., 2000). We hypothesized that Jagged1
expression by demyelinated axons may be prolonged in older
animals, delaying differentiation of Notch1-expressing OPCs.
To test this, we investigated expression patterns of Notch and
its ligands during remyelination in young and old adult rats,
exploiting the trigeminal system, where changes in neuronal
gene expression in the trigeminal ganglion can be correlated
with focal demyelination in the trigeminal tract (Fig. 1).
In further experiments, we used Plp-creER Notch1
lox/lox
transgenic mice to investigate remyelination following cupri-
zone intoxication. This overcame the phenotypic lethality of
null homozygotes (Swiatek et al., 1994; Huppert et al., 2000)
enabling the specific targeting of Notch ablation to OPC line-
age cells during adult remyelination.
Materials and methods
Creation of demyelinating lesions
Procedures were undertaken in accordance with legislation on animal
use in host institutions. Stereotaxic ethidium bromide (EB) injection
was used to create focal areas of primary demyelination in the tri-
geminal tract of adult female Sprague–Dawley rats (Fig. 1A and B)
(Shields et al., 1999; Woodruff and Franklin, 1999). Young (aged
8–10 weeks) and old (9-month ex-breeder) rats were used to gen-
erate rapidly or slowly remyelinating lesions, respectively (Shields
et al., 1999; Sim et al., 2002b). Young adult rats injected with saline
served as controls.
Tissue processing
Animals were killed at 10 (n = 5 young, 4 old), 21 (n = 7 young, 5 old),
24 (n = 5 old animals only), 28 (n = 5 young, 4 old) and 40 (n =
2 young, 4 old) days after lesion induction, together with saline
controls (n = 2 or 3 in each group). Animals were killed by pento-
barbitone injection. Brains were removed, the separated hindbrain
snap-frozen by immersion in isopentane at 30
C, and stored at
70
C. Transverse sections (12 mm) were cut by cryostat and
thaw-mounted onto poly-
L-lysine-coated slides. Air-dried sections
were fixed for 5 min in cold buffered 4% paraformaldehyde, rinsed
twice in PBS for 1 min, dehydrated in 70% ethanol for 5 min, then
stored at 4
C in 96% ethanol (in situ hybridization), or at 70
Cin
airtight boxes with desiccant (immunolabelling).
In situ hybridization
Digoxigenin (DIG)-labelled riboprobes were made from linearized
plasmid cDNA templates (gifts from Professor G. Weinmaster)
for Notch1 (SN6-7; Weinmaster et al., 1991), Notch2 (H10-6;
Weinmaster et al., 1992), Notch3 (5
0
PCR3
0
; Lindsell et al., 1995)
and Jagged1 (SN3ED; Lindsell et al., 1995). In situ hybridization
was performed as described (Fruttiger et al., 1999). RNA hybrids
were visualized immunohistochemically with alkaline phosphatase-
conjugated anti-DIG antibody (Roche Diagnostics). 5% v/v polyvinyl
alcohol was added to the final colour reaction to increase sensitivity.
Anti-sense and sense probes were run under standard conditions on
both experimental and control tissues.
Fig. 1 (A) The trigeminal tract lesion model allows histological assessment of remyelinating lesions in the trigeminal tract (black area
lateral to grey area representing the trigeminal nucleus), and changes in gene expression in the cell bodies of the demyelinated axons in the
trigeminal ganglion (TG). (B) Bilateral demyelinating lesions were generated by injection of EB into the trigeminal tract (outlined by
interrupted lines). (C) Axons in the trigeminal tract (TT) have their neuronal cell bodies exclusively in the trigeminal ganglion enabling
clear localization and sampling.
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Quantification
Notch1-positive cells were counted by an observer, blinded to the
animals’ identities, using a Nikon E600 microscope at 200 magni-
fication. Four to ten transverse sections from the centre of each tri-
geminal tract lesion were sampled. Mean lesion areas were measured
from three adjacent solochrome cyanin-stained serial sections using
MCID 4.0 image analysis software. MCID 4.0 was also used to count
cell nuclei within the demyelinated area. A mean density (cells/mm
2
)
was calculated for each animal based on measurement of both lesions
(where bilateral), and then individual animal means were used to
calculate group means 6 SEM.
Immunolabelling
For Notch1 immunolabelling, polyclonal rabbit anti-rat antiserum
against a 120 kDa band corresponding to the extracellular and trans-
membrane domains of Notch1 (a gift from Dr V. Taylor) was used.
Detection utilized tyramide signal amplification (TSA Biotin System,
NEN Life Science Products). Following quenching and blocking,
sections were incubated with Notch1 antibody at 4
C overnight
[1 : 3000 in Tris/sodium chloride/blocking agent (TNB) buffer].
Primary antibody was detected with HRP (horseradish peroxidase)-
conjugated goat anti-rabbit antibody (DAKO; 1 : 100 in TNB), and
signal amplified using biotinyl tyramide reagent (1 : 50 in amplification
diluent) for 10 min at room temperature. Biotinylated tyramide was
detected using streptavidin–FITC (fluorescein-isothiocyanate) 1 : 100
in TNB (1 h at room temperature). Notch1 labelling using this antibody
and Notch1 mRNA detection by in situ hybridization using
riboprobe SN6-7 localized similarly in serial sections of foetal rat
brain (Fig. 2A and E).
For Jagged1 immunolabelling, cryostat sections were pre-treated
with sodium dodecyl sulphate (SDS) for antigen retrieval (Brown
et al., 1996; Robinson and Vandre, 2001). Sections were rehydrated,
permeabilized and treated with 0.5% SDS for 5 min, before blocking
and incubation with anti-Jagged1 antibody (4
C overnight, Santa
Cruz, sc-6011; 1 : 100). Bound antibody was detected using donkey-
anti-goat IgG-FITC antibody (Jackson Immunochemicals; 1 : 100).
Specificity was confirmed by pre-absorption with Jagged-specific
peptides (Fig. 5A–C), which abolished labelling of SDS-treated
foetal tissues and adult cerebellum. Jagged1 antibody labelling and
mRNA detection by in situ hybridization using riboprobe SN3ED
localized similarly in ectodermal placodes in serial sections of foetal
rat tissue (Fig. 5D and E) (Shawber et al., 1996).
For dual labelling experiments, Jagged1 or Notch1 labelling was
followed by a range of primary antibodies: NG-2 (AB5320) 1 : 200
Chemicon; glial fibrillary acidic protein (GFAP) (Z0334) 1 : 200
DAKO; neurofilament (SMI31) 1 : 3000 Sternberger Monoclonals
Inc.; P0 1 : 100 (a gift from Dr J. Archelos); ED-1 (MAB1435) 1 : 400,
Chemicon; CD11b (Ox42, MCA275G) 1 : 100, Serotec. TRITC (tetra-
rhodaminine isothiocyanate)-conjugated donkey anti-rabbit or goat
anti-mouse antibodies (Jackson; 1 : 100) were used. Hoechst 33342
(Molecular Probes) stained nuclei. Sections were imaged convention-
ally (Nikon E600 microscope, Coolpix 9000 digital camera) and by
confocal microscopy (Leica confocal with TCS NT software), and
images processed using Adobe Photoshop 6.0.
RNA extraction
Ganglia were removed, snap-frozen immediately in isopentane and
stored at 70
C. Total RNA was extracted with TRIzol Reagent
(Gibco BRL). Ganglia were homogenized with a glass homogenizer
(Jencon Uniform) or minipestle (Kontes Pellet Pestle Motor). Extrac-
tions were stored at 70
C.
Reverse transcription–polymerase chain reaction
(RT–PCR)
RNA was pretreated with amplification grade DNase1 (Gibco BRL),
and reverse transcribed using the Superscript First-Strand Synthesis
System (Gibco BRL). Thirty-five cycles of PCR were used to detect
Notch ligand mRNA using Platinum Taq DNA Polymerase (Gibco
BRL) and previously published or novel primers for rat Jagged1
(Wang et al., 1998), Jagged2, Delta1 and Delta3. Cyclophilin
(Semple-Rowland et al., 1995) was to control for RNA integrity
using 25 cycles of PCR. Products were extracted (Concert Gel Extrac-
tion System, Gibco BRL) and sequenced to confirm identity with rat
Genbank sequences (Jagged1 NM
019147, Jagged2 U70050, Delta1
NM
032063, Delta3 AF084576).
Fig. 2 Notch1 expression in the normal foetal and adult CNS. (A and E ) Foetal brain (box in A denotes region depicted in E). (B, D, F and
H) Normal trigeminal tract. (C and G) Granule layer of cerebellum and cerebellar white matter. Similar patterns of expression were
observed using either anti-Notch1 polyclonal antibody (E–G) or anti-sense riboprobe for Notch1 (A–C). Dual labelling with anti-NG-2
antibody (D and H) identified Notch1-positive/NG-2-positive OPC in the normal adult trigeminal tract. Images are shown with Hoechst
33342 nuclear label. Scale bars: A = 1 mm; B (same as for C) and F (same as for G) = 100 mm; D =50mm; E = 200 mm; H =8mm.
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Transgenic mice
Plp-creER mice (Leone et al., 2003) were used to generate Plp-creER
Notch1
lox/lox
mice (Genoud et al., 2002). PLP-expressing oligoden-
drocyte lineage cells in these mice excise floxed Notch alleles in the
presence of tamoxifen. ‘Knock-out’ mice were compared with ‘con-
trol’ mice containing floxed Notch but lacking the Plp-creER con-
struct. Additional ‘knock-out’ and ‘heterozygote’ (Notch
lox/wt
) R26R
reporter mice (Soriano, 1999) enabled the mapping of Plp expression
in the presence or absence of a normal Notch1 allele.
Cuprizone administration and recombination
Two independent experiments were performed. Mice were fed 0.2%
cuprizone in-feed for 5 weeks, and groups sacrificed at the end of week
5, then 7 and 14 days later, as described (Stidworthy et al., 2003). Two
equivalent tamoxifen protocols were used (Leone et al., 2003). In the
first experiment, 1 mg of tamoxifen was injected on 15 consecutive
days, from the end of the third week of cuprizone (Stidworthy et al.,
2003). In the second, 1 mg of tamoxifen was injected twice daily for
5 days from the day after cuprizone withdrawal.
Analysis of remyelination
Following perfusion with 4% glutaraldehyde, analysis of corpus
callosum remyelination used an identical protocol to that described
previously (Stidworthy et al., 2003).
Histological analysis of mouse tissues
X-gal histochemistry, PDGFaR (platelet-derived growth factor a
receptor) in situ hybridization, immunofluorescence and TUNEL
staining used published protocols (Genoud et al., 2002). Antibodies
against NG-2 and phosphorylated histone H3 (Upstate Biotechnol-
ogy; 1 : 100) were used to label OPC and proliferating cells. Apoptosis
was analysed by TUNEL staining using biotin-labelled UTP and
FITC-conjugated streptavidin (Roche Diagnostics). NG-2 labelling
intensity was measured from images of the body and splenium of the
corpus callosum using NIH Image 1.62 software (Genoud et al., 2002).
TUNEL-positive, Hh3-positive and PDGFaR-positive cells were
counted within the sagittally sectioned midline corpus callosum.
Results
Notch1 mRNA and protein are expressed in
normal adult rat cerebellum and hindbrain
Having first confirmed specific binding of both Notch1 ribop-
robe and antibody using embryonic rat brain (Fig. 2A and E)
(Lindsell et al., 1996; Shawber et al., 1996) we examined
Notch1 expression in normal adult CNS. Notch1 mRNA
and protein were detected in small round glial cells throughout
axon tracts of the white matter, both singly and in chains,
suggestive of interfascicular oligodendrocytes (Fig. 2B, C, F
and G). Some cells were confirmed as NG-2þ OPCs in the
trigeminal tract (Fig. 2D and H). Purkinje cells, cells in the
granular layer of the cerebellum, and the majority of neurons in
brainstem and cerebellar nuclei also expressed Notch1 mRNA
and protein. Signal was detected in the choroid plexus, pia
mater of the meninges and a minor population of cells
surrounding small blood vessels. The sense probe gave only
scant deposits in cells in the granular layer of the cerebellum.
Notch1 mRNA and protein are expressed
in remyelinating lesions of young and old rats
The reduced rate of remyelination in older animals is due in part
to a delay in differentiation of recruited OPCs (Sim et al.,
2002b). We hypothesized that this may reflect differences
in the expression of Notch1, addressing this by comparing
Notch1 expression patterns following EB-induced demy-
elination in the trigeminal tract of young adult and old adult
female rats.
Compared with normal white matter, increases in numbers
of Notch1 mRNAþ cells were observed at all time-points
from 10 to 40 days after lesion induction, in both young and
old animals (Fig. 3A and E–L). At day 10, positive cells
were located predominantly around the lesion margins
(Fig. 3E and I), but from 20 days were distributed throughout
the remyelinating area. In situ hybridization with Notch2
and Notch3 probes labelled only a few scattered cells in the
lesions (Fig. 3C and D). There was no labelling with sense
riboprobes (Fig. 3B).
Protein distribution corresponded closely with that of
mRNA expression (Fig. 3M–P). Cells expressing Notch1 pro-
tein accumulated around the lesion periphery at 10 days but
was subsequently found towards the lesion centre. By 40 days
the predominant Notch1þ component comprised a dense
cluster of cells in the central portion of the lesion.
Notch1 is expressed by adult oligodendrocyte
progenitors, subpopulations of oligodendrocytes,
Schwann cells, macrophages and astrocytes
within remyelinating lesions
Dual labelling studies using antibodies directed against NG-2
(an OPC marker), GFAP (an astrocyte marker), ED-1 (a micro-
glia/macrophage marker), RIP (an oligodendrocyte marker)
and P0 (a myelinating Schwann cell marker) were performed
to identify Notch1þ cells. Despite the hypercellularity of the
lesions, making quantification of individually labelled cells
difficult, this revealed that the majority of cells in the lesions
were Notch1þ /NG-2þ (Fig. 4A). Very few NG-2 cells did not
also express Notch1.
A population of Notch1þ /RIPþ cells was found, initially
located around the lesion periphery but more centrally from
day 20 (Fig. 4D). At later survival times most RIPþ cells at the
periphery of the lesion did not express Notch1. We have shown
previously that, in the EB model, oligodendrocyte remyelina-
tion begins at the edge of the lesion and progresses towards the
centre (Woodruff and Franklin, 1999; Sim et al., 2000). The
relationship we found between Notch1 and RIP immuno-
reactivity suggested that Notch1 was expressed by RIPþ
oligodendrocytes newly generated by OPCs, but subsequently
lost from mature myelinating cells.
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At day 20, Notch1 mRNAþ cells were found as clusters in
the centre of the lesion in both young and old animals. At later
time-points these clusters were larger in size, eventually
forming centrally located areas of intense signal (Fig. 3G–H
and K–L). This pattern of expression closely resembled the
behaviour of myelinating Schwann cells previously described
in this model (Woodruff and Franklin, 1999; Sim et al ., 2002a).
Immunolabelling revealed these areas to be both Notch1þ
Fig. 3 Notch1 mRNA and protein expression in remyelinating white matter lesions of young and old adult rats. (A, E–L) In situ
hybridization using anti-sense riboprobe for Notch1 mRNA on coronal cryostat sections of lesions from an old rat 24 days after lesioning
(A), and from young (E–H) and old (I–L) adult rats at 10, 20, 28 and 40 days after lesioning. (C and D) In situ hybridization for Notch2
(N2) and Notch3 (N3) on coronal cryostat sections of lesions from old adult rats 24 days after lesioning. (B) In situ hybridization using
sense riboprobe for Notch1 of lesion from old adult rat at 28 days. (M–P) Immunolabelling using anti-Notch1 polyclonal antibody
with Hoechst 33342 nuclear label on coronal cryostat sections of remyelinating lesions from young adult rats at 10, 20, 28 and 40 days
after lesioning. Scale bars: A =50mm; B and E–L = 200 mm; C and D and M–P = 100 mm. Interrupted lines indicate lesion margins.
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and P0þ and were presumably myelinating Schwann cells
(Fig. 4E and F).
After the Notch1þ /NG-2þ and Notch1þ /P0þ cells,
the next most numerous dual-labelled cells were those
expressing Notch1 and ED-1 (Fig. 4C). These cells were
distributed across the lesions at earlier time-points, and
later found mainly around the edges of lesions. A small
number of Notch1þ /GFAPþ astrocyte processes were
observed at lesion margins and surrounding blood vessels
(Fig. 4B).
Fig. 4 (A–F) Notch1 protein labelling using anti-Notch1 polyclonal antibody (FITC secondary) on coronal cryostat sections of
remyelinating lesions (interrupted lines indicate the lesion margins, asterisks denote the later remyelinating core) with: (A) anti-NG-2,
arrows indicate clumps of NG-2þ /Notch1þ progenitors, arrowhead indicates capillary endothelial labelling; (B) anti-GFAP, arrowheads
indicate GFAPþ /Notch1þ astrocytes at lesion margins and around blood vessels; (C) anti-ED-1, arrows delineate the remyelinating
corona containing ED-1þ /Notch1þ macrophages indicated by arrowheads; (D) anti-RIP, arrows and arrowheads delineate areas of RIPþ /
Notch1þ or RIP-/Notch1þ oligodendrocyte lineage cells, respectively; (E and F) anti-P0: Notch1þ /P0- cells predominate across the
lesion at 20 days (E), whilst Notch1þ /P0þ Schwann cells clump in the lesion centres by 28 days (F). Merged images are shown with
Hoechst 33342 nuclear label. Lesions illustrated are 20 (B, D and E), 28 (A and E)or40(C) days after induction in young (B–F) or old (A)
adult rats. (E) and (F) are serial sections corresponding to Fig. 3F and G, respectively. Scale bars: A–F = 100 mm. (G) Quantification of
Notch1 mRNA expression during remyelination in young and old rats. Control values were derived from saline-injected young animals.
Values are expressed as group means 6SEM at each time-point in each age group. Nuclear counts indicate the total density of nucleated
cells within the lesion area.
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The lesional density of Notch1-expressing
cells is not significantly different between
young and old animals
We next assessed whether there were quantitative differences
in the patterns of Notch1 expression between young and old
animals. Lesion area and total nucleated cell number were
measured from solochrome cyanin-stained adjacent serial sec-
tions using an automated image analysis system. Notch1
mRNAþ cells were numerous at all time-points examined
between 10 and 40 days after lesion generation (Fig. 4G).
Although the identity of the cells expressing Notch1 mRNA
was not established, the densities of Notch1 expressing cells
were not significantly different between the two groups at any
time-point despite differences in the rate of remyelination (Sim
et al., 2002b).
mRNAs for the Notch ligands Jagged1 and
Jagged2, but not Delta1 or Delta3, are
expressed by trigeminal ganglia with normal,
demyelinated and remyelinating trigeminal tracts
The axons in the trigeminal tract have their neuronal cell bodies
in an accessible peripheral trigeminal ganglion, and a single
demyelinating lesion will affect axons located only within that
ganglion (Fig. 1C). Consequently, by generating demyelinat-
ing lesions within the tract and collecting the ipsilateral
trigeminal ganglion, we could be certain to sample the relevant
neuronal population. RT–PCR analysis of homogenates of
trigeminal ganglia from histologically confirmed demyelin-
ated tracts, as well as saline-injected and sham-operated
controls, and normal young adult rats revealed constitutive
expression of Jagged1 and Jagged2 mRNA within neurons
extending axons into the trigeminal tract (Fig. 5, table).
No Delta1 or Delta3 mRNA was detected at any time. No
significant differences were apparent between young and
old animals, or between different survival times. Only half
of the ganglia were positive 10 days after lesion induction,
but this was not a statistically significant difference by Fisher’s
exact test when compared with controls.
Patterns of Jagged1 mRNA and protein expressed
in normal adult cerebellum and hindbrain are
complementary to those of Notch1
To establish whether Notch ligands were expressed in adult
brain regions in which we had examined Notch1 expression we
undertook in situ hybridization using a Jagged1-specific anti-
sense riboprobe. First, we verified the specificity of the probe
in embryonic rat brain against previously published data
(Fig. 5E) (Lindsell et al., 1995, 1996; Shawber et al., 1996).
In the adult, Jagged1 mRNA was detected in cells in the mole-
cular and granular layers of the cerebellum, in some Purkinje
cells and neurons in the brainstem nuclei (not shown) and
interspersed through white matter tracts (Fig. 5G, I and K).
Using Jagged1-specific antibodies, verified using blocking
peptides (Fig. 5A–C), Jagged1þ cells were observed through-
out myelinated axon tracts, both as small NG-2þ cells (Fig. 5 J
and M) and in rows of cells likely to be interfascicular oligo-
dendrocytes (Fig. 5F). In non-myelinated areas, such as the
molecular layer of the cerebellum (Fig. 5A and H) and major
nuclei of the brainstem and cerebellum (not shown), Jagged1
expression was widespread. In myelinated tracts, Jagged1
expression consistent with its expression by myelinated
axons was also present, but the intensity was considerably
less than that in non-myelinated areas (Fig. 5A, J and L).
Intense Jagged1 labelling was also observed in a subset of
cells in the cerebellar granule layer (Fig. 5B and F) consistent
with published RT–PCR data (Solecki et al., 2001), as well as
in Purkinje cells, the choroid plexus, the endothelia of many
blood vessels, and the pia mater.
Jagged1 mRNA and protein are expressed
predominantly by cells recruited to remyelinating
lesions rather than by demyelinated axons
Within areas of demyelination, in situ hybridization revealed a
Jagged1 mRNA labelling pattern similar to that observed for
Notch1 (Fig. 6A–G). At 10 days, expression was predomi-
nantly in cells around the periphery of remyelinating lesions.
At later time-points, Jagged1 expressing cells were distributed
across the lesion, becoming concentrated centrally at 40 days.
There were no clear differences between young and old ani-
mals. Sense probes did not give a signal (Fig. 6H).
Immunohistochemistry revealed widespread, albeit low
level, expression of Jagged1 protein within remyelinating
lesions at all time-points in both young and old rats
(Fig. 6I–P). The intensity of labelling was initially diminished
when compared with the unlesioned tract, particularly at the
earliest survival (day 10) and in the centre of the lesions. At
days 10 and 20 there was an increase in Jagged1þ cells at the
edge of the lesion and, at later survival times, with greater
abundance towards the lesion centre. Dual labelling studies
with antibodies against NG-2, GFAP, ED-1, Ox42 (CD11b,
a microglia/macrophage marker), P0 and phosphorylated
neurofilament (present within axons) indicated that many
Jagged1þ cells were also NG-2þ (Fig. 6K and N).
Jagged1þ /GFAPþ hypertrophic astrocyte processes were
observed surrounding blood vessels and towards the lesion
edge (Fig. 6L and O). Small numbers of cells expressed
both ED-1 and Jagged1 (Fig. 6P). This may have resulted
from the ingestion of Jagged1-labelled debris by macrophages.
To resolve this, we also labelled cells with the membrane-
associated macrophage marker Ox42, and were able to demon-
strate cellular colocalization of Ox42 and Jagged1 by confocal
microscopy (Fig. 6M). A further subpopulation of cells
labelled with both Jagged1 and P0, indicating that myelinating
Schwann cells also express Jagged1 (Fig. 6I). The intensity of
Jagged1 labelling of axon profiles was reduced in lesions
compared with the normal tract (Fig. 6J). This reduction in
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Fig. 5 Jagged1 and Jagged2, but not Delta1 and Delta3, mRNA are expressed in trigeminal ganglion neurons constitutively and during
remyelination. Table: RT-PCR for Notch ligand mRNA on trigeminal ganglia derived from histologically confirmed demyelinated
trigeminal tracts. Each value represents a single ganglion (derived from 3 normal or >4 lesioned animals). The reductions in Jagged1 and 2
seen at 10 days in both groups were not statistically significant when compared with controls by Fisher’s exact test. (A–M) Identical
patterns of expression were observed using anti-Jagged1 polyclonal antibody (A–D, F, H and J) and anti-sense riboprobe for Jagged1 (E,
G, I and K) on coronal cryostat sections of foetal or adult CNS. (A–C) Expression detected in the adult cerebellum (A) and inset (B) was
abolished by preabsorption of Jagged1 antibody with a 5-fold excess of specific peptide (C). (D and E) Patterns of Jagged1 protein (D)
and mRNA expression (E) corresponded in serial sections of foetal otic placode tissue. (F–K) Corresponding patterns of protein and mRNA
expression were also found in cerebellar white matter and granular layers (F and G), cerebellar molecular layer (H and I), and
normal trigeminal tract (J and K). (L) Large diameter myelinated fibres in the trigeminal tract, labelled with anti-phosphorylated
neurofilament antibodies expressed Jagged1 on the axoplasmic surface (arrows). (M) Jagged1 antibody labelled NG-2þ OPCs. Images
are shown with Hoechst 33342 nuclear label. Scale bars: B and C = 100 mm; D and E =25mm; F–I and K = 100 mm; J and L =50mm
(L inset = 5 mm); M =8mm.
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intensity, together with changes in axon morphology and neu-
rofilament phosphorylation, made it difficult to unequivocally
colocalize Jagged1 and neurofilament even with high-
resolution confocal microscopy.
Histological and ultrastructural measures of
remyelination show no significant differences
between control and Notch1 conditional
knock-out mice
Since there was no qualitative or quantitative difference in
patterns of expression of Notch1 and Jagged1 between
young and old animals, we inferred that Notch1 signalling
was not a major determinant of the delayed OPC differen-
tiation during slow remyelination in old rats (Sim et al.,
2002b). To address further the role of Notch signalling during
remyelination, we used adult Notch1 conditional knock-out
mice in which demyelination was induced using cuprizone
(Stidworthy et al., 2003). Plp-creER Notch1
lox/lox
(‘knock-
out’) mice were compared with controls lacking the Plp-
creER allele and thus expressing normal levels of Notch1
(Genoud et al., 2002; Leone et al., 2003). Remyelination du-
ring the 2 weeks after cuprizone withdrawal was assessed
by the ranking of sections of the corpus callosum (Fig. 7A
and B). In this method, the highest rank (1st) was given to
Fig. 6 Jagged1 mRNA and protein expression in remyelinating lesions. (A–G) In situ hybridization using anti-sense riboprobe for Jagged1
mRNA of remyelinating lesions from young and old adult rats at 10, 28 and 40 days (A–C and E–G), and old rat at 24 days (D). (H) In situ
hybridization using sense riboprobe for Jagged1 mRNA. (I–Q) Jagged1 protein labelling using anti-Jagged1 polyclonal antibody (FITC
secondary) on coronal cryostat sections of remyelinating lesions with: (I) anti-P0; (J) anti-phosphorylated neurofilament (SMI), box region
of probable axonal Jagged1 colocalization; (K and N ) anti-NG-2; (L and O) anti-GFAP; (M) anti-Ox42; (P) anti-ED-1. (J and L–P)
Stacked confocal images. (I and K) Epifluorescent images. (J–P) Lesions depicted are 10 day in young (L and O), 20 day in young (J, K
and N) and 20 day in old (M and P) rats. Merged images are shown with Hoechst 33342 nuclear label. (I) Serial section to B. Scale
bars: A–C and E–H = 200 mm; D =50mm; I = 100 mm; J and M =10mm; K and L =20mm; N and O =4mm; P =8mm.
1936 M. F. Stidworthy et al.
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the animal exhibiting the highest proportion of remyelination.
If it was not possible to differentiate between two animals using
this method then they were given the same rank. Two inde-
pendent experiments were performed; the first using age-
matched mice of mixed gender, the second using male mice
only. Time-points were chosen to coincide with maximal OPC
recruitment to areas of demyelination (Matsushima and Mor-
ell, 2001). In the first experiment (Fig. 7A) a single daily
injection of tamoxifen was given from the beginning of
week 4 of cuprizone treatment, continuing until the animals
were sacrificed. Following ranking, groups were compared
using the Mann–Whitney test. No significant differences
were observed between groups at either time-point or location,
between males or females, or within female-only groups. Com-
parison of males at 14 days identified more myelin in control
mice, with a P value of 0.1, the minimum permissible P value
with the available group sizes. A second experiment thus used
males only. Induction of recombination was by twice-daily
tamoxifen injection from the day after cuprizone ceased, a
protocol previously demonstrated to have equivalent or super-
ior recombination efficiency (Leone et al., 2003). This timing
removed the possibility that OPC recruited during the demye-
linating phase were eliminated by differentiation in the pre-
sence of cuprizone, obscuring a true difference in the rate of
remyelination. Rankings were performed and revealed no dif-
ferences between the control and knock-out groups (Fig. 7B).
To exclude the possibility that ranking was confounded by
either small axon size or the degree of inflammation, propor-
tions of myelinated and unmyelinated fibres in the body of
the corpus callosum and the dorsal hippocampal region of
the splenium were assessed ultrastructurally as previously
described (Stidworthy et al., 2003). Proportions of myelinated
and unmyelinated fibres followed predicted trends as remye-
lination proceeded, but there was no significant difference
between the proportions in control or knock-out groups at
any time (Fig. 7C). Such analysis would not identify differ-
ences in the remyelination rate manifest in the thickness
of partially formed sheaths. Therefore, ultrastructural
Fig. 7 Histological and ultrastructural measures of early remyelination are not significantly different between Notch knock-out and control
mice. (A and B) Myelination rank data for experiment 1 (A) using age-matched mice of mixed gender, and for experiment 2 (B) using male
mice only. Comparison of the rank sums in each experiment using the Mann–Whitney test yielded no significant differences between
groups. (C) Proportions of myelinated and unmyelinated axons show expected trends as remyelination proceeds, but there is no difference
between knock-out and control animals. Table: Mean G ratio measurements for normal mice (n = 4), and for knock-out and control
mice at 6 and 7 weeks in experiment 1. There is no significant difference between any of the groups.
Notch-jagged and CNS remyelination 1937
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measurements of G ratio (the ratio of axon diameter to mye-
linated fibre diameter) were made at two locations in the corpus
callosum (Stidworthy et al., 2003). Individual means were
calculated from at least 100 axons from each animal, and
used to generate group means for knock-out, control and
untreated animals (Fig. 7, table). Means were compared by
ANOVA. This revealed no significant differences in G ratio
between the groups at any time-point.
Expression patterns of a LacZ reporter gene,
NG-2, and markers of mitosis and apoptosis
are similar between Notch1 knock-out mice
and Notch1 heterozygote mice
To confirm that numbers and distributions of recombined
Plp-positive cells were similar between mice in which
Notch1 was ablated, and those in which a normal Notch1 allele
remained, the distribution of LacZ reporter-expressing cells
was compared between cuprizone-treated, age-matched
female Notch1 knock-out animals and heterozygous animals
expressing Plp-creER in combination with one normal
Notch1 allele and the R26R allele (Leone et al., 2003).
Distribution of LacZ-positive cells in both groups was similar
to that described previously within myelinated and grey matter
areas (Leone et al., 2003). In the demyelinated corpus callo-
sum, patchy labelling was observed which paralleled the
distribution of myelination and demyelination as previously
characterized (Stidworthy et al., 2003) (Fig. 8A–D). No
qualitative or quantitative differences were apparent between
the two groups in the distribution or number of LacZ-positive
cells, indicating that tamoxifen treatment and Cre-mediated
recombination per se do not result in differences in Plp-
positive OPC recruitment. Furthermore, LacZ expression is
likely to denote recombination at all lox sites within a
particular cell, and hence ablation of both Notch1 alleles in
Plp-creER Notch1
lox/lox
mice. This conclusion was further
supported by immunolabelling of serial sections from both
groups for NG-2. Both were qualitatively similar, and
yielded identical labelling intensities in the splenium and
Fig. 8 Patterns of OPC recruitment and proliferation are similar in the corpus callosum of Notch1 knock-out and Notch1
lox/wt
heterozygote
mice, despite a reduction in Notch1 expression. (A–D) Recruitment of recombined Plp-expressing cells, labelled with the marker gene
LacZ, is similar into areas of demyelination in both heterozygote (A and C) and knock-out (B and D) mice. Arrow indicates myelinated
rostral commissure labelled with X-gal. (E and F) Dual labelling of the remyelinating corpus callosum with antibodies against NG-2 and
Notch1. NG-2 and Notch1 colocalize in areas of remyelination in heterozygote animals (E), whereas there are areas of NG-2þ cell
recruitment without Notch1 labelling in knock-out mice (F). (G–I) Similar numbers of PDGFaRþ cells are recruited into the corpus
callosum of heterozygote (G) and knock-out (H) mice, and there is no difference in the intensity of NG-2 labelling intensity between the
two groups (I). (J) The number of proliferating cells in the remyelinating corpus callosum (identified by phosphorylated histone h3
expression) is not significantly different between heterozygote and knock-out mice. Scale bars: A–D = 1 mm; E and F = 100 mm;
G and H =50mm.
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body of the corpus callosum when quantified by a method
similar to that described previously (Genoud et al., 2002)
(Fig. 8I). Comparison of OPC labelling by in situ
hybridization for PDGFaR was similar (Fig. 8G and H). To
identify differences in proliferation or cell death as a conse-
quence of Notch1 ablation, serial sections of corpus
callosum were labelled using an antibody against phosphoryl-
ated histone h3 (Hh3), a proliferation marker, and by the
TUNEL method, a marker of apoptotic cell death. No signific-
ant differences in counts of Hh3-positive cells within the
corpus callosum were detected between the groups at either
time-point (Fig. 8J), implying that cellular proliferation is
independent of genotype. Very few TUNEL-positive cells
were seen and there was no difference between the groups
(data not shown). Detailed histological, ultrastructural and
immunohistochemical examination thus indicated no differ-
ences in the extent of remyelination, the proportion of remye-
linated axons, the rate at which axons were remyelinated, or in
OPC recruitment, proliferation and cell death within the corpus
callosum of Notch1 knock-out mice and mice expressing
Notch1.
Notch1 is expressed in remyelinating lesions of
the mouse corpus callosum, and dual NG-2/
Notch1 labelling segregates differently
between heterozygote and knock-out animals
Finally, sections of corpus callosum from each of the groups
were labelled simultaneously with antibodies against Notch1
and NG-2. In control animals, Notch1 positivity was found
both in the presence and absence of colocalized NG-2 positiv-
ity (Fig. 8E). In knock-out animals, however, the bulk of the
Notch1 positivity did not overlap with areas of NG-2 labelling
(Fig. 8F), further indicating the efficiency of recombination in
the Plp-creER model.
Discussion
To study the role of the Notch–Jagged pathway in remyelina-
tion we have examined the expression of Notch and Jagged in
an experimental model of demyelination that undergoes com-
plete remyelination (Woodruff and Franklin, 1999; Penderis
et al., 2003). First, we compared expression in young animals
with that in old animals, where the slower rate of remyelina-
tion is largely due to a delay in OPC differentiation (Shields
et al., 1999; Sim et al., 2000, 2002b). If this delay were
attributable to Notch–Jagged signalling we would predict
divergence in expression profiles between the two age groups,
particularly at the later time-points, when remyelination in
young animals is complete, but continuing in older animals
(Shields et al., 1999; Sim et al., 2000, 2002b). Secondly, we
used an inducible knock-out mouse, in which Notch1 ablation
was confined to Plp-expressing cells in the presence of
tamoxifen, to compare remyelination in the absence of
Notch1.
Our findings of Notch1 expression in adult oligodendrocyte
lineage cells correspond to those described in the adult optic
nerve (Wang et al., 1998) and the adult forebrain (Irvin et al.,
2001). We found that resting adult astrocytes do not express
Notch1, suggesting Notch1 function in astrocytes is predomin-
antly confined to lineage commitment in development
(Morrison et al., 2000; Ge et al., 2002). However, following
demyelination, expression was observed in small numbers of
astrocytes at the lesion margins. Reports on neuronal expres-
sion are conflicting. Some demonstrate Notch1 in neurons
of the adult cerebellum and neocortex (Higuchi et al., 1995;
Berezovska et al., 1998; Mikami et al., 2001; Stump et al.,
2002), and enteric nervous system (Sander et al., 2003). Others
have failed to demonstrate significant expression in neurons in
the adult striatum or in neuron-enriched embryonic cultures
(Irvin et al., 2001). This may reflect anatomical variability, or
differences in the sensitivities of methods employed. In our
study there was good correlation in neuronal expression of both
mRNA and protein by independent methods.
Using RT–PCR we demonstrated that two Notch ligands,
Jagged1 and Jagged2, are expressed constitutively in the tri-
geminal ganglion, subsequently confirming expression of
Jagged1 on myelinated axons using immunolabelling. The
constitutive expression of Jagged1 on myelinated fibres con-
trasts with the developing optic nerve, in which axonal Jagged1
expression fell below the limits of detection as myelina-
tion proceeded (Wang et al., 1998). We did, however, confirm
Jagged1 expression in NG-2-positive OPCs, and in oligoden-
drocytes, consistent with previous findings (Wang et al., 1998).
The presence of Notch1 in post-mitotic neurons and oligo-
dendrocytes, together with the ligand Jagged1 in axons and
OPCs, suggests an ongoing role for Notch–Jagged signalling
in the adult.
Hypotheses based on developmental recapitulation in adult
regenerative processes often prove helpful. If regulation of
remyelination in the adult trigeminal tract were to recapitulate
the developmental pattern in the optic nerve, one would expect
OPCs to express Notch1 and levels of Jagged1 expression to
increase on demyelinated axons, subsequently declining as
axons remyelinate. We confirmed that Notch1 is expressed
by adult NG-2-positive OPCs recruited into remyelinating
lesions. Although we did not unequivocally demonstrate func-
tional activation of Notch1 in this study we think this likely
to be the case since Notch1 expression is associated with
increased expression of the Notch-associated transcription
factor Hes5 (Kondo and Raff, 2000) in mouse models of
toxin-induced demyelination (unpublished data). These cells
populated the lesion from the periphery, a pattern previously
described for PDGFaR-positive OPCs (Sim et al., 2002b).
Throughout remyelination, RT–PCR confirmed Jagged1 and
Jagged2 expression in trigeminal ganglia. However, the intens-
ity of Jagged1 protein expression by demyelinated axons was
low. Thus, in contrast to development, demyelinated axons are
not a major source of Jagged1 in our experimental model or in
multiple sclerosis tissue (John et al., 2002), and other cell types
such as macrophages and reactive astrocytes provide a more
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abundant source. In addition, we noted Jagged1 expression by
both NG-2-positive adult OPCs (suggesting that adjacent
OPCs may exert mutually inhibitory effects on one another)
and adult oligodendrocytes, as well as by myelinating Schwann
cells. Jagged1 was originally cloned from a neonatal rat
Schwann cell cDNA library (Lindsell et al., 1995), but expres-
sion during remyelination has not been reported. The alterna-
tive sources of ligand appear likely to overwhelm the axonal
Jagged1 component, by virtue not only of their quantitative
predominance, but also of their close temporal and spatial
association with the Notch1-expressing population. The
macrophage and reactive astrocyte presence is especially pro-
minent during the early phases of remyelination, gradually
subsiding as remyelination progresses. This suggests a
mechanism in which Jagged1 expression by inflammatory
cells and reactive astrocytes provides signals that by prevent-
ing premature differentiation of OPCs maintains them in a
mitogen responsive state during the initial recruitment phase
of remyelination. Consistent with this concept is the protracted
macrophage presence in toxin lesions in old animals, which
may contribute to the delayed OPC differentiation (Hinks and
Franklin, 2000).
Using Notch1 knock-out mice, we were unable to identify
differences during remyelination after cuprizone intoxication.
The time-points were chosen to coincide with maximal OPC
recruitment and differentiation within demyelinated areas of
the corpus callosum (Matsushima and Morell, 2001). We also
used protocols previously shown to induce high levels of
recombination of floxed loci in Plp-creER mice (Leone
et al., 2003). Using a range of histological measures of remye-
lination (Stidworthy et al., 2003), as well as immunolabelling
techniques to identify OPC, cellular proliferation and apopto-
sis, we were unable to identify differences in the extent of
remyelination, the proportion of remyelinated axons, the
rate at which axons were remyelinated, or in OPC recruitment,
proliferation and cell death within the corpus callosum. These
findings support the conclusion that Notch signalling, either
via Jagged or other ligands such as F3/Contactin (Hu et al.,
2003) is not rate-determining during remyelination of toxin-
induced demyelination. It would also seem that Notch signal-
ling does not play a major role in determining OPC recruit-
ment, proliferation or cell death, during the early stages of
remyelination.
This study has allowed us to draw several conclusions
regarding the role of Notch–Jagged signalling in CNS remye-
lination. First, since remyelination can proceed to completion
despite widespread Notch–Jagged expression, our findings
indicate that Notch–Jagged signalling in adult OPCs does
not imply that remyelination will fail. Secondly, although simi-
lar signalling pathways may be used during remyelination the
principal cellular protagonists may differ compared with
developmental myelination. Lastly, since there were no quan-
titative differences in Notch1 expressing cells in slow and
rapidly remyelinating lesions, or in the remyelination that
occurred in mice in which Notch1 was excised in OPCs and
in control mice, we conclude that Notch–Jagged signalling is
not a rate-limiting determinant of remyelination in rodent
models of demyelination.
Acknowledgements
The authors wish to thank Paddy Mannion, Anil Kalupahana
and Mike Peacock for their assistance and Gerry Weinmaster
and Verdon Taylor for kind donations of reagents. The project
was funded by the Wellcome Trust, Research into Ageing, a
Neuropathology and Applied Neurobiology Bursary, and the
European Commission specific RTD programme ‘Quality of
life and Management of Living Resources’, QLK6-CT-2000-
00179. MFS held a Wellcome Trust Research Training
Fellowship. U.S. is supported by the Swiss National Science
Foundation and the National Center of Competence in
Research ‘Neural Plasticity and Repair’.
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