1 MRC Laboratory for Molecular Cell Biology and Cell Biology Unit, University
College London, London WC1E 6BT, UK
2 Biochemistry Laboratory, IDI-IRCCS, c/o Tor Vergata University, Via
Montpellier, 1, 00133 Roma, Italy
3 Breakthrough Breast Cancer Centre, London Institute of Cancer Research, 237
Fulham Road, London SW3 6JB, UK
4 Wolfson Institute for Biomedical Research, University College London, London
WC1E 6BT, UK
5 MRC Toxicology Unit, Hodgkin Building, Leicester University, Lancaster Road,
Leicester LE1 9HN, UK
Author for correspondence (e-mail:
billon{at}unice.fr)
Accepted 10 December 2003
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SUMMARY |
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Key words: Differentiation, Oligodendrocyte, p53, p63, p73, Rat
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Introduction |
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We have been studying the stopping mechanisms in the oligodendrocyte cell
lineage in the rodent optic nerve. Oligodendrocyte precursor cells (OPCs)
migrate from the brain into the developing rat optic nerve before birth
(Small et al., 1987). After a
period of proliferation, most OPCs stop dividing and terminally differentiate
into oligodendrocytes (Temple and Raff,
1986
), which then myelinate the axons in the nerve. The first
oligodendrocytes appear in the rat optic nerve around birth and then increase
in number for the next six weeks (Barres et
al., 1992
; Miller et al.,
1985
; Skoff et al.,
1976
).
The normal timing of oligodendrocyte differentiation can be reconstituted
in cultures of perinatal rat optic nerve cells
(Raff et al., 1985). Clonal
analyses performed with single (Temple and
Raff, 1986
) or purified (Barres
et al., 1994
) OPCs show that the progeny of an individual OPC stop
dividing and differentiate at approximately the same time, even if separated
and cultured in different microwells, suggesting that a cell-intrinsic
mechanism operates in the OPCs to help limit their proliferation and initiate
differentiation after a certain period of time or number of cell divisions.
The finding that OPCs cultured at 33°C divide more slowly but stop
dividing and differentiate sooner, after fewer divisions, than when they are
cultured at 37°C suggests that the intrinsic mechanism does not operate by
counting cell divisions but instead measures time in some other way
(Gao et al., 1997
). The timing
mechanism depends in part on the progressive increase in cyclic-dependent
kinase (Cdk) inhibitor p27/kip1
(Casaccia-Bonnefil et al.,
1997
; Durand et al.,
1998
; Durand et al.,
1997
) and the progressive decrease in the inhibitor of
differentiation 4 (Id4) protein (Kondo and
Raff, 2000
).
Although the timer is cell-intrinsic, it is not cell autonomous. It
requires extracellular signals to operate normally. The mitogen PDGF, for
example, is one of these signals (Noble et
al., 1988; Pringle et al.,
1989
; Raff et al.,
1988
). In the absence of PDGF, cultured OPCs prematurely stop
dividing and differentiate into oligodendrocytes within 1-2 days
(Noble and Murray, 1984
;
Temple and Raff, 1985
). It
seems probable that a lack of sufficient PDGF is responsible for the
differentiation of some OPCs in vivo, especially early in development
(Calver et al., 1998
;
van Heyningen et al., 2001
).
It is unclear, however, how PDGF withdrawal induces OPCs to differentiate.
Other extracellular signals are also required for the normal operation of
the intrinsic timer in cultured OPCs, including hydrophobic signals such as
thyroid hormone (TH) or retinoic acid (RA)
(Ahlgren et al., 1997;
Barres et al., 1994
;
Gao et al., 1998
). When
purified postnatal day 7 (P7) rat OPCs are cultured in the presence of PDGF
but in the absence of TH and RA, most of the cells keep dividing and do not
differentiate (Ahlgren et al.,
1997
; Barres et al.,
1994
; Tang et al.,
2001
). If TH is added after 8 days, however, most of the cells
stop dividing and differentiate within 4 days
(Barres et al., 1994
). These
findings and others (Bögler and Noble,
1994
) suggest that the intrinsic timer consists of at least two
components: a timing component, which depends on PDGF and measures time
independently of TH or RA, and an effector component, which can be regulated
by TH and RA and stops cell division and initiates differentiation when time
is up. Thus, TH and RA can induce OPCs to differentiate only when the OPCs
have reached a certain stage of maturation
(Gao et al., 1998
), whereas
PDGF withdrawal induces OPCs to differentiate at any stage of maturation,
whether TH or RA is present or not (Ahlgren
et al., 1997
; Barres et al.,
1994
; Gao et al.,
1998
).
Although it is unclear whether RA regulates oligodendrocyte development in
vivo, it has long been known that TH does. Myelination, for example, is
delayed in hypothyroid animals (Dussault
and Ruel, 1987; Rodriguez-Pena
et al., 1993
) and accelerated in hyperthyroid animals
(Marta et al., 1998
;
Walters and Morell, 1981
).
Moreover, perinatal hypothyroidism decreases the number of oligodendrocytes in
the optic nerve of the rat (Ibarrola et
al., 1996
) and mouse (Ahlgren
et al., 1997
). Thus, it seems probable that the TH-regulated
intrinsic timer is responsible for the differentiation of at least some OPCs
in vivo, especially postnatally, when TH levels are rising and OPCs are
becoming more responsive to TH (Gao et
al., 1998
). But TH is not required for oligodendrocyte
development, as even in its absence some OPCs eventually differentiate into
oligodendrocytes both in vivo (Ahlgren et
al., 1997
) and in vitro
(Ibarrola et al., 1996
;
Ahlgren et al., 1997
). Because
TH influences the timing of differentiation in several cell lineages, it is
probable that it plays a part in coordinating the timing of differentiation in
different tissues during vertebrate development: TH coordinates the onset of
myelination in the central and peripheral parts of the auditory nerve, for
example (Knipper et al.,
1998
).
It remains unclear how TH or RA triggers OPC differentiation. Both act by
binding to nuclear receptors that are members of the same superfamily of
ligand-regulated transcription factors
(Evans, 1988;
Mangelsdorf et al., 1995
). We
showed previously that the TH receptor TR
1 is required for the normal
timing of oligodendrocyte development in vitro and in vivo, but the downstream
effectors of this differentiation pathway are still uncertain
(Billon et al., 2002
). One part
of the pathway probably involves E2F-1, as TH rapidly inhibits the expression
of E2F-1 in purified rat OPCs; the E2F-1 promoter contains a negative
TRE (called a Z-element) that binds thyroid hormone receptors (TRs), which
directly activate E2F-1 transcription in the absence of TH and
repress it in the presence of TH
(Nygård et al., 2003
).
As E2F-1 promotes progression from G1 into S phase of the cell cycle
(Helin, 1998
), its repression
by TH is likely to contribute to the cell-cycle withdrawal and differentiation
of OPCs in response to TH (Nygård et
al., 2003
). On a slower timescale, TH also influences the
expression of other genes that would be expected to help induce OPC to exit
the cell cycle and differentiate: by 16 hours, for example, it stimulates an
increase in mRNAs that encodes various Cdk inhibitors, and, by 24 hours, it
decreases the level of cyclin D1 and D2 proteins
(Tokumoto et al., 2001
).
There is also evidence that the p53 family of proteins plays a part in RA-
and TH-induced OPC differentiation. This differentiation is blocked if
purified OPCs are infected by a retroviral vector encoding a dominant-negative
form of p53 that inhibits both p53 and other members of the p53 family
(Tokumoto et al., 2001). It
has also been reported that a dominant-negative form of p53 inhibits
spontaneous oligodendrocyte differentiation in mixed cultures of neonatal rat
brain cells (Eizenberg et al.,
1996
). It is still uncertain, however, which p53 family members
are important for OPC differentiation or how they promote this
differentiation.
p53, p63 and p73 proteins share considerable structural and functional
homology (Yang et al., 2002).
They all function as transcription factors and regulate the expression of
similar groups of genes by directly binding to identical DNA response elements
in the promoter. Whereas the p53 gene encodes one major protein, both
the p63 and p73 genes contain two separate promoters that
direct the expression of two functionally distinct types of protein from each
gene (reviewed by Irwin and Kaelin,
2001
; Levrero et al.,
2000
; Melino et al.,
2002
). One type of protein, denoted TAp63 or TAp73, has an acidic
N-terminus, which is homologous to the transactivation domain of p53. In the
second type of protein, denoted
Np63 or
Np73, the N-terminus is
truncated and lacks the transactivation domain. Alternative splicing at the
3' end of the p63 and p73 RNA transcripts generates
additional complexity by creating both TA and
N proteins with different
C-termini. Whereas the TA isoforms are able to activate gene expression, the
N isoforms cannot and instead can exert a dominant-negative effect on
p53 and the TA forms of p63 and p73 (Grob
et al., 2001
). In principle, this dominant-negative effect could
involve competition between the
N and TA isoforms for DNA response
elements, formation of nonfunctional oligomers between the
N and TA
isoforms, or both (Melino et al.,
2002
).
Despite the functional similarities of p53, p63 and p73 proteins, deletion
of the individual genes in mice has very different outcomes, suggesting that
each gene has distinct roles in vivo. p53-deficient mice have an unstable
genome and an increased incidence of cancer, but they generally develop
normally (Donehower et al.,
1992), although a small proportion have defects in neural tube
closure (Armstrong et al.,
1995
; Sah et al.,
1995
). In contrast, the p63 and the p73 genes
are both critical for normal development. p63 is required for epidermal
development. Mice lacking all forms of p63 have no skin or limbs
(Mills et al., 1999
;
Yang et al., 1999
), and
anti-sense inhibition of
Np63 in developing zebrafish indicates that it
is this isoform of p63 that is critical for epidermal and fin development in
the fish (Bakkers et al., 2002
;
Lee and Kimelman, 2002
). Mice
deficient in all forms of p73 have severe defects in neural development,
including hydrocephalus, hippocampal dysgenesis, loss of Cajal-Retzius
neurons, and defects in pheromone sensory pathways; they also suffer from
chronic infection and inflammation (Yang
et al., 2000
). The neuronal loss in these mice may be because of
the loss of
Np73, which has been shown to prevent neuronal
apoptosis by antagonizing p53 in both sympathetic and cortical neurons
(Pozniak et al., 2002
;
Pozniak et al., 2000
). A role
for p73 in neural differentiation is suggested by the findings that RA-induced
differentiation of a mouse neuroblastoma line depends on p73, and
overexpression of p73 in these cells induces their differentiation in the
absence of RA (De Laurenzi et al.,
2000
).
In the present study, we have used RT-PCR, immunofluorescence, retrovirus-mediated gene transfer, and p53-/- mice to investigate the expression and function of the individual p53 family proteins in the developing oligodendrocyte lineage. Our findings suggest that both p53 and p73, but not p63, normally play a part in OPC differentiation.
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Materials and methods |
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UV irradiation was performed with a UV cross-linker at 50 J/m2. Cells were returned to the incubator for 8 hours before they were analysed.
RT-PCR analysis
Cells were harvested with trypsin and processed immediately for RT-PCR
analysis. Total RNA was prepared using an RNeasy purification kit (Quiagen).
cDNAs were synthetised using Superscript-II Reverse Transcriptase (Invitrogen)
according to the supplier's instructions and were used as templates for the
PCR reaction.
The following oligonucleotide primers were synthesised. For
glyceraldehyde-3-phosphate dehydrogenase (G3PDH), the
5' primer was 5'-ACC ACA GTC CAT GCC ATC AC-3', and the
3' primer was 5'-TCC ACC ACC CTG TTG CTG TA-3'; for
p53, the 5' primer was 5'-GCT TTG AGG TTC GTG TTT GTG
CC-3', and the 3' primer was 5'-AGT CAT AAG ACA GCA AGG AGA
GGG G-3'; for TAp73, the 5' primer was 5'-AGG GTC
TGT CGT GGT ACT TTG ACC-3', and the 3' primer was 5'-GGT TGT
TGC CTT CTA CAC GGA TGA G-3'; for Np73, the 5'
primer was 5'-CAC GAG CCT ACC ATG CTT TAC-3', and the 3'
primer was 5'-GGT TGT TGC CTT CTA CAC GGA TGA G-3'; for
TAp63, the 5' primer was 5'-CTT ACA TCC AGC GTT TCA
A-3', and the 3' primer was 5'-GTT AGG GCA TCG TTT CAC
A-3'; for
Np63, the 5' primer was 5'-TGG AAA
GCA ATG CCC AGA CTC-3', and the 3' primer was 5'-CAA CCT GTG
GTG GCT CAT AAG G-3'.
The RT-PCR reactions were performed as follows: 25 µl of reaction
mixture contained 200 pg of template cDNA, 300 nM of 5' and 3' PCR
primers, 0.2 mM dNTP, 1.25 mM MgCl2, 10 mM Tris-HCl (pH 9.0), 50 mM
KCl, 0.1% Triton X-100, and 1.25 units of Taq DNA polymerase (Promega). The
reaction mixture was denatured for 3 minutes at 95°C. The PCR parameters
were 95°C for 40 seconds for the denaturing step, 58°C (p53),
59.5°C (TAp73 and Np73) or 55°C
(TAp63,
Np63 and GAPDH) for 35 seconds for
the annealing step, and 72°C for 35 seconds for the elongation step. The
PCR products were electrophoresed in a 1.6% agarose gel and stained with EtBr.
The number of PCR cycles was 35 for p53, 37 for TAp73 and
Np73, 39 for TAp63 and
Np63, and 25
for G3PDH.
Retrovirus vectors
To identify infected cells, we used the pBird vector, which expresses the
cDNA encoding enhanced green fluorescent protein (GFP), controlled by the CMV
promoter (Tokumoto et al.,
2001). To express human p53, p53DN (R175H)
(Kern et al., 1992
),
p53DD (Shaulian et al.,
1992
), TAp73 (De
Laurenzi et al., 1998
),
Np73
(Grob et al., 2001
), mouse
TAp63 (GeneBank accession y19234) and
Np63 (GeneBank
accession y1923) transgenes, we cloned the respective cDNAs into the pBird
vector to create retroviral vectors that expressed the transgenes under the
control of the Moloney murine leukemia virus long terminal repeat promoter, as
well as the GFP transgene under the control of the CMV promoter. Recombinant
retroviruses were produced and concentrated as previously described
(Kondo and Raff, 2000
).
Infection of rat OPCs with retroviral vectors
After 2 days in culture in the presence of PDGF, purified rat OPCs were
infected with concentrated retroviral supernatant for 3 hours, washed, removed
from the culture flask with trypsin, and replated on PDL-coated Nunc slide
flasks in the presence of PDGF. After 1 day, the cells were either maintained
in PDGF alone or switched to either PDGF plus TH (triiodothyronine, 40 ng/ml),
PDGF plus RA (all-trans, 1 µM), or medium that did not contain
PDGF. The latter three conditions were used to induce OPC differentiation. The
percentage of GFP-positive cells that had differentiated into oligodendrocytes
was determined at different time points, using a Leica inverted fluorescence
microscope and morphological criteria
(Temple and Raff, 1986). In
some experiments, the percentage of oligodendrocytes was confirmed by fixing
the cells and staining them with a monoclonal anti-galactocerebroside (GC)
antibody (Raff et al., 1978
;
Ranscht et al., 1982
), as
described below.
Mouse optic nerve cells
P53+/- mice
(Donehower et al., 1992) were
bred at the animal facility at the Institute of Cancer Research, London. Mice
were sacrificed at P7 and genotyped by RT-PCR. Optic nerves were removed,
dissociated with papain, and the cells were cultured on PDL-coated Nunc slide
flasks as previously described (Billon et
al., 2002
). They were cultured for 2 days in PDGF alone and then
infected for 3 hours as described above. After another 2 days in PDGF alone,
they were either maintained in PDGF alone or switched to either PDGF plus TH
or to medium that did not contain PDGF for an additional 1-3 days. They were
then fixed and stained for GC, as described below. In some cases, the optic
nerve cells were counted in a haemocytometer, cultured on PDL-coated glass
coverslips for 3-4 hours, before staining them for GC, NG2, or glial
fibrillary acidic protein (GFAP), as described below.
Immunocytochemistry
All treatments were performed at room temperature. Cells were fixed in 2%
paraformaldehyde in PBS for 5 minutes. After washing with PBS, they were
incubated for 30 minutes in normal goat serum to block non-specific staining.
They were then incubated for 1 hour in a mouse monoclonal anti-GC antibody
(Ranscht et al., 1982)
(supernatant, diluted 1/5) or rabbit anti-NG2 antibodies (Chemicon, diluted
1/100). Cells were then washed in PBS and incubated for 1 hour in
Texas-Red-coupled goat anti-mouse IgG (GC) or goat anti-rabbit IgG (NG2)
antibodies (Jackson Labs, diluted 1/100) and bisbenzamide (Sigma, 5 ng/ml) to
stain the nuclei.
For GFAP, p53, p63 and p73 staining, the cells were fixed in
paraformaldehyde as above and then permeabilised in 0.1% triton in normal goat
serum for 30 minutes. The cells were then incubated for 1 hour in a mouse
monoclonal anti-GFAP antibody (Sigma G3893, diluted 1/100), rabbit anti-p53
antibodies (CM5, gift from Alison Sparks, University of Dundee, diluted
1/10,000), a mouse monoclonal anti-p63 antibody (BD Pharmingen; diluted
1/100), or rabbit anti-p73 antibodies (P73N, raised against an N-terminal
peptide in TAp73 that is not present in Np73; gift from Susan Bray,
University of Dundee; diluted 1/10 000). Cells were then washed in PBS and
incubated for 1 hour in fluorescein-coupled goat anti-mouse IgG (GFAP, p63),
or anti-rabbit IgG (p53 and p73) antibodies (Jackson Labs, diluted 1/100) and
bisbenzamide. In some experiments, cells were double stained for GC and p73.
In this case, GC staining was performed first, followed by permeabilisation
and p73 staining. Coverslips were mounted in Citifluor mounting medium
(CitiFluor, UK) and examined with a Zeiss Axioskop fluorescence microscope. In
all cases, no staining was seen when the fluorescent anti-IgG antibodies were
used on their own.
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Results |
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Expression of p53, p63 and p73 in the rat oligodendrocyte lineage
We first used RT-PCR to examine the expression of p53, TAp63,
Np63, TAp73 and
Np73 mRNAs in cultured OPCs
purified from the P7 rat optic nerve. The purified cells were expanded for 10
days in the presence of PDGF and the absence of TH and RA. As shown in
Fig. 1, we could detect all of
these mRNAs.
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|
To determine whether p73 was expressed in OPCs in vivo, we purified OPCs from P7 rat optic nerves, cultured them on PDL-coated glass coverslips for 2 hours, and stained them for p73. As for the OPCs expanded for 10 days in PDGF, all of the freshly isolated OPCs expressed p73 in their nucleus (not shown).
Effects of p53 or dominant-negative p53 transgenes in rat OPCs
In a previous study (Tokumoto et al.,
2001), when purified OPCs were infected with a retroviral vector
encoding a dominant-negative form of human p53 (R175H, which we shall refer to
as p53DN) (Kern et al., 1992
),
the infected cells failed to stop dividing and differentiate in response to TH
or RA, although they stopped dividing and differentiated normally in response
to PDGF withdrawal. This mutant form of p53, however, has been shown to
inhibit the transcriptional activity of p63 and p73, as well as of p53
(Gaiddon et al., 2001
;
Strano et al., 2000
).
We therefore repeated these experiments with a dominant-negative form of
p53 (p53DD) (Shaulian et al.,
1992) that lacks the central core domain, which is thought to
mediate the interaction of wild-type p53 with p73
(Gaiddon et al., 2001
). This
form of p53 therefore should specifically inhibit p53 and not p73. We cultured
purified rat OPCs in PDGF without TH and RA for 2 days and then infected them
with either a control retroviral vector (pBird), which encodes GFP only, or a
retroviral vector that encodes both GFP and either p53DN (pBird-p53DN) or
p53DD (pBird-p53DD). After a further day in culture in PDGF without TH and RA,
we either maintained the cells in these conditions or induced them to
differentiate by switching them to PDGF plus TH, PDGF plus RA, or medium that
did not contain PDGF. After three days, we determined the percentage of
GFP-positive cells with morphological features characteristic of
oligodendrocytes. In some experiments, we confirmed the oligodendrocyte
identity of these cells by staining for GC (not shown). As shown in
Fig. 3, both the p53DN and the
p53DD vectors completely blocked TH- and RA-induced differentiation. Neither
of them, however, inhibited spontaneous differentiation in the presence of
PDGF without TH or RA or the differentiation induced by PDGF withdrawal, as
shown previously for P53DN (Tokumoto et
al., 2001
). These findings suggest that p53 is involved in OPC
differentiation induced by TH or RA in culture, but not in OPC differentiation
induced by PDGF withdrawal.
|
Effects of TAp63 or Np63 transgenes in rat OPCs
To test whether the expression of transgenes encoding either the
full-length TAp63 or dominant-negative Np63 isoforms of p63 would
affect OPC differentiation, we repeated the experiments just described but
used retroviral vectors that encode either GFP and TAp63 (pBird-TAp63) or GFP
and
Np63 (pBird-
Np63) and cultured the cells as described in
Fig. 3. As shown in
Fig. 4, the expression of
either the TAp63 or
Np63 transgene did not significantly affect either
the spontaneous differentiation of OPCs cultured in PDGF without TH or RA or
the differentiation induced by TH, RA or PDGF withdrawal. Thus, p63 is
unlikely to play a part in OPC differentiation, at least in culture.
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|
Effects of p73N transgene on p53-/- mouse OPC differentiation
As Np73 would be expected to inhibit the transcriptional activity of
p53, as well as that of TAp73, it was important to determine whether
Np73 could inhibit OPC differentiation in the absence of p53. We
therefore tested the effect of the
Np73 transgene on cultures
of P7 optic nerve cells prepared from wild-type or p53-/-
mice. We infected the cells with the pBird-
Np73 retroviral vector and
cultured them in either PDGF without TH and RA, in PDGF with TH, or without
PDGF. After 1-3 days, we stained the cultures for GC to determine the
proportion of GFP-positive cells that had differentiated into GC-positive
oligodendrocytes.
As shown in Fig. 6,
expression of the Np73 transgene significantly decreased both
spontaneous differentiation (Fig.
6A) and the differentiation induced by either TH
(Fig. 6B) or PDGF withdrawal
(Fig. 6C) in both wild-type and
p53-/- cultures. Thus, p53 is not required for the
Np73 transgene to inhibit OPC differentiation in vitro.
Interestingly, however, the level of induced differentiation in
p53-/- cultures was slightly, but reproducibly, less than
that in wild-type cultures (see Fig.
6B,C).
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Discussion |
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We can detect mRNAs for each of the three families in purified OPCs by
RT-PCR, raising the possibility that all three may be involved in
oligodendrocyte development. However, although we can detect mRNA for both p63
and Np63 in OPCs and p63 protein in the nucleus of almost all OPCs and
oligodendrocytes in culture, it seems unlikely that p63 is involved in OPC
differentiation. The expression of transgenes that encode either TAp63 or
dominant-negative
Np63 in purified rat OPCs has no detectable effect on
OPC differentiation in culture - either on spontaneous differentiation or on
differentiation induced by TH, RA or PDGF withdrawal.
In contrast, several lines of evidence suggest a crucial role for p73 in
OPC differentiation. First, the only change in the three family member
proteins that we observe when OPCs differentiate is in p73. Whereas p73
staining is seen exclusively in the nucleus in OPCs, it is seen in both the
nucleus and the processes of oligodendocytes. The mechanism and functional
significance of this change in p73 distribution remain to be determined. As
the anti-p73 antibodies that we used recognise TAp73 isoforms but not
Np73 isoforms, it is probable that it is one or more TAp73 isoforms
that redistributes when OPCs differentiate. As the antibodies do not
distinguish between the various C-terminus isoforms of TAp73, which are
generated by alternative splicing at the 3' end of the p73 RNA
transcript, we do not know which isoforms are expressed in OPCs or
oligodendrocytes, or which ones redistribute upon OPC differentiation. The
second line of evidence suggesting an important role for p73 in OPC
differentiation is that the expression of a transgene encoding TAp73 in
purified OPCs increases the spontaneous differentiation of OPCs in the
presence of PDGF and the absence of TH and RA, as well as the differentiation
of OPCs induced by treatment with TH or RA. This is not seen with transgenes
encoding either p53 or TAp63. The third line of evidence is that the
expression of a transgene encoding
Np73 in purified OPCs inhibits all
forms of OPC differentiation in culture, including spontaneous differentiation
and differentiation induced by either PDGF withdrawal or treatment with TH or
RA. This is the only dominant-negative p53 family member that we tested that
inhibits all OPC differentiation in culture. Although
Np73 would be
expected to act as a dominant-negative inhibitor of all three p53 family
members, it inhibits both spontaneous and induced OPC differentiation in
cultures of p53-/- mouse optic nerve cells, indicating
that the inhibition does not depend on the inhibition of p53. As
Np63
does not inhibit OPC differentiation, it is unlikely that the
Np73
inhibition of OPC differentiation depends on the inhibition of TAp63. Thus, we
conclude that
Np73 inhibits all forms of OPC differentiation by
blocking TAp73 isoforms and that one or more of these isoforms is required for
normal OPC differentiation, at least in culture. It will be important to
confirm this conclusion in p73-deficient mice, which have severe
neurological defects, including congenital hydrocephalus, hippocampal
dysgenesis, and abnormalities in pheromone sensory pathways
(Yang et al., 2000
).
Oligodendrocyte development and myelination were not specifically addressed in
the report on these mice (Yang et al.,
2000
).
Although we can only detect p53 by immunocytochemistry in a small fraction
of OPCs and oligodendrocytes in culture, this does not necessarily exclude a
role for p53 in oligodendrocyte development. Indeed, two lines of evidence
suggest that p53 may be involved in OPC differentiation. First, the expression
of either of two transgenes encoding mutant, dominant-negative forms of p53 in
purified OPCs inhibits both TH- and RA-induced OPC differentiation, although
not spontaneous or PDGF-withdrawal-induced differentiation, as reported
previously (Tokumoto et al.,
2001). Although one of these mutant forms of p53 (p53DN) would be
expected to act as a dominant-negative inhibitor of all three family members,
the other (p53DD) lacks the central core domain and would be expected to
inhibit p53 specifically (Gaiddon et al.,
2001
; Shaulian et al.,
1992
). Second, we find a decrease in the number of
oligodendrocytes and an increase in the number of OPCs in the P7 optic nerve
of p53-/- mice compared with wild-type mice, consistent
with the possibility that p53 plays a part in OPC differentiation in vivo.
Similar results have recently been obtained independently in the developing
p53-/- optic nerve by Dean Tang and his colleagues; in
addition, they found that the numbers of oligodendrocytes and OPCs normalized
in the p53-/- optic nerves by P21 (Lubna Patrawala and
Dean Tang, University of Texas, personal communication). Together, these data
strongly suggest that p53-/- OPCs have a delayed
differentiation, at least in the optic nerve.
Although the CNS is thought to develop normally in most
p53-/- mice (Donehower
et al., 1992), a small proportion have defects in neural tube
closure (Armstrong et al.,
1995
; Sah et al.,
1995
). A detailed study of oligodendrocyte development and
myelination remains to be done in developing p53-/- mice.
Interestingly, p53 has been shown to play an important part in the
differentiation of neural and mesoderm cells in Xenopus embryos
(Wallingford et al., 1997
). It
physically and functionally interacts with Smads in the activin and BMP
signalling pathways to induce the expression of homeobox genes involved in
mesoderm formation in Xenopus
(Takebayashi-Suzuki et al.,
2003
).
In some respects, our results with p53 conflict with those of Eizenberg et
al. (Eizenberg et al., 1996),
who reported that p53 protein is highly expressed in brain-derived OPCs and
translocates from the cytoplasm to the nucleus when these cells differentiate
into oligodendrocytes in culture. Using three different antibodies, including
the antibody used in their study (not shown), we see relatively little p53
staining in OPCs and oligodendrocytes and cannot detect any in the cytoplasm
of either OPCs or oligodendrocytes. The reasons for these discrepancies are
unclear. However, Eizenberg et al. did find that a dominant-negative form of
p53 (p53DD) inhibited OPC differentiation in their culture system, consistent
with our present and previous (Tokumoto et
al., 2001
) findings.
We previously suggested that there may be at least two independent
intracellular pathways leading to cell-cycle arrest and differentiation in
OPCs - one that is activated by TH and RA and is p53-family-dependent and
another that is activated by PDGF withdrawal and is p53-family-independent
(Tokumoto et al., 2001). Our
present findings are consistent with this hypothesis for p53 but suggest that
p73 may be a key player in both pathways. As discussed previously, it is
probable that oligodendrocytes develop by both pathways in vivo
(Tokumoto et al., 2001
):
whereas a lack of sufficient PDGF may be responsible for the differentiation
of some OPCs, especially early in development
(Calver et al., 1998
;
van Heyningen et al., 2001
),
TH is more likely to influence OPC differentiation postnatally, when TH levels
are rising and OPCs are becoming more responsive to TH
(Gao et al., 1998
).
More work on p53-deficient mice and p73-deficient mice will be required to
establish roles for p53 and p73 in OPC differentiation in vivo. It will also
be important to determine how these proteins act to promote OPC cell-cycle
withdrawal and differentiation. Both proteins promote the transcription of
genes that encode cell-cycle inhibitory proteins such as p21/Cip1 and p27/Kip1
(Blint et al., 2002;
Fontemaggi et al., 2002
),
which probably help OPCs to exit the cell cycle and differentiate
(Casaccia-Bonnefil et al.,
1997
; Durand et al.,
1998
; Durand et al.,
1997
; Tokumoto et al.,
2001
), but it is highly probable that p53 and p73 regulate the
transcription of other genes involved in OPC cell-cycle withdrawal and
differentiation. It will also be important to determine the relative
contributions of TAp73 and
Np73 isoforms, especially as OPCs are the
first normal mammalian cells in which p73 has been implicated in
differentiation.
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ACKNOWLEDGMENTS |
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Footnotes |
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