1’ 1 2’ 2 2’ 2 1 1’ Fbxw7 f/f Fbxw7 ∆N Fbxw7 f/f Fbxw7 ∆N Fbxw7 b a floxed locus Fbxw7 f deletion Fbxw7 ∆ loxP-site Cre-mediated recombination 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 (Fbxw7 f ) and excised upon crossing Fbxw7 f/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 Fbxw7 f/f and Fbxw7 ∆N E18.5 heads. Scale bars: 100 μm. Supplementary Figure 1 Nature Neuroscience: doi:10.1038/nn.2644
19
Embed
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)
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
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
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
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