First published online December 1, 2003
doi: 10.1242/10.1242/dev.00871
Development 130, 6599-6609 (2003)
Published by The Company of Biologists 2003
FGF-dependent generation of oligodendrocytes by a hedgehog-independent pathway
Siddharthan Chandran1,*,
Hidemasa Kato2,
Dianne Gerreli3,
Alastair Compston1,
Clive N. Svendsen4 and
Nicholas D. Allen2,
1 Cambridge Centre for Brain Repair, University of Cambridge, ED Adrian
Building, Forvie Site, Robinson Way, Cambridge CB2 2PY, UK
2 The Babraham Institute, Babraham, Cambridge CB2 4AT, UK
3 Neural Development Unit, Institute of Child Health, 30 Guilford Street, London
WC1N 1EH, UK
4 Waisman Center Stem Cell Research Program, University of Wisconsin-Madison,
1500 Highland Avenue, Madison, WI 53705, USA
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Fig. 1. Immunomicrograph showing the CNS potential of E14 rat dorsal spinal
cord-derived clones. (A) A single clone can generate
ß-tubulin+ neurones, O4+ oligodendrocytes and
GFAP+ astrocytes. (B) Hedgehog signalling is not necessary for the
generation of oligodendrocytes from FGF2-treated E14 rat dorsal cultures.
Addition of cyclopamine or KAAD during the 7 day exposure to FGF2 does not
significantly reduce numbers of oligodendrocytes generated upon mitogen
withdrawal, plating and subsequent differentiation for a further 7 days
(P>0.05). (C) Immunofluorescent micrograph showing representative
labelling of differentiating cultures for O4-, GC- and MBP-positive cells from
E14 dorsal-derived cultures following treatment with FGF2 in the presence of
cyclopamine (1 µM).
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Fig. 2. SHH-knockout mice have latent in vitro oligodendrocyte potential. (A) Whole
E12.5 SHH-null spinal cords were examined for O4 or GC expression before
(primary) or after (expanded) exposure to FGF2. Primary cultures contain no
O4+ or GC+ cells, but following FGF2 stimulation there
are significant numbers of oligodendrocyte lineage cells
(*=P<0.01, **=P<0.05). (B)
Immunofluorescent micrograph showing representative labelling of
Hoechst+, O4+ and GC+ cells in E12.5 null
spinal cord cultures after (expanded) exposure to FGF2. O4+ cells
(red), and O4 and GC co-labelled cell (yellow; arrow) are shown. (C)
Immunomicrograph showing O4+ oligodendrocyte derived from SHH null
cultures following treatment with FGF2 in the presence of cyclopamine (1
µM). (D) Clonal analysis of SHH null cultures. Immunomicrograph showing
monoclonal derivation of tripotential and bipotential clones containing
ß-tubulin+ neurones (green), O4+ oligodendrocytes
(red) and GFAP+ astrocytes (blue) from E12.5 SHH null-derived
FGF2-treated single cell cultures.
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Fig. 3. (A) Induction of oligodendrocyte-related genes in isolated mouse E12.5
dorsal and whole E12.5 SHH null cord following FGF2 treatment in the presence
or absence of cyclopamine (1 µM). Untreated dorsal wild-type and whole SHH
null spinal cord show no Olig2 and Nkx2.2 expression.
Following FGF2 expansion, induction of Olig2 and Nkx2.2 is
apparent with significant upregulation of Olig1. Ihh message is
present in dissociated untreated wild-type cultures and in SHH-null expanded
cultures. Significantly, absence of the V3 and pMN genes Nkx2.9 and
Nkx6.1 is evident in all conditions. (B) Immunomicrographs showing
OLIG2- and NKX2.2-positive cells in dorsal-derived neurospheres grown in the
presence of cyclopamine (1 µM), and fixed and stained at 7 days in vitro.
Arrow indicates co-positive OLIG2 and NKX2.2 cells.
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Fig. 4. FGF2-mediated oligodendrocyte induction is dependent on MAP kinase
signalling and is inhibited by BMP4. (A) Isolated E14 rat dorsal spinal cord
cultures were examined for expression of oligodendrocytes (O4), astrocytes
(GFAP), neurones (ß-tubulin) and smooth muscle actin (SMA) at 4-days
post-plating, following exposure to FGF2 and BMP4 (0.1-10 ng/ml), MEK
inhibitor (UO126) or PI3K pathway inhibitor (LY294002). BMP-treated cultures
show a dose-dependent reduction of oligodendrocytes and astrocytes, and an
induction of SMA. UO126-treated cultures show a similar reduction in
oligodendrocytes (*=P<0.01;
**=P<0.05). (B) Immunomicrograph showing
oligodendrocyte (O4) and astrocyte (GFAP) generation following 96 hours of (I)
FGF2 and (II) FGF2 + BMP4 (10 ng/ml) treatment of E14 dorsal cultures.
BMP4-treated cultures have no oligodendrocytes and reduced numbers of
astrocytes following differentiation in control media after withdrawal of
4-day FGF2 and BMP4 treatment; compare with cultures treated with FGF2 alone.
Immunomicrographs showing (III) neuronal (ß-tubulin) generation following
FGF2 stimulation of E14 dorsal cultures, and (IV) smooth muscle actin-positive
staining cells derived from FGF2 and BMP4 (10 ng/ml) treated cultures.
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Fig. 5. Expression of Bmp4, noggin and Egfr genes in isolated
mouse E12.5 dorsal spinal cord cultures following FGF2 treatment in the
presence or absence of cyclopamine (1 µM). BMP4 expression is evident in
both primary and FGF2-treated cultures. Noggin expression is downregulated
upon FGF2 treatment, in contrast to the induction of EGFR.
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Fig. 6. Summary of SHH-dependent and - independent induction of oligodendrocytes.
SHH-dependent: generation of oligodendrocytes in vivo is confined to the pMN
(and V3) region, which earlier in development produces motoneurones and which
is characterised by the expression of the class II gene Nkx6.1 prior
to induction of Olig2. Subsequent co-expression of NKX2.2 in the pMN
OLIG2-expressing domain is associated with the generation of migratory OPCs.
SHH-independent: neural stem cells derived from the developing spinal cord
that are Nkx6.1, Olig2 and Nkx2.2 negative can generate
Olig2- and Nkx2.2-positive progeny following FGF2 treatment
in the absence of SHH signalling, and without induction of the V3 or pMN
associated genes Nkx2.9 and Nkx6.1. This differentiation is
MAP kinase dependent and can be opposed by BMP4.
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© The Company of Biologists Ltd 2003
