fbw7 controls neural stem cell differentiation and ... · iz cp vz svz iz cp a supplementary figure...
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1’
1
2’
2
2’
2
1
1’
Fbxw7f/f
Fbxw7∆N
Fbxw7f/f
Fbxw7∆N
Fbxw7
b
a
floxed locusFbxw7f
deletion
Fbxw7∆
loxP-site
Cre-mediatedrecombination
exon4 exon5 exon6
exon
Fbw7 controls neural stem cell differentiation and progenitor apoptosis via
Notch and c-Jun
Joerg D Hoeck, Anett Jandke, Sophia M Blake, Emma Nye, Bradley Spencer-Dene,
Sebastian Brandner and Axel Behrens
Supplementary Figure 1 Efficient Fbxw7 deletion in Fbxw7∆N mice. (a) Schematic representation of the
targeting construct before and after Cre recombination. Exon 5 of the Fbxw7 allele is flanked by loxP-sites
(Fbxw7f) and excised upon crossing Fbxw7f/f mice to transgenic mice expressing Cre recombinase. Conditional
knockout of Fbxw7 in the brain occurs by expressing Cre under the control of a Nestin promoter (Fbxw7∆N).
(b) Fbxw7 (exon 2–5 specific probe) in situ hybridisation with Giemsa counterstain (blue). Rectangles mark
comparable regions of the cortex (1, 1’) and the eye (2, 2’) shown below in high magnification for Fbxw7f/f
and Fbxw7∆N E18.5 heads. Scale bars: 100 µm.
Supplementary Figure 1
Nature Neuroscience: doi:10.1038/nn.2644
Fbxw7f/f Fbxw7∆N Fbxw7f/f Fbxw7∆N Fbxw7f/f Fbxw7∆N
1 2 3
fb
mb
1 2 3
fb
a
b
Supplementary Figure 2
Supplementary Figure 2 Cortex size in the Fbxw7∆N brain. (a) Schematic representation of the E18.5 mouse forebrain (fb;
green) and midbrain (mb; red) dorsal view. Comparable sagittal sections of Fbxw7f/f and Fbxw7∆N E18.5 heads were taken
alongside the lateral-medial axis (1, 2, 3) and are shown in (b) stained with hematoxylin and eosin (H&E). Scale bars: in 1,
580 µm; in 2, 730 µm; in 3, 870 µm.
Nature Neuroscience: doi:10.1038/nn.2644
Fbxw7f/f Fbxw7f/fFbxw7∆N Fbxw7∆N
Cerebellum Thalamus
Supplementary Figure 3
a b
Supplementary Figure 3 Decreased cellularity in the Fbxw7∆N brain. H&E staining of
the E18.5 (a) cerebellum and (b) thalamus from Fbxw7f/f and Fbxw7∆N mouse embryos.
Rectangles mark comparable regions shown below in high magnification. Scale bars:
50 µm.
Nature Neuroscience: doi:10.1038/nn.2644
VZ
SVZ
IZ
CP
VZ
SVZ
IZ
CP
VZ
SVZ
IZ
CP
VZ
SVZ
IZ
CP
VZ
SVZ
IZ
CP
VZ
SVZ
IZ
CP
Fbxw7f/f Fbxw7∆N
E14.5 Fbxw7f/f Fbxw7∆NE16.5
Fbxw7f/f Fbxw7∆NE18.5
Ki67 Ki67 Ki67
b
c
dSupplementary Figure 4
Fbxw7f/f Fbxw7∆N
E10.5
a
Ki67
0
20
40
60
80
100
e
Fbxw7f/fFbxw7∆N
E10.5 E14.5 E16.5 E18.5
K
i67+
cel
ls
Per
cent
age
of
Supplementary Figure 4 Loss of Fbw7 does not affect proliferation in vivo. DAB staining for the S-phase marker Ki67 on representative
sections of the Fbxw7f/f and Fbxw7∆N cortex at (a) E10.5, (b) E14.5, (c) E16.5 and (d) E18.5. Cells are counterstained with hematoxylin.
Scale bars: 50 µm. (e) Quantification of Ki67-positive cells in the E10.5 Fbxw7f/f and Fbxw7∆N cortex and in the SVZ of the E14.5,
E16.5 and E18.5 Fbxw7f/f and Fbxw7∆N cortex. n = 3.
Error bars represent the s.e.m. CP: cortical plate, IZ: intermediate zone, SVZ: subventricular zone, VZ: ventricular zone.Nature Neuroscience: doi:10.1038/nn.2644
**
Dcx Dcx Dcx
Fbxw7f/f Fbxw7∆N
Fbxw7f/f Fbxw7∆N
Fbxw7f/f Fbxw7∆N
E18.5
E16.5
E14.5
**
VZ
SVZ
IZ
CP
VZ
SVZ
IZ
CP
VZ
SVZ
IZ
CP
VZ
SVZ
IZ
CP
VZ
SVZ
IZ
CP
VZ
SVZ
IZ
CP
Supplementary Figure 5
a
b
d
c
0
10
20
30
40
Fbxw7f/fFbxw7∆N
*
E18.5 E14.5E16.5
D
cx+
cel
ls
Per
cent
age
of
Supplementary Figure 5 Reduced number of neuroblasts in the Fbxw7∆N cortex. DAB staining for Doublecortin (Dcx) in the (a) E18.5, (b) E16.5 and (c) E14.5
Fbxw7f/f and Fbxw7∆N cortex. Black rectangles mark areas shown in high magnification in panels at the top. Cells are counterstained with hematoxylin. Scale bars:
50 µm. (d) Quantification of Dcx-positive cells in the SVZ of the E18.5 (n = 4), E16.5 (n = 3) and E14.5 (n = 3) Fbxw7f/f and Fbxw7∆N cortex.
Error bars represent the s.e.m. Statistical significance: *: p ≤ 0.05; **: p ≤ 0.01 (unpaired t-test). CP: cortical plate, IZ: intermediate zone, SVZ: subventricular
zone, VZ: ventricular zone.Nature Neuroscience: doi:10.1038/nn.2644
Fbxw7f/f Fbxw7∆N Fbxw7f/f Fbxw7∆N
Fbxw7f/f Fbxw7∆N Fbxw7f/f Fbxw7∆N
S100 NG2
NG2S100
b
f
0
1
2
3
0
5
10
15
20
25
0
5
10
15
0
5
10
15
d
c e
g
h
i
IZ/CP IZ/CP
Fbxw7f/fFbxw7∆N
Fbxw7f/fFbxw7∆N
Fbxw7f/fFbxw7∆N
Fbxw7f/fFbxw7∆N
n.s.
n.s.n.s.
n.s.
SupplementaryFigure 6
GFAPGFAPDNA
Fbxw7f/f
Fbxw7∆N
E18.5
VZSVZ
IZ
CP
VZSVZ
IZ
CP
a
Supplementary Figure 6 Loss of Fbw7 does
not affect gliogenesis.
(a) Immunohistochemistry for GFAP (red) on
representative sections of the Fbxw7f/f and
Fbxw7∆N E18.5 cortex. DNA (blue) was
counterstained with DAPI. Scale bars: 50 µm.
DAB staining for (b) S100 and (d) NG2 in the
CP of the E18.5 Fbxw7f/f and Fbxw7∆N cortex.
Red arrowheads denote positive cells. Cells
are counterstained with hematoxylin. Scale
bars: 50 µm. Quantification of (c) S100-positive
and (e) NG2-positive cells in the IZ and the
CP of the E18.5 Fbxw7f/f and Fbxw7∆N cortex.
n = 3. DAB staining for (f) S100 and (h) NG2
on Fbxw7f/f and Fbxw7∆N neurosphere sections.
Red arrowheads denote positive cells. Cells
are counterstained with hematoxylin. Scale
bars: 50 µm. Quantification of (g) S100-positive
and (i) NG2-positive cells in Fbxw7f/f and
Fbxw7∆N neurospheres. n = 3. Error bars
represent the s.e.m. Statistical significance:
n.s.: not significant (unpaired t-test). CP:
cortical plate, IZ: intermediate zone, SVZ:
subventricular zone, VZ: ventricular zone.
S
100+
cel
ls
Per
cent
age
of
S10
0+ c
ells
P
erce
ntag
e of
N
G2+
cel
ls
Per
cent
age
of
NG
2+ c
ells
P
erce
ntag
e of
Nature Neuroscience: doi:10.1038/nn.2644
Msi1Msi1DNA BLBP
BLBPDNA Nestin
NestinDNA NeuN
NeuNDNA
Fbxw7f/f
Fbxw7∆N
VZ
ML
VZ
ML
E18.5
Supplementary Figure 7
Supplementary Figure 7 Increased stem cell and decreased neuronal marker expression
in the Fbxw7∆N tectum. From left to right: Immunohistochemistry for Musashi1 (Msi1; red),
BLBP (red), Nestin (red) and NeuN (green) on representative sections of the Fbxw7f/f and
Fbxw7∆N E18.5 tectum. DNA (blue) was counterstained with DAPI. ML: mantle layer, VZ:
ventricular zone. Scale bars: 50 µm.
Nature Neuroscience: doi:10.1038/nn.2644
Fbxw7f/f
Fbxw7∆N
pH3/DNA
Supplementary Figure 8
0
20
40
60
80
100
d1 d3 d5 d70
0.5
1.0
1.5
2.0
Fbxw7f/f
Fbxw7∆N
cell
divi
sion
s
per
day
C
FS
E-in
tens
ity
(in %
)
time (in days)
Fbxw7f/f
Fbxw7∆N
CFSE
CFSE
ud7 d5 d3 d1
ud7 d5 d3 d1
Per
cent
age
of M
ax
P
erce
ntag
e of
Max
a
b
Fbxw7f/f
Fbxw7∆N
c d
Supplementary Figure 8 Loss of Fbw7 does
not affect proliferation in vitro.
(a) Immunocytochemistry for serine 10
phosphorylated histone H3 (pH3; red) on Fbxw7f/f
and Fbxw7∆N neurosphere sections. DNA (blue)
was counterstained with DAPI. Arrowheads
denote pH3-positive cells. Scale bars: 50 µm.
( b ) F A C S h i s t o g r a m s s h o w i n g
carboxyfluorescein diacetate succinimidyl ester
(CFSE) intensity in Fbxw7f/f and Fbxw7∆N
neurosphere cultures 1 (d1), 3 (d3), 5 (d5) and
7 (d7) days after CFSE staining. u: unstained
control. (c) Histogram showing the loss of CFSE
intensity (in %) over time (in days).
(d) Histogram showing cell division rates of
Fbxw7f/f and Fbxw7∆N neurosphere cells based
on the loss of CFSE intensity. Error bars represent
the s.e.m.
Nature Neuroscience: doi:10.1038/nn.2644
DNA
Fbxw7f/f
Fbxw7∆N
Supplementary Figure 9
Fbxw7f/f
Fbxw7∆N
DNA
a
b
Supplementary Figure 9 Fbxw7∆N neurospheres exhibit differentiation defect. (a) Phase
contrast pictures of Fbxw7f/f and Fbxw7∆N neurosphere cultures under differentiation
conditions. (b) Stainings for DNA (blue) with Hoechst 33342 on Fbxw7f/f and Fbxw7∆N
neurosphere cultures after 5 d of differentiation. Scale bars: 50 µm.
Nature Neuroscience: doi:10.1038/nn.2644
BLBP
BLBP
Vimentin
Vimentin
Vimentin/BLBP/DNA
Vimentin/BLBP/DNA
Fbxw7f/f
Fbxw7∆N
Vimentin/BLBP/DNA
Vimentin/BLBP/DNA
Supplementary Figure 10
CD133
CD133
CD133/DNA
Fbxw7f/f
Fbxw7∆N
CD133/DNA
CD133/DNA CD133/DNA
a
b
Supplementary Figure 10 Increased stem cell marker expression in Fbxw7∆N neurosphere
differentiation cultures. Immunocytochemistry for (a) Vimentin (green) and BLBP (red)
and (b) CD133 (green) on Fbxw7f/f and Fbxw7∆N neurosphere cultures after 5 d under
differentiation conditions. White squares mark areas shown in high magnification in panels
on the right. DNA (blue) was counterstained with Hoechst 33342. Scale bars: 100 µm.
Nature Neuroscience: doi:10.1038/nn.2644
Fbxw7f/f Fbxw7∆N
e f
a
b
c
d
Supplementary Figure 11
0102030
40506070
Fbxw7f/fFbxw7∆N
*
0
1
2
3
4
5
6
7
8 Fbxw7f/f Fbxw7∆N
Jun Fbxw7 Hes5
F
old
chan
ge
Fbxw7f/f Fbxw7∆N
Fbxw7f/f Fbxw7∆N
Fbw7
p-c-Jun
NICD1
p-c-Myc
p-CyclinE
β-Actin
Fbxw
7f/f
Fbxw
7∆N
Nestin/DNA Nestin/DNA
Nestin/DNA Nestin/DNA
N
estin
+ c
ells
P
erce
ntag
e of
Supplementary Figure 11 Loss of Fbw7 leads to
similar results in adherent NSC cultures in comparison
to neurosphere cultures. (a) Phase contrast pictures
of Fbxw7f/f and Fbxw7∆N adherent NSC cultures under
self-renewal conditions. Scale bars: 50 µm.
Immunocytochemistry for Nestin (green) on Fbxw7f/f
and Fbxw7∆N adherent NSC cultures (b) under self-
renewal conditions and (c) after 5 d under
differentiation conditions. Scale bars: 50 µm.
(d) Quantification of Nestin-positive cells in Fbxw7f/f
and Fbxw7∆N adherent NSC cultures after 5 days
under differentiation conditions. (e) Western blot
analysis of Fbw7, serine 73 phosphorylated c-Jun (p-
c-Jun), activated Notch1 (NICD1), threonine 58/serine
62 phosphorylated c-Myc (p-c-Myc), threonine 395
phosphorylated cyclinE (p-CyclinE) and β-Actin on
protein lysates from Fbxw7f/f and Fbxw7∆N adherent
NSC cultures (cropped images, full-length blots are
presented in Supplementary Fig. 15). (f) Quantitative
real-time PCR analysis of Fbxw7, Jun and Hes5
transcripts in cells from Fbxw7f/f and Fbxw7∆N
adherent NSC cultures. The data are normalised to
Gapdh and represented as fold change over RNA
levels in Fbxw7f/f adherent NSCs, which is set to 1.
Error bars represent the s.e.m. Statistical significance:
*: p ≤ 0.05 (unpaired t-test).
Nature Neuroscience: doi:10.1038/nn.2644
Fbxw7f/f
Fbxw7∆N
Nestin
Nestin Nestin/DNA
Nestin/DNA Nestin/DNA
Nestin/DNA
Supplementary Figure 12
Supplementary Figure 12 Increased number of immature cells in Fbxw7∆N neurosphere
cultures after prolonged time under differentiation conditions. Immunocytochemistry for
Nestin (green) on Fbxw7f/f and Fbxw7∆N neurosphere cultures after 11 d of differentiation.
White squares mark areas shown in high magnification in panels on the right. DNA (blue)
was counterstained with Hoechst 33342. Scale bars: 100 µm.
Nature Neuroscience: doi:10.1038/nn.2644
Map2/O4/Cx43/DNA
Map2/O4/Cx43/DNA
O4
O4
Map2
Map2
Fbxw7f/f
Fbxw7∆N
Cx43
Cx43
DNA
DNA
Supplementary Figure 13
Supplementary Figure 13 Loss of Fbw7 does not affect neurosphere multipotentiality.
Immunocytochemistry for Map2 (green), O4 (red) and Connexin43 (Cx43; magenta) on
Fbxw7f/f and Fbxw7∆N neurosphere cultures after 5 d of differentiation. DNA (blue) was
counterstained with Hoechst 33342. Scale bars: 50 µm.
Nature Neuroscience: doi:10.1038/nn.2644
Supplementary Figure 14
α α
α α
β β
β β
γ γ
γ γ
Brain
Skeletal Muscle Liver
Rel
ativ
e ab
unda
nce
of F
bxw
7 is
ofor
ms
0
0.5
1.0
1.5
2.0
2.5
3.0
0
0.2
0.4
0.6
0.8
1.0
1.2
Rel
ativ
e ab
unda
nce
of F
bxw
7 is
ofor
ms
0
0.2
0.4
0.6
0.8
1.0
1.2
Rel
ativ
e ab
unda
nce
of F
bxw
7 is
ofor
ms
0
0.2
0.4
0.6
0.8
1.0
1.2 R
elat
ive
abun
danc
eof
Fbx
w7
isof
orm
s
Neurospheres
Supplementary Figure 14 Fbxw7 isoform expression. Quantitative real-time PCR analysis
showing the relative abundance of Fbxw7 isoforms α, β and γ in Fbxw7f/f neurospheres,
adult brain, skeletal muscle and liver normalised to Gapdh expression. Expression of Fbxw7
α is set to 1. Error bars represent the standard deviation (s.d.).
Nature Neuroscience: doi:10.1038/nn.2644
Fbw7
83c-Jun
p-c-Jun
38
NICD1
52
p-c-Myc76
p-CyclinE52
β-Actin
38
Supplementary Figure 15
a
Supplementary Figure 15 Full-length Western Blots. Full-length Western Blots from (a) Fig. 5a and (b) Supplementary Fig. 11e. Molecular weight
standards are shown in kDa.
b
76
Fbw7
p-c-Jun
p-c-Myc
55
NICD176
p-CyclinE55
β-Actin
31
Nature Neuroscience: doi:10.1038/nn.2644
Fbxw7f/f Fbxw7∆N Fbxw7∆N; Jun∆N/+
VZ VZ
ML ML
Supplementary Figure 16
Supplementary Figure 16 Decreased cellularity in Fbxw7∆Ν brains is c-Jun-dependent.
H&E staining of comparable regions of the Fbxw7f/f, Fbxw7∆Ν and Fbxw7∆N; Jun∆N/+ E18.5
midbrain. Rectangles mark the area of the tectum shown below in high magnification. ML:
mantle layer, VZ: ventricular zone. Scale bars: 200 µm.
Nature Neuroscience: doi:10.1038/nn.2644
Fbxw7f/f
Fbxw7∆N
Fbxw7∆N;Jun∆N/+
0
5
10
15
20
25
30
35
***
n.s.
Supplementary Figure 17
P
erce
ntag
e of
Nes
tin+
cel
ls
Supplementary Figure 17 Differentiation defect in Fbxw7∆Ν neurosphere cultures is not
c-Jun-dependent. Histogram showing the percentage of Nestin-positive cells in Fbxw7f/f
(n = 8), Fbxw7∆N (n = 11) and Fbxw7∆N; Jun∆N/+ (n = 2) neurosphere cultures after 5 d of
differentiation. Error bars represent the s.e.m. Statistical significance: n.s.: not significant;
***: p ≤ 0.001 (unpaired t-test).
Nature Neuroscience: doi:10.1038/nn.2644
Map2/DNAMap2/DNA
Map2/DNAMap2/DNA
Map2/DNAMap2/DNA
Fbxw7f/f
Fbxw7∆N
Fbxw7∆N; Notch1∆/+
0
5
10
15
20
25
Fbxw7f/f
Fbxw7∆N
Fbxw7∆N; Notch1∆/+
**
*
c
d
NeuN
Fbxw7f/f Fbxw7∆N Fbxw7∆N; Notch1∆/+
Fbxw7f/f
Fbxw7∆N
Fbxw7∆N;Notch1∆/+
**
*
0
10
20
30
40
50
VZ
SVZ
IZ
CP
VZ
SVZ
IZ
CP
a
b
Supplementary Figure 18
IZ/CP
N
euN
+ c
ells
P
erce
ntag
e of
M
ap2+
cel
ls
Per
cent
age
of
Supplementary Figure 18 Notch downregulation rescues neuronal numbers. (a) DAB staining for NeuN on the E18.5 Fbxw7f/f, Fbxw7∆N and Fbxw7∆N; Notch1∆/+ cortex.
Cells are counterstained with hematoxylin. Scale bars: 50 µm. (b) Quantification of NeuN-positive cells in the IZ and the CP of the E18.5 Fbxw7f/f, Fbxw7∆N and Fbxw7∆N;
Notch1∆/+ cortex (n = 3). (c) Immunocytochemistry for Map2 (green) on Fbxw7f/f, Fbxw7∆N and Fbxw7∆N; Notch1∆/+ neurosphere cultures after 5 d under differentiation
conditions. White squares mark areas shown in high magnification in panels on the right. DNA (blue) was counterstained with Hoechst 33342. Scale bars: 50 µm. (d)
Histogram showing the percentage of Map2-positive cells in Fbxw7f/f (n = 3), Fbxw7∆N (n = 4) and Fbxw7∆N; Notch1∆/+ (n = 5) neurosphere cultures after 5 d under differentiation
conditions. Error bars represent the s.e.m. Statistical significance: *: p ≤ 0.05; **: p ≤ 0.01 (unpaired t-test). CP: cortical plate, IZ: intermediate zone, SVZ: subventricular
zone, VZ: ventricular zone.
Nature Neuroscience: doi:10.1038/nn.2644
a
0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Fbxw7f/f +DMSO Fbxw7∆N +DMSOFbxw7f/f +1 µM DAPT Fbxw7∆N +1 µM DAPT
F
old
chan
ge
Hes5 Hey1 Hes1
b
Fbxw7f/f
+ 1µM DAPT
Fbxw7∆N
+ 1µM DAPT
Fbxw7f/f
+DMSO
Fbxw7∆N
+DMSO
Nestin
Nestin
Nestin
Nestin Nestin/DNA Nestin/DNA
Nestin/DNA Nestin/DNA
Nestin/DNA Nestin/DNA
Nestin/DNA Nestin/DNA
c
Fbxw7f/f Fbxw7∆N
+DMSO +1 µM DAPT
0
5
10
15
20
25
30
35 ***
n.s.
**
Supplementary Figure 19
N
estin
+ c
ells
P
erce
ntag
e of
Supplementary Figure 19 Inhibition of Notch signalling alleviates the block in stem cell differentiation. (a) Quantitative
real-time PCR analysis of Hes5, Hey1 and Hes1 transcripts in Fbxw7f/f and Fbxw7∆N neurospheres treated with DMSO
(control) or 1 µM DAPT. The data are normalised to Gapdh and represented as fold change over RNA levels in Fbxw7f/f
+ DMSO neurospheres, which is set to 1. (b) Immunocytochemistry for Nestin (green) on Fbxw7f/f and Fbxw7∆N neurosphere
cultures after 5 d under differentiation conditions treated with DMSO (control) or 1 µM DAPT. White squares mark areas
shown in high magnification in panels on the right. DNA (blue) was counterstained with Hoechst 33342. Scale bars: 100
µm. (c) Histogram showing the percentage of Nestin-positive cells in Fbxw7f/f and Fbxw7∆N neurosphere cultures after
5 d under differentiation conditions treated with DMSO (control) or 1 µM DAPT. n = 5. Error bars represent the s.e.m.
Statistical significance: n.s.: not significant; **: p ≤ 0.01; ***: p ≤ 0.001 (unpaired t-test).Nature Neuroscience: doi:10.1038/nn.2644
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