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Transient deregulation of canonical Wnt signaling in developing pyramidal neurons leads to dendritic defects and impaired behavior

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Transient deregulation of canonical Wnt signaling in developing pyramidal neurons leads to dendritic defects and impaired behavior

VIALE, Beatrice, et al.

Abstract

During development, the precise implementation of molecular programs is a key determinant of proper dendritic development. Here, we demonstrate that canonical Wnt signaling is active in dendritic bundle-forming layer II pyramidal neurons of the rat retrosplenial cortex during dendritic branching and spine formation. Transient downregulation of canonical Wnt transcriptional activity during the early postnatal period irreversibly reduces dendritic arbor architecture, leading to long-lasting deficits in spatial exploration and/or navigation and spatial memory in the adult. During the late phase of dendritogenesis, canonical Wnt-dependent transcription regulates spine formation and maturation. We identify neurotrophin-3 as canonical Wnt target gene in regulating dendritogenesis. Our findings demonstrate how temporary imbalance in canonical Wnt signaling during specific time windows can result in irreversible dendritic defects, leading to abnormal behavior in the adult.

VIALE, Beatrice,

et al

. Transient deregulation of canonical Wnt signaling in developing

pyramidal neurons leads to dendritic defects and impaired behavior.

Cell Reports

, 2019, vol.

27, no. 5, p. 1487-1502.e6

PMID : 31042475

DOI : 10.1016/j.celrep.2019.04.026

Available at:

http://archive-ouverte.unige.ch/unige:126064

Disclaimer: layout of this document may differ from the published version.

1 / 1

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Cell Reports, Volume27

Supplemental Information

Transient Deregulation of Canonical Wnt Signaling in Developing Pyramidal Neurons Leads to

Dendritic Defects and Impaired Behavior

Beatrice Viale, Lin Song, Volodymyr Petrenko, Anne-Laure Wenger Combremont, Alessandro Contestabile, Riccardo Bocchi, Patrick Salmon, Alan Carleton, Lijia An, Laszlo Vutskits, and Jozsef Zoltan Kiss

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Branch order

****

Apical dendrites

1 13

Number of branches 00 5 10 15

2 3 4 10 12

***

9

****

8 7

**

6

5 11

C Basal dendrites

Branch order 0

2 4 8

Number of branches 0

6 7 1 2

***

3

**

4 5 6

Figure S1

β-cateninControl

GFP LY

A GFP/β-catenin

E18 E21

LY analysisP21

β-catenin

pTF hPGK GFP E2A rtTA GFP

UBI

****

Apical dendrites branch points

0 100 180

120 140 160

0 100 160

Apical dendrites length (µm)

120 140

**

β-catenin Control

Basal dendrites length (µm) **

0 100 160

120 140

***

Basal dendrites branch points

0 100 180

120 140 160

B

0 10

Basal dendrites branch points

8

4 ns

****

6

2 ns

**

0 800

Basal dendrites length (µm)

600 400 200

***

ns

**

0 50

Apical dendrites branch points

40 30 20 10

**

ns

**

0 2500 2000 1500 1000 500

**

Apical dendrites length (µm)

dnTCF4/∆DVL2 E21

LY analysisP21 E18

D

∆DVL2dnTCF4

E

∆DVL2

pTF hPGK GFP E2A rtTA dnTCF4

pTF hPGK GFP E2A rtTA

∆DVL2 dnTCF4 Control

G

F GFP/dnTCF4

E21

LY analysisP21 E18

S1

****

0 3000

1000 2000

Apical dendrites length (µm) **

0 1500

Basal dendrites length (µm)

1000

500

****

0 15

Basal dendrites branch points10 5

Control S1 dnTCF4 S1

****

0 30

Apical dendrites branch points20 10

GFP UBI

dnTCF4

pTF hPGK GFP E2A rtTA

dnTCF4Control

S1

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Figure S1. Wnt GOF increases dendritic complexity in RSC. Relative to Figure 1.

(A) Timeline of the experiment and plasmid used. Coronal slices of P21 brains electro- porated at E18 with control plasmid (top panel) or β-catenin (expressed from E21) (lower panel) showing cells iontophoretically injected with Lucifer Yellow (LY) and their neurolucida reconstructions.

(B) Quantification of total length and number of branch points in apical and basal den- drites of β-catenin-expressing neurons as a percentage of control cells at P21. Histo- grams represent mean ± SEM of n=4 brains for control, n=4 brains for β-catenin from at least 2 independent experiments (Student’s t-test).

Absolute values:

AD, apical dendrites; BD, basal dendrites

(C) Quantification of the total branch number for each branch order of control and β-cat- enin-expressing neurons at P21. Data represent mean ± SEM of n=4 brains for control, n=4 brains for β-catenin from at least 2 independent experiments (Two-way ANOVA followed by Bonferroni post-test).

(D) Timeline of the experiment. Neurolucida reconstructions of dnTCF4-expressing neu- rons and ΔDVL2-expressing neurons at P21 in RSC.

(E) Plasmids used. Quantification of total length and number of branch points in apical and basal dendrites of control, dnTCF4-expressing neurons and ΔDVL2-expressing neurons at P21. Histograms represent mean ± SEM of n=4 brains for control, n=5 brains for dnTCF4, n=3 brains for ΔDVL2 from at least 2 independent experiments (One-way ANOVA followed by Bonferroni post-test).

(F) Timeline of the experiment. Neurolucida reconstructions of control and dnTCF4-ex- pressing neurons at P21 in S1.

(G) Plasmids used. Quantification of total length and number of branch points in apical and basal dendrites of control and dnTCF4-expressing neurons at P21 in S1. Histo- grams represent mean ± SEM of n=6 brains for control, n=6 brains for dnTCF4 from at least 2 independent experiments (Student’s t-test).

** P<0.01; *** P<0.001; **** P<0.0001

Scale bar = 200 μm (A top panel), 50 μm (A bottom panel and C), 40 μm (F), 20 μm (A right panel, D)

Control β-catenin

Mean SEM Mean SEM

AD length (µm) 1097 31.63 1503 79.15 AD branch points 17.42 0.9335 25.02 0.6377 BD length (µm) 316.7 43.9 411.5 19.06 BD branch points 3.947 0.6078 5.798 0.3096

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Figure S2. Dendritic defect induced by Wnt LOF does not depend on altered cell fate nor perturbed migration. Relative to Figure 2.

(A) Timeline of the experiment. Canonical Wnt activity reporter (TOPdGFP) and TOM co-expression at P7 and P11, corresponding to 0 and 4 days after the end of dnTCF4 induction, respectively. Quantification of TOPdGFP/TOM intensity ratio (dnTFC4/con- trol) shows that canonical Wnt activity levels are restored after 4 days without dnTCF4 induction. Histograms represent mean ± SEM of n=4 brains for P7, n=4 brains for P11 from at 2 independent experiments (Student’s t-test).

(B) Timeline of the experiment. Coronal slices of P56 brains electroporated at E18 with tomato (TOM) or dnTCF4 (GFP) showing positive SatB2 immunofluorescent staining.

(C) Timeline of the experiment. Neurolucida reconstructions of control cells and neurons expressing dnTCF4 from P4 to P7 analyzed at P21.

(D) Quantification of total length and number of branch points in apical and basal den- drites of control and dnTCF4-expressing neurons at P21, with dnTCF4 expression restricted from P4 to P7. Histograms represent mean ± SEM of n=11 brains for control, n=6 brains for dnTCF4 from at least 2 independent experiments (Student’s t-test).

* P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001 Scale bar = 20 μm (A, B); 50 μm (C)

B

ControldnTCF4

P56 TOM

GFP

SatB2 TOM/dnTCF4

E18 E21 P56

immuno P7

C GFP/dnTCF4

E18 P4

LY analysisP21 P7

dnTCF4 P4-P7 Control

Figure S2

D

****

0 1500

500 1000

Apical dendrites length (µm) ****

0 20

Apical dendrites branch points

15 10 5

0 500

Basal dendrites length (µm)

400 300 200 100

**

0 5

Basal dendrites branch points

4 3 2 1

***

Control dnTCF4

A

dnTCF4 Control

P7P11

TOM/dnTCF4+TOPdGFP

E18 E21 P11

analysis analysisP7

*

0 1.5

0.5 1

Wnt activity ratio (dnTCF4/control) P7 P11

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Figure S3. The early phase of dendritogenesis is activity-independent. Relative to Figure 2.

(A) Timeline of the experiment and plasmid used. Neurolucida reconstructions of control cells and neurons expressing Kir2.1 from E21 to P7 analyzed at P7. Quantification of total length and number of branch points in apical and basal dendrites of control and Kir2.1-expressing neurons at P7. Histograms represent mean ± SEM of n=10 brains for

ns

0 15

Basal dendrites branch points10 5 ns

0 400

Basal dendrites length (µm)

300 200 100 ns

0 15

Apical dendrites branch points10 5 ns

0 600

200 400

Apical dendrites length (µm)

Control Kir2.1

A GFP/Kir2.1

E21

LY analysisP7 E18

GFP/hM3Dq+CNO P0

LY analysisP7 E18

B Control

hM3Dq+CNO

ns

0 15

Basal dendrites branch points10 5 ns

0 800

200 400

Apical dendrites length (µm)

600

ns

0 15

Apical dendrites branch points10 5

ns

0 400

Basal dendrites length (µm)

300 200 100

Control dnTCF4

dnTCF4+hM3Dq+CNO

ns

0 600

200 400

Apical dendrites length (µm) *** **

0 15

Apical dendrites branch points10 5

* *ns

0 400

Basal dendrites length (µm)

300 200 100

nsns

0 15

Basal dendrites branch points10 5 GFP/dnTCF4/dnTCF+hM3Dq+CNO

E18 P0

LY analysisP7

C

Figure S3

hM3Dq

pTF hPGK GFP E2A rtTA Kir2.1

pTF hPGK GFP E2A rtTA

Kir2.1

Control hM3Dq+CNO

dnTCF4+hM3Dq+CNO dnTCF4

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control, n=5 brains for Kir2.1 from at least 2 independent experiments (Student’s t-test).

(B) Timeline of the experiment and plasmid used. Neurolucida reconstructions of hM3Dq-expressing neurons at P7, with hM3Dq activation (CNO injections) from P0 to P7. Quantification of total length and number of branch points in apical and basal den- drites of control and hM3Dq-expressing neurons at P7. Histograms represent mean ± SEM of n=10 brains for control, n=4 brains for hM3Dq+CNO from at least 2 independent experiments (Student’s t-test).

(C) Timeline of the experiment. Neurolucida reconstructions of neurons expressing dnTCF4 from E21 to P7 and cells that co-express dnTCF4 and hM3Dq (activated from P0 to P7) (dnTCF4+hM3Dq+CNO) at P7. Quantification of total length and number of branch points in apical and basal dendrites of control, dnTCF4-expressing neurons and cells that co-express dnTCF4 and hM3Dq (dnTCF4+hM3Dq+CNO) at P7. Histograms represent mean ± SEM of n=10 brains for control, n=4 brains for dnTCF4, n=3 brains for dnTCF4+hM3Dq+CNO from at least 2 independent experiments (One-way ANOVA followed by Bonferroni post-test).

Controls in histograms in A, B, C, and dnTCF4 in histograms in C are the same as in Figure 4G.

* P<0.05; ** P<0.01; *** P<0.001 Scale bar = 20 μm (A, B, C).

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Figure S4. During the late phase, spine development is influenced by activity. Rel- ative to Figure 3.

(A) Timeline of the experiment. Confocal images of representative apical dendritic seg- ments of P30 control cells and neurons expressing hM3Dq (activated from P21 to P30).

Quantification of number of spines per μm of control cells and neurons expressing hM3Dq activated with CNO from P21 to P30. Histograms represent mean ± SEM of n=6 brains for control (1810 spines for apical dendrite; 1110 spines for basal dendrites), n=3 brains for hM3Dq+CNO (1645 spines for apical dendrite; 1298 spines for basal den- drites) from at least 2 independent experiments (Student’s t-test).

(B) Timeline of the experiment. Confocal images of representative apical dendritic seg- ments of P30 neurons expressing dnTCF4 from P21 to P30 and cells that co-express dnTCF4 and hM3Dq (activated from P21 to P30). Quantification of number of spines per μm of control, dnTCF4-expressing neurons and cells that co-express dnTCF4 and hM3Dq (dnTCF4+hM3Dq+CNO) at P30 (activation of dnTCF4 and hM3Dq from P21 to P30). Histograms represent mean ± SEM of n=6 brains for control (1810 spines for apical dendrite; 1110 spines for basal dendrites), n=6 brains for dnTCF4 (1356 spines for apical dendrite; 1061 spines for basal dendrites), n=3 brains for dnTCF4+hM3Dq+C- NO (608 spines for apical dendrite; 396 spines for basal dendrites) from at least 2 inde- pendent experiments (One-way ANOVA followed by Bonferroni post-test).

Image of control in A and image of dnTCF4 in B are the same as in Figure 3C.

* P<0.05; *** P<0.001 Scale bar = 5 μm (A, B).

Control dnTCF4

dnTCF4+hM3Dq+CNO ns

Apical dendrites

0 1.0

0.6 0.8

Nb of spines/µm

0.4 0.2

* nsns

Basal dendrites

0 1.0

0.6 0.8

Nb of spines/µm

0.4 0.2 GFP/hM3Dq+CNO

P21

LY analysisP30 E18

A GFP/dnTCF4/dnTCF+hM3Dq+CNO

P21 E18

LY analysisP30

B

Control hM3Dq+CNO

***

0 1.5

1.0

Nb of spines/µm

0.5

Basal dendrites

***

0 1.5

1.0

Nb of spines/µm

0.5

Apical dendrites

Figure S4

hM3Dq

Apical Dendrite Control

Apical Dendrite dnTCF4

hM3Dq dnTCF4

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Figure S5. In vitro validation of shNT3 and NT3 LOF between P7 and P15. Relative to Figure 4.

(A) Timeline of the experiment and plasmids used. Quantification of NT3 mRNA expres- sion of HEK293T/17 cell cultures transfected at DIV1 with either a plasmid encoding for rat NT3 (NT3) alone or with both NT3 plasmid and dox-inducible shNT3 plasmid (NT3+shNT3 –dox when the expression of the shRNA was not induced; NT3+shNT3 +dox when shRNA expression was induced). Histogram represents mean ± SEM of n=3 experiments (One-way ANOVA followed by Bonferroni post-test).

(B) Timeline of the experiment. Neurolucida reconstructions of control cells and neurons expressing shNT3 from P7 to P15 analyzed at P15.

(C) Quantification of total length and number of branch points in apical and basal den- drites of control and shNT3-expressing neurons at P15. Histograms represent mean ± SEM of n=3 brains for control, n=3 brains for shNT3 from at least 2 independent experi- ments (Student’s t-test).

*** P<0.001; **** P<0.0001 Scale bar = 20 μm (B).

NT3/NT3+shNT3dox

qPCRDIV4 DIV1

A

Figure S5

shNT3

pTF hPGK GFP E2A rtTA NT3

UBI hPGK GFP

ns****

Relative NT3 mRNA expression 0 1.0

0.5

***

85% 19%

NT3

NT3+shNT3 -dox NT3+shNT3 +dox

Control shNT3 GFP/shNT3

E21

LY analysisP15 E18

B C

shNT3 Control

P7

0 500 1000 1500

Apical dendrites length (µm) ns

ns

0 25

Apical dendrites branch points

10 5 20 15

ns

0 400

Basal dendrites length (µm)

300 200 100

ns

0 15

Basal dendrites branch points10 5

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50 pA 500 ms

dnTCF4

50 pA

Control

500 ms

Figure S6. The electrophysiological properties of dnTCF4 neurons are normal.

Relative to Figure 6.

(A-J) Comparison of electrophysiological properties as well as spontaneous and minia- ture postsynaptic currents of RSC layer II GFP+ neurons from acute slices of control and dnTCF4 electroporated animals.

C P21 sEPSCs

dnTCF4

500 ms 10 pA

Control

500 ms 10 pA

ns

Amplitude(pA)

0 5 10 ns

0 200 400 600

IEI(ms)

D P21 sIPSCs ns

0 20 40

Amplitude(pA)

ns

0 500 1000

IEI(ms)

50 pA 500 ms

Control

50 pA 500 ms

dnTCF4

A

Figure S6

ns

0 1000 1500 2000

IEI(ms)

500

ns

Amplitude(pA)

0 20 40 60 ns 80

0 200 400 600

IEI(ms)

ns

Amplitude(pA)

0 10

20 ns

0 500 1000 1500

IEI(ms)

ns

Amplitude(pA)

0 10 ns 20

0 500 1000

IEI(ms)

ns

Amplitude(pA)

0 20 40 60 80 ns

0 50 100 150

Rheobase (pA)

ns

0 20 40 60 80

Capacitance (pF)

ns

0 0.1 0.2 0.3 0.4

Input resistance (G)

ns

-80 -70 -50

-60

RMP (mV)

Control dnTCF4

P21 B

E P21 mEPSCs F P21 mIPSCs

ns

0 500 1000 1500

IEI(ms)

ns

0 1000 1500 2000

500

IEI(ms)

ns

0 20 40 60 80

Amplitude(pA)

ns

0 10 20

Amplitude(pA)

G P45 sEPSCs H P45 sIPSCs I P45 mEPSCs J P45 mIPSCs P45

ns

0 0.1 0.2 0.3 0.4

Input resistance (G)

ns

-80 -70 -50

-60

RMP (mV)

ns

0 10 20 30 40

Capacitance (pF)

ns

0 50 100 150

Rheobase (pA)

10 pA 500 ms

dnTCF4

10 pA 500 ms

Control

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(A) Graphs comparing typical electrophysiological properties between dnTCF4-ex- pressing neurons and control cells at P21. Histograms represent mean + SEM of n=12 and 9 cells for control and dnTCF4, respectively, (Mann-Whitney test). RMP= resting membrane potential.

(B) Same as in (A) at P45. Histograms represent mean + SEM of n=7 cells/condition, (Mann-Whitney test).

(C) Example of spontaneous EPSCs recording together with quantification of interev- ent-interval (IEI) and amplitude of the events at P21. Histograms represent mean + SEM of n=6 cells/condition, (Mann-Whitney test).

(D) Same as C but for spontaneous inhibitory postsynaptic currents. Histograms repre- sent mean + SEM of n=7 cells/condition, (Mann-Whitney test).

(E) Example of miniature EPSCs recording together with quantification of interevent-in- terval (IEI) and amplitude of the events at P21. Histograms represent mean + SEM of n=8 cells/condition, (Mann-Whitney test).

(F) Same as E but for miniature IPSCs. Histograms represent mean + SEM of n=6 and 8 cells for control and dnTCF4, respectively (Mann-Whitney test).

(G) Quantification of interevent-interval and amplitude of spontaneous EPSCs at P45.

Histograms represent mean + SEM of n=7 cells/condition, (Mann-Whitney test).

(H) Same as G for spontaneous IPSCs at P45. Histograms represent mean + SEM of n=5 and 6 cells for control and dnTCF4, respectively, (Mann-Whitney test).

(I) Quantification of interevent-interval and amplitude of miniature EPSCs at P45. Histo- grams represent mean + SEM of n=7 cells/condition, (Mann-Whitney test).

(J) Same as I for miniature IPSCs at P45. Histograms represent mean + SEM of n=7 and 8 cells for control and dnTCF4, respectively, (Mann-Whitney test).

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Figure S7. dnTCF4 electroporated animals show no defect in motor activity. Rela- tive to Figure 7.

(A) Extent of the electroporated area for behavioral experiments.

(B) Barnes Maze Test showing no difference in number of errors/latency and in percent of time spent in the target quadrant during the habituation phase. Histograms represent mean ± SEM of n=24 animals for control, n=18 animals for dnTCF4 (Student’s t-test).

(C) Cylinder Test showing no motor defect. Histogram represents mean ± SEM of n=31 animals for control, n=24 animals for dnTCF4 (Student’s t-test).

(D) Footprint Test showing no motor defect. Histograms represent mean ± SEM of n=23 animals for control, n=24 animals for dnTCF4 (Student’s t-test).

ns

nb rearing/min

0 20 15 10 5

caudal

rostral

dnTCF4 Control

Footprint Test Cylinder Test

C

stride length

step width stance length

D A

ns

Nb errors/latency

0 0.5

0.3 0.4

0.2 0.1

ns

% time in target quadrant 0 200 150 100 50

Habituation

B

Barnes Maze Test

Forelimb

ns

Length/weight (cm/g)

0 0.02

0.01

Step Width

ns

Length/weight (cm/g)

0 0.10

0.04

Stride Length

0.02 0.08 0.06

ns

Length/weight (cm/g)

0 0.05

0.02

Stance Length

0.01 0.04 0.03

Figure S7

Control dnTCF4

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