Fbw7 controls neural stem cell differentiation and ... · IZ CP VZ SVZ IZ CP a Supplementary Figure 6 Loss of Fbw7 does not affect gliogenesis. (a) Immunohistochemistry for GFAP (red)

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 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.


Fbxw7f/f Fbxw7f/fFbxw7∆N Fbxw7∆N

Cerebellum Thalamus


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.


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


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

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3

0

5

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25

0

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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


Msi1Msi1DNA BLBP

BLBPDNA Nestin

NestinDNA NeuN

NeuNDNA

Fbxw7f/f

Fbxw7∆N

VZ

ML

VZ

ML

E18.5


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.


Fbxw7f/f

Fbxw7∆N

pH3/DNA


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.


DNA

Fbxw7f/f

Fbxw7∆N


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.


BLBP

BLBP

Vimentin

Vimentin

Vimentin/BLBP/DNA

Vimentin/BLBP/DNA

Fbxw7f/f

Fbxw7∆N

Vimentin/BLBP/DNA

Vimentin/BLBP/DNA


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.


Fbxw7f/f Fbxw7∆N

e f

a

b

c

d


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


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).


Fbxw7f/f

Fbxw7∆N

Nestin

Nestin Nestin/DNA


Nestin/DNA


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.


Map2/O4/Cx43/DNA

Map2/O4/Cx43/DNA

O4

O4

Map2

Map2

Fbxw7f/f

Fbxw7∆N

Cx43

Cx43

DNA

DNA


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.



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

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ativ

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nce

of F

bxw

7 is

ofor

ms

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ativ

e ab

unda

nce

of F

bxw

7 is

ofor

ms

0

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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.).


Fbw7

83c-Jun

p-c-Jun

38

NICD1

52

p-c-Myc76

p-CyclinE52

β-Actin

38


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


Fbxw7f/f Fbxw7∆N Fbxw7∆N; Jun∆N/+

VZ VZ

ML ML


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.


Fbxw7f/f

Fbxw7∆N

Fbxw7∆N;Jun∆N/+

0

5

10

15

20

25

30

35

***

n.s.


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).


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

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50

VZ

SVZ

IZ

CP

VZ

SVZ

IZ

CP

a

b


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.


a

0

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1.0

1.5

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2.5

3.0

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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




c

Fbxw7f/f Fbxw7∆N

+DMSO +1 µM DAPT

0

5

10

15

20

25

30

35 ***

n.s.

**


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

Documents

Fbw7 controls neural stem cell differentiation and ... · IZ CP VZ SVZ IZ CP a Supplementary Figure 6 Loss of Fbw7 does not affect gliogenesis. (a) Immunohistochemistry for GFAP (red)