First published online 20 February 2008
doi: 10.1242/dev.015370
Development 135, 1271-1281 (2008)
Published by The Company of Biologists 2008
Ascl1 is required for oligodendrocyte development in the spinal cord
Michiya Sugimori1,
Motoshi Nagao1,
Carlos M. Parras2,
Hiroko Nakatani2,
Mélanie Lebel3,
François Guillemot3 and
Masato Nakafuku1,4,5,*
1 Division of Developmental Biology, Cincinnati Children's Hospital Research
Foundation, 3333 Burnet Avenue, Cincinnati, OH 45229, USA.
2 Biologie des Interactions Neurones/Glie, Unite Mixte de Recherche INSERM
U-711, UPMC Hopital de la Salpetriere, 75651 Paris cedex 13, France.
3 Division of Molecular Neurobiology, National Institute for Medical Research,
The Ridgeway, Mill Hill, London NW7 1AA, UK.
4 Departments of Pediatrics and Neurosurgery, University of Cincinnati College
of Medicine, 125 Eden Avenue, Cincinnati, OH 45267, USA.
5 Solution Oriented Research for Science and Technology, Japan Science and
Technology Agency, 3-4-15, Nihonbashi, Chuo-ku, Tokyo 103-0027, Japan.
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Fig. 1. Expression of Ascl1 at late embryonic stages. Transverse sections of
rat spinal cord at the brachial level at E16.5 (A-D) and E18.5
(E-I). Molecules stained in red and green are shown at the top. Broken
lines indicate the boundary between the VZ and MZ or the forming white matter
of the spinal cord. Boxes in A and E show the locations of the areas shown in
other panels. Insets show double-labeled cells in the dorsomedial white matter
(unlabelled boxed areas in A,E) or the ventral white matter (B-D,F).
Arrowheads indicate double-labeled cells, whereas arrows in I indicate
separate Olig2+ and Nkx2-2+ cells. FP, floor plate; MZ,
mantle zone; VZ, ventricular zone. Scale bars: 200 µm in A,E; in D,H and I,
50 µm for B-D,F-H and I.
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Fig. 2. Co-expression of Ascl1, Olig2 and Nkx2-2 in differentiating OLPs at
perinatal stages. Images show staining of rat spinal cord for Ascl1, Olig2
and Nkx2-2, together with GalC and MBP at E20.5 (A-H) and P0
(I-O). Boxes in E and M indicate the locations of the areas shown in
other panels. D shows a triple-positive cell (merged image on the left).
Insets and arrowheads indicate double-positive cells. Asterisks in I indicate
Olig2+/Ascl1- cells. Broken lines indicate the boundary
between the VZ and MZ. Scale bars: in C,G and L, 50 µm for A-C, F,G and
I-L; 100 µm in E,H,N,O; 200 µm in M.
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Fig. 3. Expression of Ascl1-GFP transgene in
PDGFR + OLPs. Images show transverse sections of
E16.5 spinal cords of Ascl1::GFP reporter mice. (A-D'')
Ascl1-GFP and PDGFR were stained in green and red, respectively, and
cell nuclei were stained with DAPI in blue. (E-H'') Co-staining
for Ascl1-GFP (green), PDGFR (blue) and endogenous Ascl1 protein (red).
Boxes indicate the locations of the areas shown in other panels. Broken lines
in B-B'' indicates the VZ. Arrowheads indicate the co-expression of GFP
and PDGFR, whereas arrows indicate
GFP-/PDGFR+ (C-C'') or
Ascl1-/GFP+/PDGFR+ cells
(F-F'',H-H''). Scale bars: 50 µm in A-A''; in
B'',C'',D'',E,G,F'' and H'', 20 µm for
B-B'',C-C'',D-D'',E,F-F'' and H-H''.
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Fig. 4. Temporal sequence of the co-expression of Ascl1, Olig2 and Nkx2-2.
(A,B) Stage-dependent increase in the number of
Ascl1+, Olig2+ and Nkx2-2+ cells (A), and
NG2+, GalC+ and MBP+ (B) cells. Cells
isolated from rat spinal cords between the upper and lower limbs were
subjected to immunostaining. Data are mean±s.d. obtained from three
independent experiments. The number of cells triple positive for Ascl1, Olig2
and Nkx2-2 was estimated based on the results of a series of double staining.
(C) Immunostaining of dissociated spinal cord cells for various markers
at E20.5. Arrowheads indicate cells double positive for respective markers.
(D) Developmental changes in the co-expression pattern of various
markers. The percentages of cells positive for a given marker (leftmost
column) that were double positive for other markers (top column) are shown.
Data are the mean of three independent experiments. Scale bars: in C, parts
c,f, 20 µm for C, parts a-f; in C, part i, 50 µm for C, parts g-i.
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Fig. 5. Expression and activity of Ascl3 and Ascl5. (A) Expression of
Aslc3 and Ascl5 in the spinal cord at E10.5 and E16.5. mRNA expression was
detected by RT-PCR using reverse-transcribed (RT+) and non-transcribed (RT-)
RNA samples as templates. Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was
used as internal control. (B-D) Expression of Ascl1 protein, and of
Ascl3 and Ascl5 mRNA in the VZ of the mouse spinal cord at E10.5. The neural
tube is outlined in C,D. Brackets indicate the position of the
Olig2+ motoneuron progenitor domain (pMN). The horizontal lines
indicate the boundary of the dorsal and ventral aspects of the VZ.
(E-G) Neurogenic and oligodendrogenic activity of Ascl3 and Ascl5.
Neurospheres derived from E13.5 spinal cords were infected with GFP
retroviruses expressing Ascl1, Ascl3 and Ascl5, and subsequently induced to
differentiate for 4 (E) or 10 (F,G) days. The percentages of GFP+
cells that differentiated into TuJ1+ neurons, O4+
oligodendrocytes and GFAP+ astrocytes were quantified
(mean±s.d., three independent experiments).
*P<0.01 compared with control virus-infected culture.
Scale bar: 100 µm.
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Fig. 6. Defect in oligodendrocyte differentiation in
Ascl1-/- spinal cords. (A-H) Co-immunostaining
of CNP, MAG and MBP with Olig2 in wild type (A-D) and
Ascl1-/- mutants (E-H). Boxed areas in A and E indicate
the regions shown in other panels. Arrowheads indicate double-positive cells.
Asterisks indicate non-specific staining outside the spinal cord. (I)
Reduction of myelin+ oligodendrocytes in
Ascl1-/- mutants at P0. Data are mean±s.d. obtained
from staining of five or six sections derived from three embryos for each
genotype. The percentage of the mutant level compared with the wild type is
shown for each marker. *P<0.01. (J-M) Expression
of GalC and NG2 (J,K) and MBP (L,M) in culture of wild-type and
Ascl1-/- embryos. Cells from E18.5 spinal cords were
cultured for 7 days. In J and K, arrows indicate
NG2-/GalC+ oligodendrocytes, whereas arrowheads indicate
NG2+/GalC+ intermediate cells. In L and M, arrows
indicate MBP+ oligodendrocytes. Cell nuclei were stained with DAPI
(blue). (N) Differentiation of MBP+ oligodendrocytes in
vitro. Culture of E18.5 spinal cords was performed either the presence (+) or
absence (-) of TH, and the percentage of MBP+ cells among total
cells was quantified at DAP1 and DAP7 (mean±s.d., three independent
experiments). Parentheses show the percentages of the mutant level compared
with the wild type. *P<0.05,
**P<0.01 compared with the wild type. Scale bars: in
A,E, 200 µm; in D,H, 50 µm for B-D,F-H; in M, 50 µm for J-M.
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Fig. 7. Defect in the co-expression of Olig2 and Nkx2-2 in
Ascl1-/- spinal cords. (A) Co-expression of
Olig2 and Nkx2-2 in vivo at E16.5. Arrowheads indicate
Nkx2-2+/Olig2+ cells in the ventral white matter. Scale
bar: 50 µm. (B,C) Expression of Nkx2-2 in Olig2+
OLPs in vitro. Spinal cord cells were isolated from either E16.5 (B) or E18.5
(C) embryos, and the expression of Nkx2-2 in Olig2+ cells was
compared between the wild type and Ascl1-/- mutants at
indicated time points. Data are mean±s.d. (n=3).
*P<0.01 compared with DAP7; %P<0.01
compared with the wild type.
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Fig. 8. Promotion of oligodendrocyte development by Ascl1. Neurospheres
derived from E13.5 spinal cords were infected with GFP retroviruses expressing
various transcription factors either alone or in combination, and subsequently
induced to differentiate for 10 days. (A-D) Overexpression of Olig2,
Nkx2-2 and Ascl1 in virus-infected cells. Cells infected with control (A),
Olig2 (B), Nkx2-2 (C) and Ascl1 (D) viruses were stained for respective
transcription factors together with GFP (both in green; see lower panels) and
oligodendrocyte markers (red). Arrows indicate transgene expression in nuclei
of cells with GFP+ soma, whereas arrowheads indicate the expression
of endogenous factors in uninfected cells with GFP- soma.
(E,F) Differentiation of virus-infected cells into
NG2+ and GalC+ cells. The images show Ascl1
virus-infected cells expressing NG2 and GalC (arrowheads). Scale bars: in D
and F, 20 µm for A-D and E,F. (G,H) Percentages of
NG2+ and GalC+ cells among virus-infected cells
(mean±s.d. from three or four independent experiments). Parentheses
indicate the fold changes compared with the control level.
*P<0.01 compared with the control virus;
%P<0.01 compared with single infections.
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Fig. 9. Combinatorial actions of Ascl1, Olig2 and Nkx2-2 in oligodendrocyte
differentiation. (A-C) The percentages of cells with three distinct
phenotypes among total virus-infected cells (mean±s.d. from three or
four independent experiments). Viruses used for infection are shown on the
left. (D) Promotion of oligodendrocyte differentiation by the
combinatorial actions of Ascl1, Olig2 and Nkx2-2. The percentages of
NG2+/GalC- (pale colors on the left),
NG2+/GalC+ (middle) and NG2-/GalC+
(dark colors on the right) in the total oligodendrocyte lineage cells are
compared between cultures infected with various viruses.
*P<0.01 compared with the control virus;
%P<0.01 compared with culture infected with Olig2 and
Nkx2-2 viruses. $P<0.01 compared with culture infected
with Ascl1 virus.
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Fig. 10. Proposed actions of Ascl1 in oligodendrocyte development. In this
model, Ascl1 regulates oligodendrocyte development at multiple steps (thick
vertical arrows). At early stages, Ascl1 promotes specification of OLPs from
multipotent progenitors (MP). At late embryonic and perinatal periods, Ascl1
regulates the co-expression of Olig2 and Nkx2-2, and subsequently cooperates
with them to promote differentiation of myelin+
oligodendrocytes.
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© The Company of Biologists Ltd 2008
