|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 9 January 2008
doi: 10.1242/dev.010454
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
expressionInstitut für Biochemie, Emil-Fischer-Zentrum, Universität Erlangen, Fahrstrasse 17, D-91054 Erlangen, Germany.
* Author for correspondence (e-mail: m.wegner{at}biochem.uni-erlangen.de)
Accepted 27 November 2007
 |
SUMMARY |
|---|
 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
expression in the mutant
oligodendrocyte precursors, arguing that PDGF receptor is under
transcriptional control of Sox9 and Sox10. Altered PDGF receptor
expression is furthermore sufficient to explain the observed phenotype, as
PDGF is both an important survival factor and migratory cue for
oligodendrocyte precursors. We thus conclude that Sox9 and Sox10 are required
in a functionally redundant manner in oligodendrocyte precursors for
PDGF-dependent survival and migration.
Key words: Sry, High-mobility group, Redundancy, SoxE, PDGF, Glia, Transgenic mice
|
INTRODUCTION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). In the developing mouse spinal cord, the vast majority of
oligodendrocytes arises as proliferative precursor cells from multipotent
neuroepithelial cells of the pMN domain in the ventral part of the ventricular
zone around 12 days post coitum (dpc). Oligodendrocyte precursors (OLPs) then
migrate to colonize the spinal cord and reach a particularly high density in
the white matter region where they start to undergo terminal differentiation
at the end of embryogenesis (Rowitch,
2004).
Several transcription factors have been identified that regulate different
steps of oligodendrocyte development including bHLH proteins of the Olig
family, homeodomain proteins of the Nkx family, the zinc-finger protein Zfp488
and HMG-domain proteins of the Sox family
(Rowitch et al., 2002;
Sohn et al., 2006;
Wang et al., 2006;
Wegner, 2001;
Wegner and Stolt, 2005).
Among Sox proteins, two highly related group E (SoxE) proteins with
pleiotropic roles in development are particularly important. Sox9, which also
influences chondrogenesis, male sex determination and neural crest development
(Akiyama et al., 2002;
Chaboissier et al., 2004;
Cheung and Briscoe, 2003),
exerts a strong effect on the specification of OLPs
(Stolt et al., 2003). Sox10,
by contrast, is a major determinant in terminal oligodendrocyte
differentiation (Stolt et al.,
2002) besides having many functions in the neural crest
(Britsch et al., 2001;
Herbarth et al., 1998;
Southard-Smith et al., 1998).
Sox8 as the third SoxE protein is only of minor importance in oligodendrocyte
development and appears to support Sox9 and Sox10 in their function
(Stolt et al., 2004;
Stolt et al., 2005).
We have previously shown that Sox9 continues to be expressed after
specification in OLPs, while Sox10 is present in OLPs long before they undergo
terminal differentiation. Sox9 and Sox10 thus jointly occur in cells of the
oligodendrocyte lineage between these events. Oligodendrocyte development
during this period progressed fairly normally in the absence of Sox10
(Stolt et al., 2002).
Likewise, the few OLPs that were specified in a Sox9-deficient spinal cord
thrived and replenished the oligodendrocyte pool during late embryogenesis
(Stolt et al., 2003). We
therefore concluded that if Sox9 and Sox10 are important for oligodendrocyte
development during the period after specification and before terminal
differentiation, they must have largely redundant functions and thus be able
to reciprocally compensate each other's loss.
To uncover novel functions for Sox9 and Sox10 in OLPs, we generated mice
that were not only deficient for Sox10, but in addition selectively lost Sox9
in OLPs after the specification event. As a result, normal numbers of OLPs
were born. However, these cells soon developed aberrantly. This involved
altered patterns of precursor cell migration and a decrease in precursor cell
numbers that went along with an increased rate of apoptosis and a severe
reduction of PDGF receptor (Pdgfra). We conclude that Sox9 and Sox10
are crucial for migration and survival of OLPs, at least partly through
regulating PDGF responsiveness.
|
MATERIALS AND METHODS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
) were
maintained as a colony or alternatively bred with mice carrying a
Sox10lacZ allele
(Britsch et al., 2001) or a
Sox10::Cre transgene (Stolt et
al., 2006) on a mixed C57Bl6J/C3H background. Sox9 deficiency in
OLPs was achieved by crossing Sox9loxP/loxP mice with
Sox9loxP/+ mice that additionally carried a
Sox10::Cre transgene. For the generation of embryos with combined
deficiency of Sox9 and Sox10 in OLPs, Sox9loxP/loxP,
Sox10lacZ/+ mice were crossed with Sox9loxP/+,
Sox10lacZ/+, Sox10::Cre mice. Embryos from 12.5 dpc to 18.5
dpc were from staged pregnancies. To obtain Sox9loxP/loxP,
Sox10lacZ/lacZ, Sox10::Cre embryos beyond 15.5 dpc, drinking
water of pregnant female mice was supplemented with 100 µg/ml
L-phenylephrine, 100 µg/ml isoproterenol and 2 mg/ml ascorbic acid from 8.5
dpc onwards (Pattyn et al.,
2000). This treatment prevented lethality due to developmental
defects in the autonomic nervous system of the double mutant unrelated to the
analyzed oligodendrocyte phenotype. All embryos older than 15.5 dpc in this
study underwent the same treatment. Comparison of treated control,
Sox10lacZ/lacZ or Sox9loxP/loxP,
Sox10::Cre embryos with untreated embryos revealed that the treatment did
not influence oligodendrocyte development in these genotypes. Because
oligodendrocyte development was identical in Sox9+/+ and
Sox9loxP/loxP genotypes, both wild-type and floxed alleles
were used in control and Sox10lacZ/lacZ embryos.
Genotyping was performed by PCR using primers a
(5'-GGGGCTTGTCTCCTTCAGAG-3'), b
(5'-TGGTAATGAGTCATACACAGTAC-3'), c
(5'-ACACAGCATAGGCTACCTG-3'), d
(5'-GTCAAGCGACCCATGAACGC-3') for Sox9; primers e
(5'-GAGGTGGGCGTTGGGCTCTT-3'), f
(5'-CAGAGCTTGCCTAGTGTCTT-3'), g
(5'-TAAAAATGCGCTCAGGTCAA-3') for Sox10; and primers h
(5'-ATGCTGTTTCACTGGTTATG-3'), i
(5'-ATTGCCCCTGTTTCACTATC-3') for the Sox10::Cre transgene
(Fig. 1A), as described
previously (Britsch et al.,
2001; Stolt et al.,
2003; Stolt et al.,
2006). For BrdU labelling, pregnant mice were injected
intraperitoneally with 100 µg BrdU (Sigma) per gram body weight 1 hour or
24 hours before embryo preparation depending on the experiment
(Stolt et al., 2003).
Preparation of spinal cord cultures and tissue sections, immunohistochemistry, TUNEL and in situ hybridization
For primary cell cultures, spinal cords were dissected from embryos at 18.5
dpc and immediately triturated into single cell suspensions. Equal numbers of
cells were seeded for each embryo into 35 mm dishes containing
polylysine-coated cover slips in DMEM containing 10% foetal calf serum. After
incubation for 3 hours, cells were adherent. For immediate analysis, cultures
from wild-type, Sox10lacZ/lacZ and
Sox9loxP/loxP, Sox10lacZ/lacZ, Sox10::Cre
embryos underwent fixation in 3% paraformaldehyde. For 48 hour cultures,
medium was switched after 3 hours to serum-free DMEM containing N2 supplement,
20 ng/ml bFGF and in some cases 20 ng/ml PDGF-AA (Strathmann Biotec). After
fixation of cells and extensive washing, cover slips were processed for
immunocytochemistry.
For the preparation of cryotome sections, genotyped, age-matched embryos
were fixed in 4% paraformaldehyde, cryoprotected in 30% sucrose and embedded
in OCT compound at -80°C. As previously described, 10 µm sections were
used for immunohistochemistry, 14 µm sections for in situ hybridization
according to standard protocols (Stolt et
al., 2003; Stolt et al.,
2002). For better comparison, all spinal cord sections were from
the forelimb level. For immunochemistry, the following primary antibodies were
used in various combinations: anti-NeuN mouse monoclonal (1:500 dilution,
Chemicon), anti-PCNA mouse monoclonal (1:100 dilution, Roche Biochemicals),
anti-Nkx2.2 mouse monoclonal (1:400 dilution, Developmental Studies Hybridoma
Bank), anti-DCC mouse monoclonal (1:1000 dilution, BD Pharmingen), anti-O4
mouse monoclonal (1:500 dilution, R&D Systems), anti-Sox10 guinea pig
antiserum [1:1000 dilution (Maka et al.,
2005)], anti-Sox9 guinea pig antiserum [1:500 dilution
(Stolt et al., 2003)],
anti-Ki67 rabbit monoclonal (1:500 dilution, Neomarkers), affinity-purified
anti-Sox9 rabbit antiserum [1:2000 dilution
(Stolt et al., 2004)],
anti-NG2 rabbit antiserum (1:1000 dilution, gift of J. Trotter,
Universität Mainz), anti-Pdgfra rabbit antiserum (1:80 dilution,
Neomarkers), anti-Olig2 rabbit antiserum (1:50,000 dilution, gift of D.
Rowitch, UCSF, San Francisco), anti-B-FABP rabbit antiserum, (1:10,000
dilution, gift of C. Birchmeier and T. Müller, MDC, Berlin), anti-Glast
rabbit antiserum (1:1000 dilution, BD Transduction Laboratories),
anti-β-galactosidase rabbit antiserum (1:500 dilution, ICN) or
anti-β-galactosidase goat antiserum (1:500 dilution, Biotrend). Detection
of immunoreactivity was with secondary antibodies conjugated to Cy2, Cy3 or
Alexa Fluor immunofluorescent dyes (Dianova and Molecular Probes).
Incorporated BrdU was visualized by an Alexa-488-coupled mouse monoclonal
antibody directed against BrdU (Molecular Probes) at a 1:20 dilution. TUNEL
assays were performed according to the manufacturer's protocol (Chemicon). In
situ hybridization was performed with DIG-labelled antisense riboprobes for
Mbp (myelin basic protein gene), Plp (proteolipid protein
gene; Plp1 - Mouse Genome Informatics) and Pdgfra
(Stolt et al., 2002). Samples
were analyzed and documented using either a Leica TCS SL confocal microscope
or a Leica inverted microscope (DMIRB) equipped with a cooled SPOT CCD camera
(Diagnostic Instruments, Sterling Heights, MI).
Chromatin immunoprecipitation
Chromatin immunoprecipitation assays were performed as described
(Schlierf et al., 2006).
Briefly, cellular protein and genomic DNA from freshly prepared spinal cords
of 4-day-old mice were crosslinked in 1% formaldehyde before chromatin
extraction and sonication to an average fragment length of 300 to 600 bp.
Immunoprecipitations were performed overnight at 4°C using polyclonal
control IgG or anti-Sox9 IgG from rabbit
(Stolt et al., 2003). DNA was
purified from precipitates after crosslink reversal and subjected to PCR. For
detection of sequence elements within the 5' flanking region of the
Pdgfra gene, the following primer pairs were used in 34 cycles of
standard PCR using an annealing temperature of 58°C:
5'-TCTGGTTGCCCATGGTGGCT-3' and
5'-AGCTCAGCCTTCTGAGTGGC-3' for positions -7999 to -7721 relative
to the start of exon 1 (N1); 5'-ACAGGCGTTGTCTGCCCAAC-3' and
5'-CTGGTCGTCCGATTCCCTCT-3' for positions -4060 to -3781 (N2);
5'-ATTTGCTTGCCTGCTCCACC-3' and
5'-CCACAAATGGTAACTTCACAGT-3' for positions -2100 to -1959 (C1);
5'-CCCTCGCTCCGTGTGTGTG-3' and
5'-ACCGTGGGGATATCAGGCTC-3' for positions -1609 to -1443 (C2);
5'-GAAGAGGTCTTGAGCCTGAG-3' and
5'-CTCCTTCTATGTCAATTTGCAAA-3' for positions -133 to +67 (C3).
|
RESULTS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
) and a
Sox10::Cre BAC transgene
(Kessaris et al., 2006;
Stolt et al., 2006)
(Fig. 1A). As Sox10 is
restricted in the CNS to cells of the oligodendrocyte lineage and starts to be
expressed immediately upon oligodendrocyte specification
(Stolt et al., 2002), Cre
recombinase activity and resulting Sox9 deletion is predicted to
occur selectively in cells of the oligodendrocyte lineage and should commence
after specification from ventricular zone cells.
To verify the expected deletion pattern, we analyzed the occurrence of Sox9
in the developing spinal cord by immunohistochemistry
(Fig. 1B-M). At 12.5 dpc,
Sox9-expressing cells are preferentially localized to the ventricular zone
(Stolt et al., 2003). Their
overall number was comparable in the spinal cord of wild-type and
Sox9loxP/loxP, Sox10::Cre embryos
(Fig. 1B,E).
As spinal cord OLPs are predominantly derived from the pMN domain, we
focused on this region of the ventral ventricular zone. Again, Sox9 expression
was unaltered in pMN domain cells of Sox9loxP/loxP,
Sox10::Cre embryos, which are marked by Olig2 expression
(Fig. 1C,F). Of the
Olig2-expressing cells, few are also marked by Sox10
(Stolt et al., 2003). These
newly specified OLPs occurred in normal numbers in
Sox9loxP/loxP, Sox10::Cre embryos and still contained Sox9
protein (Fig. 1D,G).
At 15.5 dpc, Sox9-positive cells were not only present in the ventricular zone, but also throughout the mantle zone in a dispersed pattern. When overall number and distribution of Sox9-positive cells in Sox9loxP/loxP, Sox10::Cre embryos were now compared with the wild type, there still was no obvious difference in the ventricular zone. However, Sox9-positive cells in the mantle zone appeared to be slightly reduced in number (Fig. 1H,K). Closer inspection revealed that Sox9 was selectively lost from cells in the mantle zone that express Olig2 and Sox10 and, thus, represent OLPs (compare Fig. 1I,J with Fig. 1L,M). The very few cells that still co-expressed Sox9 and Sox10 were predominantly found in close vicinity to the ventricular zone and thus likely corresponded to newly specified, late-born OLPs (data not shown). We thus conclude that Sox9 is both effectively and selectively lost from OLPs in spinal cords of Sox9loxP/loxP, Sox10::Cre embryos by 15.5 dpc. A more detailed analysis revealed that OLPs in the mantle zone were already devoid of Sox9 at 14.5 dpc (Fig. 2I-P). Even at 13.5 dpc, the Sox9-expressing OLPs amounted to a mere 10-15% of the whole OLP population in the mantle zone (Fig. 2A-H).
|
|
Even at 18.5 dpc, Sox9-deficient OLPs still exhibited normal distribution
throughout the spinal cord and were present in normal numbers as evident from
Sox10, Olig2 and Pdgfra expression patterns (compare
Fig. 3C,G,K with
Fig. 3D,H,L). At 18.5 dpc,
several OLPs in the marginal zone of the spinal cord furthermore downregulate
Pdgfra expression and start to activate myelin gene expression as part of
their terminal differentiation program
(Stolt et al., 2002). Again,
no difference was detected between the Sox9flox/flox,
Sox10::Cre and the wild-type spinal cord in the onset of myelin gene
expression as evident from in situ hybridization with Plp- or
Mbp-specific probes (Fig.
3M,N and data not shown). We therefore conclude, that in stark
contrast to the specification event, Sox9 is dispensable in OLPs for their
further timely development during embryogenesis.
Abnormal OLP distribution and reduced number in the combined absence of Sox9 and Sox10
The apparently normal development of Sox9-deficient OLPs could be explained
by the ability of Sox10 to compensate for the loss of Sox9. A reciprocal
compensatory mechanism has previously been invoked to explain the unaltered
development of Sox10-deficient OLPs until they undergo terminal
differentiation (Stolt et al.,
2002). To clarify whether Sox9 and Sox10 are indeed functionally
redundant in OLPs, we generated Sox9loxP/loxP,
Sox10lacZ/lacZ, Sox10::Cre embryos.
|
|
At 13.5 dpc, alterations in OLP distribution
(Fig. 5E-H), but not in OLP
number (Fig. 5V) became evident
in the double mutant using either Olig2 or β-galactosidase as a marker.
This altered distribution was even more pronounced at 14.5 dpc and 15.5 dpc
(Fig. 5I-P). Whereas OLPs
spread throughout the mantle zone and became more or less equally distributed
throughout the wild-type spinal cord with a slightly lower density in the
dorsal than in the ventral half (Fig.
5I,K,M,O), Olig2- and β-galactosidase-positive cells in the
Sox9loxP/loxP, Sox10lacZ/lacZ, Sox10::Cre
spinal cord were preferentially localized around the pMN domain and in the
ventral region of the spinal cord, as if they had a decreased ability to
migrate away from their region of origin
(Fig. 5J,L,N,P). In contrast to
the situation at 13.5 dpc, OLP numbers were now also reduced in
Sox9loxP/loxP, Sox10lacZ/lacZ, Sox10::Cre
embryos. Quantification revealed a 46-62% reduction in
β-galactosidase-positive cells and a similar decrease in Olig2-positive
cells (Fig. 5V,W and data not
shown). By contrast, no statistically significant alteration of OLP numbers
was observed in Sox9loxP/loxP, Sox10::Cre embryos or
Sox10lacZ/lacZ littermates, indicating that their
reduction is specific to the double mutant
(Fig. 5W and data not shown).
OLP numbers in Sox10lacZ/lacZ embryos had previously been
reported to be normal in one study (Stolt
et al., 2002), but reduced in another
(Liu et al., 2007). Although
the reason for this discrepancy is not clear at the moment, it could be caused
by differences in genetic backgrounds.
Neither OLP numbers nor their distribution recovered to the wild-type situation at 18.5 dpc with Olig2- and β-galactosidase-positive cells still being preferentially localized in the ventral part of the spinal cord (Fig. 5Q-T) and numbers of Olig2-positive cells being reduced by 42% (Fig. 5W). Very few OLPs ever reached the dorsal-most region of the spinal cord.
|
), we
determined the fraction of Ki67-immunoreactive cells that were also labelled
with BrdU. Again, we detected no difference between wild type and mutant at
15.5 dpc and 18.5 dpc (Fig.
6B), indicating that OLPs in Sox9loxP/loxP,
Sox10lacZ/lacZ, Sox10::Cre spinal cords have an unaltered cell
cycle length. We also examined Ki67-immunoreactivity and BrdU incorporation at 15.5 dpc after a single BrdU pulse 24 hours earlier. By determining the fraction of BrdU-labelled cells that were no longer Ki67 positive, a measure was obtained for the rate of cell cycle exit. As this fraction was similar between wild type and double mutant, cell cycle exit was not significantly altered in Sox9loxP/loxP, Sox10lacZ/lacZ, Sox10::Cre spinal cords (Fig. 6C).
Differences were, however, detectable in the rate of apoptosis at 15.5 dpc
and 18.5 dpc (Fig. 6D-G). In
the Sox9loxP/loxP, Sox10lacZ/lacZ, Sox10::Cre
spinal cord, there were 
60% more TUNEL-positive cells than in the wild
type (Fig. 6H). TUNEL-positive
cells were preferentially localized to those areas where OLPs were
preferentially found in the Sox9loxP/loxP,
Sox10lacZ/lacZ, Sox10::Cre spinal cord
(Fig. 6D-G). Direct analysis
furthermore revealed that OLPs had a significantly increased rate of apoptosis
in the double mutant (Fig. 6I).
Taking the unaltered rates of oligodendrocyte specification and proliferation
into account, the lower number of Sox9/Sox10 double deficient OLPs is thus
probably caused by reduced survival rates.
No transdifferentiation of OLPs in the combined absence of Sox9 and Sox10
OLPs in the Sox9loxP/loxP, Sox10lacZ/lacZ,
Sox10::Cre spinal cord have so far been operationally defined as those
cells that are positive for Olig2 and express β-galactosidase from the
mutant Sox10 locus. To characterize these cells in further detail, we
first asked whether there is aberrant expression of neuronal, astroglial or
radial glial markers in these Sox9/Sox10 double-deficient OLPs. In
co-immunohistochemistry (30 sections from two independent embryos), we did not
detect NeuN or Tuj1 expression in Olig2-positive cells of the
Sox9loxP/loxP, Sox10lacZ/lacZ, Sox10::Cre
spinal cord at 15.5 dpc and 18.5 dpc, and very few Glast or glutamine
synthetase-expressing Olig2-positive cells. As the latter were also present in
the wild type, mutant OLPs did not aberrantly express neuronal and astroglial
markers (Fig. 7A,B,D,E and data
not shown). There was also no significant overlap between β-galactosidase
and B-FABP expression (Fig.
7C,F), arguing that Sox9/Sox10 double-deficient OLPs are similarly
negative for radial glia markers as wild-type precursors
(Stolt et al., 2004).
|
|
By contrast, the relative number of Olig2-expressing cells that were also positive for Nkx2.2 was reduced to 50% (Fig. 8D). In the mouse spinal cord, Nkx2.2 expression commences fairly late in OLPs, which are predominantly localized in the marginal zone (Fig. 8E), and may rely on marginal zone signals. As proportionately fewer OLPs have entered the marginal zone in Sox9loxP/loxP, Sox10lacZ/lacZ, Sox10::Cre spinal cords at 18.5 dpc, the lower relative number of Nkx2.2-expressing OLPs may be caused by the altered migration pattern observed in the mutant genotype. Despite this reduction, Nkx2.2 was still detectable in OLPs.
|
|
) and is
thus not specific to the double deficient OLPs analyzed in this study.
Severely reduced Pdgfra expression in OLPs in the combined absence of Sox9 and Sox10
Taking the fairly normal expression of most OLP markers into account, we
did not expect dramatic changes in Pdgfra occurrence. We analyzed wild-type,
single mutant and double mutant spinal cords from 13.5 dpc to 18.5 dpc by in
situ hybridization, and from 14.5 dpc to 18.5 dpc by immunohistochemistry for
Pdgfra expression. Compared with the wild type, there was no significant
difference in the single mutants regarding appearance, number or distribution
of Pdgfra-positive cells at any of the analyzed time points by in situ
hybridization or immunohistochemistry (Fig.
3I-L, Fig.
4A-C,E-G, Fig.
9A-C,E-G, Fig.
10A-C and data not shown). However, to obtain in situ
hybridization signals for the single mutants that were comparable in intensity
with the wild type, staining reactions had to be extended, especially at the
earlier stages. This indicated that Pdgfra transcript levels were
reduced in OLPs from the single mutants, as already reported for the
Sox10-deficient OLPs (Stolt et al.,
2002).
Pdgfra expression was much more dramatically altered in Sox9loxP/loxP, Sox10lacZ/lacZ, Sox10::Cre spinal cords. Already at 13.5 dpc, very few Pdgfra-positive cells remained (compare Fig. 10A-C with Fig. 10D). These Pdgfra-positive cells were furthermore localized in close vicinity to the ventricular zone and thus probably corresponded to newly specified OLPs that had not yet lost Sox9. From 14.5 dpc onwards, Pdgfra was no longer detected in the double mutant either on the transcript (compare Fig. 10E,I,M with Fig. 10F,J,N) or on the protein level (compare Fig. 10G,K,O with Fig. 10H,L,P; see also Fig. 4D,H and Fig. 9D,H). Even at 18.5 dpc, Pdgfra expression had not recovered, indicating that Pdgfra expression was severely compromised in the double mutant and is probably under the control of Sox9 and Sox10 in spinal cord OLPs.
Chromatin immunoprecipitation experiments furthermore revealed that Sox9 is bound to elements C1, C2 and C3 within the proximal 5' flanking region of the Pdgfra gene that are conserved among amniotes (http://ecrbrowser.dcode.org), but not to non-conserved regions N1 and N2 in the distal 5' flanking part (Fig. 10Q). These results are compatible with a direct Pdgfra regulation by SoxE proteins. In primary cultures from spinal cords of 18.5 days old Sox9loxP/loxP, Sox10lacZ/lacZ, Sox10::Cre embryos treated with saturating amounts of PDGF-AA, OLP numbers exhibited a PDGF-dependent increase after 48 hours, arguing that at least under these conditions PDGF-AA retained mitogenic activity on Sox9/Sox10 double deficient OLPs (Fig. 10R).
|
|
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
), whereas the related Sox10 is required for their
differentiation into mature oligodendrocytes
(Stolt et al., 2002). The
presence of Sox9 and Sox10 in OLPs has furthermore led us to postulate
additional roles for both Sox proteins during lineage progression. To address
this issue, we deleted Sox9 in already specified OLPs, thereby leaving Sox9
function during specification intact. The resulting loss of Sox9 did not lead
to significant alterations in the development of spinal cord OLPs in the
corresponding mouse mutant. Defects became detectable only after the combined
loss of Sox9 and Sox10. Taken together with the already reported normal
development of OLPs in Sox10-deficient mice until terminal differentiation
(Stolt et al., 2002), we
conclude that Sox9 and Sox10 have additional functions in developing OLPs, but
exert them in a functionally redundant manner. Our analysis focused on
pMN-derived OLPs. However, our findings can very probably be extrapolated to
the dorsally derived OLPs of the spinal cord
(Richardson et al., 2006),
because SoxE expression is very similar in all spinal cord
oligodendrocytes.
Interestingly, many OLP markers continued to be expressed in the absence of
Sox9 and Sox10. These included Olig2, DCC, NG2, O4 and Nkx2.2, although the
latter was expressed in proportionately fewer OLPs. Even expression of
β-galactosidase from the mutant Sox10 locus continued in OLPs in
the absence of Sox9 and Sox10. Although our results do not exclude an earlier
essential role for Sox9 in the induction of Sox10 expression similar to what
has been observed in the early neural crest
(Cheung et al., 2005), neither
Sox9 nor Sox10 are essential for maintained expression from the Sox10
locus in OLPs. Whether Sox9 and Sox10 are substituted in this function by
Sox8, which is closely related and also expressed in OLPs
(Stolt et al., 2004;
Stolt et al., 2005), is
unclear at present. Alternatively, SoxE proteins are completely dispensable
for maintaining Sox10 expression in OLPs.
OLPs not only continued to express many of their characteristic markers, they also failed to ectopically express other markers that are normally found in neurons, astroglia or radial glia. Thus, there is no reason to assume that OLPs in the absence of Sox9 and Sox10 have been partially transformed into different CNS cell types. On the contrary, OLPs seem to preserve their identity in the absence of Sox9 and Sox10.
On this background, our failure to detect Pdgfra in most OLPs at 13.5 and
all OLPs at later stages is particularly intriguing and argues that Pdgfra
signalling is one of the main pathways by which Sox9 and Sox10 regulate OLP
lineage progression. Although our study proves that Sox9 and Sox10 are
genetically upstream of Pdgfra in spinal cord OLPs, it does not allow a firm
conclusion of whether Pdgfra represents a direct or an indirect
target gene. In vivo binding of Sox9 to the proximal 5' flanking region
of the Pdgfra gene, especially to the core promoter supports the
assumption that Sox9 directly activates Pdgfra expression. It has to
be stressed, however, that the 5' flanking region is not sufficient for
expression in OLPs (Reinertsen et al.,
1997). Sox9 binding to this region may therefore be relevant to
aspects of Pdgfra expression other than oligodendroglial expression. Full
clarification of this issue has to await a better understanding of
Pdgfra gene regulation, in particular the identification of the
regulatory regions that are responsible for oligodendroglial expression. So
far, they have only been mapped to a 380 kb region
(Sun et al., 2000).
Pdgfra remained detectable in OLPs after Sox9 deletion, indicating that
Sox10 occurrence in OLPs is sufficient to permit Pdgfra expression,
although at reduced levels. The same Sox10, however, fails to maintain
Pdgfra expression during terminal differentiation and thereafter in
mature oligodendrocytes. In a reciprocal manner, several myelin genes have
been shown to be upregulated by Sox10 with the onset of terminal
differentiation, but are not expressed earlier in OLPs, despite the fact that
Sox10 is already present (Bondurand et al.,
2001; Schlierf et al.,
2006; Stolt et al.,
2002).
This argues that the activity of Sox10, and probably also of Sox9, has to
be modulated in a stage-specific manner during oligodendrocyte development.
Most likely, Sox10 requires additional transcription factors that are
differentially present in OLPs and in mature oligodendrocytes. The reliance on
partner transcription factors is a general property of Sox proteins
(Kamachi et al., 2000;
Wegner, 2005). We have
recently shown that the presence of Sox5 and Sox6 in OLPs prevents a premature
Sox10-dependent activation of myelin gene expression
(Stolt et al., 2006). Sox5 and
Sox6 may also be involved in allowing Sox10-dependent Pdgfra
activation.
The role of PDGF signalling in oligodendrocyte development has been
intensely studied both in animal models and cell culture systems. On cultured
OLPs, PDGF-AA acts as a mitogen (Noble et
al., 1988; Richardson et al.,
1988), a survival factor
(Barres et al., 1992) and a
chemoattractant (Armstrong et al.,
1990). Thus, it is very likely that the altered migration pattern
that we observed for OLPs in the combined absence of Sox9 and Sox10, and the
increased apoptosis are direct consequences of the changed Pdgfra
expression in these mice. Supporting evidence comes from the close resemblance
of OLP defects in Sox9/Sox10 double-deficient mice and in mice harbouring
Pdgfra mutants with eliminated PI3 kinase-dependent or Src-dependent
downstream signalling (Klinghoffer et al.,
2002). Klinghoffer and colleagues also found altered migration and
reduction of OLP numbers, although the cause for the latter is not clear, as
both proliferation and apoptosis were found to be normal. Interestingly, both
the Sox9/Sox10 double-deficient and Pdgfra mutant phenotypes are,
however, less severe than the one caused by PDGF-AA loss, in which OLP numbers
are reduced to less than 10% of the wild type and OLP proliferation is
severely disturbed (Fruttiger et al.,
1999). This phenotypic divergence is surprising, as PDGF-AA is
thought to act preferentially on OLPs through Pdgfra
(Hoch and Soriano, 2003).
The mitogenic function of PDGF-AA on OLPs has been impressively confirmed
in both loss-of-function and gain-of-function studies in mice
(Calver et al., 1998;
Fruttiger et al., 1999).
Interestingly, Sox9/Sox10 double-deficient OLPs proliferated normally in vivo
(where PDGF-AA levels are limiting) and exhibited a mitogenic response to
PDGF-AA in mixed spinal cord cultures (where PDGF-AA is present in saturating
amounts). PDGF-AA thus retained its mitogenic activity on Sox9/Sox10
double-deficient OLPs. The mitogenic effect of PDGF-AA may therefore not be
exclusively mediated by Pdgfra, but additionally by yet unknown pathways.
Alternatively, Pdgfra expression may not be completely turned off in
OLPs in the combined absence of Sox9 and Sox10, but simply below our detection
limits. Low residual levels may then be sufficient for mediating the mitogenic
effect of PDGF-AA, but not for the survival effect in vivo.
Whatever the exact mechanism, this study clearly shows that in addition to
their respective functions during OLP specification and terminal
differentiation, Sox9 and Sox10 additionally influence survival and migration
of OLPs through their effect on PDGF signalling. Comparable influences on
survival and migration have already been described for Sox10 in the neural
crest and its derivatives (Kim et al.,
2003; Maka et al.,
2005; Paratore et al.,
2001), but never before in the oligodendrocyte lineage.
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Akiyama, H., Chaboissier, M.-C., Martin, J. F., Schedl, A. and
de Crombrugghe, B. (2002). The transcription factor Sox9 has
essential roles in successive steps of the chondrocyte differentiation pathway
and is required for expression of Sox5 and Sox6. Genes
Dev. 16,2813
-2828.
Armstrong, R. C., Harvath, L. and Dubois-Dalcq, M. (1990). Type 1 astrocytes and oligodendrocyte-type 2 astrocyte glial progenitors migrate toward distinct molecules. J. Neurosci. Res. 27,400 -407.[CrossRef][Medline]
Barres, B. A., Hart, I. K., Coles, H. S. R., Burne, J. F., Voyvodic, J. T., Richardson, W. D. and Raff, M. C. (1992). Cell death in the oligodendrocyte lineage. J. Neurobiol. 23,1221 -1230.[CrossRef][Medline]
Bondurand, N., Girard, M., Pingault, V., Lemort, N., Dubourg, O.
and Goossens, M. (2001). Human Connexin 32, a gap junction
protein altered in the X-linked form of Charcot-Marie-Tooth disease, is
directly regulated by the transcription factor SOX10. Hum. Mol.
Genet. 10,2783
-2795.
Britsch, S., Goerich, D. E., Riethmacher, D., Peirano, R. I.,
Rossner, M., Nave, K. A., Birchmeier, C. and Wegner, M.
(2001). The transcription factor Sox10 is a key regulator of
peripheral glial development. Genes Dev.
15, 66-78.
Calver, A. R., Hall, A. C., Yu, W.-P., Walsh, F. S., Heath, F. S., Betsholtz, C. and Richardson, W. D. (1998). Oligodendrocyte population dynamics and the role of PDGF in vivo. Neuron 20,869 -882.[CrossRef][Medline]
Chaboissier, M.-C., Kobayashi, A., Vidal, V. I. P.,
Lützkendorf, S., van de Kant, H. J. G., Wegner, M., de Rooij, D. G.,
Behringer, R. R. and Schedl, A. (2004). Functional analysis
of Sox8 and Sox9 during sex determination in the mouse.
Development 131,1891
-1901.
Chenn, A. and Walsh, C. A. (2002). Regulation
of cerebral cortical size by control of cell cycle exit in neural precursors.
Science 297,365
-369.
Cheung, M. and Briscoe, J. (2003). Neural crest
development is regulated by the transcription factor Sox9.
Development 130,5681
-5693.
Cheung, M., Chaboissier, M.-C., Mynett, A., Hirst, E., Schedl, A. and Briscoe, J. (2005). The transcriptional control of trunk neural crest induction, survival, and delamination. Dev. Cell 8,179 -192.[CrossRef][Medline]
Fruttiger, M., Karlsson, L., Hall, A. C., Abramsson, A., Calver, A. R., Boström, H., Willetts, K., Bertold, C.-H., Heath, F. S., Betsholtz, C. et al. (1999). Defective oligodendrocyte development and severe hypomyelination in PDGF-A knockout mice. Development 126,457 -467.[Abstract]
Herbarth, B., Pingault, V., Bondurand, N., Kuhlbrodt, K.,
Hermans-Borgmeyer, I., Puliti, A., Lemort, N., Goossens, M. and Wegner, M.
(1998). Mutation of the Sry-related Sox10 gene in Dominant
megacolon, a mouse model for human Hirschsprung disease. Proc.
Natl. Acad. Sci. USA 95,5161
-5165.
Hoch, R. V. and Soriano, P. (2003). Roles of
PDGF in animal development. Development
130,4769
-4784.
Kamachi, Y., Uchikawa, M. and Kondoh, H. (2000). Pairing SOX off: with partners in the regulation of embryonic development. Trends Genet. 16,182 -187.[CrossRef][Medline]
Kessaris, N., Fogarty, M., Iannarelli, P., Grist, M., Wegner, M. and Richardson, W. D. (2006). Competition waves of oligodendrocytes in the forebrain and postnatal elimination of an early embryonic lineage. Nat. Neurosci. 9, 173-179.[CrossRef][Medline]
Kim, J., Lo, L., Dormand, E. and Anderson, D. J. (2003). SOX10 maintains multipotency and inhibits neuronal differentiation of neural crest stem cells. Neuron 38, 17-31.[CrossRef][Medline]
Klinghoffer, R. A., Hamilton, T. G., Hoch, R. and Soriano, P. (2002). An allelic series at the PDGFalphaR locus indicates unequal contributions of distinct signaling pathways during development. Dev. Cell 2, 103-113.[CrossRef][Medline]
Liu, Z., Hu, X., Cai, J., Liu, B., Peng, X., Wegner, M. and Qiu, M. (2007). Induction of oligodendrocyte differentiation by Olig2 and Sox10: evidence for reciprocal interactions and dosage-dependent mechanisms. Dev. Biol. 302,683 -693.[CrossRef][Medline]
Maka, M., Stolt, C. C. and Wegner, M. (2005). Identification of Sox8 as a modifier gene in a mouse model of Hirschsprung disease reveals underlying molecular defect. Dev. Biol. 277,155 -169.[CrossRef][Medline]
Noble, M., Murray, K., Stroobant, P., Waterfield, M. D. and Riddle, P. (1988). Platelet-derived growth factor promotes division and motility and inhibits differentiation of the oligodendrocyte/type-2 astrocyte progenitor cell. Nature 333,560 -565.[CrossRef][Medline]
Paratore, C., Goerich, D. E., Suter, U., Wegner, M. and Sommer, L. (2001). Survival and glial fate acquisition of neural crest cells are regulated by an interplay between the transcription factor Sox10 and extrinsic combinatorial signaling. Development 128,3949 -3961.[Medline]
Pattyn, A., Goridis, C. and Brunet, J. F. (2000). Specification of the central noradrenergic phenotype by the homeobox gene Phox2b. Mol. Cell. Neurosci. 15,235 -243.[CrossRef][Medline]
Reinertsen, K. K., Bronson, R. T., Stiles, C. D. and Wang, C. (1997). Temporal and spatial specificity of PDGF alpha receptor promoter in transgenic mice. Gene Expr. 6, 301-314.[Medline]
Richardson, W. D., Pringle, N., Mosley, M. J., Westermark, B. and Dubois-Dalcq, M. (1988). A role for platelet-derived growth factor in normal gliogenesis in the central nervous system. Cell 53,309 -319.[CrossRef][Medline]
Richardson, W. D., Kessaris, N. and Pringle, N. (2006). Oligodendrocyte wars. Nat. Rev. Neurosci. 7,11 -18.[CrossRef][Medline]
Rowitch, D. H. (2004). Glial specification in the vertebrate neural tube. Nat. Rev. Neurosci. 5, 409-419.[Medline]
Rowitch, D. H., Lu, Q. R., Kessaris, N. and Richardson, W. D. (2002). An `oligarchy' rules neural development. Trends Neurosci. 25,417 -422.[CrossRef][Medline]
Schlierf, B., Werner, T., Glaser, G. and Wegner, M. (2006). Expression of Connexin47 in oligodendrocytes is regulated by the Sox10 transcription factor. J. Mol. Biol. 361, 11-21.[CrossRef][Medline]
Sohn, J., Natale, J., Chew, L. J., Belachew, S., Cheng, Y.,
Aguirre, A., Lytle, J., Nait-Oumesmar, B., Kerninon, C., Kanai-Azuma, M. et
al. (2006). Identification of Sox17 as a transcription factor
that regulates oligodendrocyte development. J.
Neurosci. 26,9722
-9735.
Southard-Smith, E. M., Kos, L. and Pavan, W. J. (1998). Sox10 mutation disrupts neural crest development in Dom Hirschsprung mouse model. Nat. Genet. 18, 60-64.[CrossRef][Medline]
Stolt, C. C., Rehberg, S., Ader, M., Lommes, P., Riethmacher,
D., Schachner, M., Bartsch, U. and Wegner, M. (2002).
Terminal differentiation of myelin-forming oligodendrocytes depends on the
transcription factor Sox10. Genes Dev.
16,165
-170.
Stolt, C. C., Lommes, P., Sock, E., Chaboissier, M.-C., Schedl,
A. and Wegner, M. (2003). The Sox9 transcription factor
determines glial fate choice in the developing spinal cord. Genes
Dev. 17,1677
-1689.
Stolt, C. C., Lommes, P., Friedrich, R. P. and Wegner, M.
(2004). Transcription factors Sox8 and Sox10 perform
non-equivalent roles during oligodendrocyte development despite functional
redundancy. Development
131,2349
-2358.
Stolt, C. C., Schmitt, S., Lommes, P., Sock, E. and Wegner, M. (2005). Impact of transcription factor Sox8 on oligodendrocyte specification in the mouse embryonic spinal cord. Dev. Biol. 281,323 -331.
Stolt, C. C., Schlierf, A., Lommes, P., Hillgärtner, S., Werner, T., Kosian, T., Sock, E., Kessaris, N., Richardson, W. D., Lefebvre, V. et al. (2006). SoxD proteins influence multiple stages of oligodendrocyte development and modulate SoxE protein function. Dev. Cell 11,697 -710.[CrossRef][Medline]
Sun, T., Jayatilake, D., Afink, G. B., Ataliotis, P., Nister, M., Richardson, W. D. and Smith, H. K. (2000). A human YAC transgene rescues craniofacial and nueral tube development in PDGFRalpha knockout mice and uncovers a role for PDGFRalpha in prenatal lung growth. Development 127,4519 -4529.[Abstract]
Wang, S. Z., Dulin, J., Wu, H., Hurlock, E., Lee, S. E.,
Jansson, K. and Lu, Q. R. (2006). An oligodendrocyte-specific
zinc-finger transcription regulator cooperates with Olig2 to promote
oligodendrocyte differentiation. Development
133,3389
-3398.
Wegner, M. (2001). Expression of transcription factors during oligodendroglial development. Microsc. Res. Tech. 52,746 -752.[CrossRef][Medline]
Wegner, M. (2005). Secrets to a healthy Sox life: lessons for melanocytes. Pigment Cell Res. 18, 74-85.[CrossRef][Medline]
Wegner, M. and Stolt, C. C. (2005). From stem cells to neurons and glia: a soxist's view of neural development. Trends Neurosci. 28,583 -588.[CrossRef][Medline]
| ||||||||||||||||||||||||||||||||||||||||||||||
/
0.001).
