1. Volume 27, Issue 5, Pages 1487-1502.e6 (April 2019)
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, Jozsef Zoltan Kiss 
Cell Reports 
Volume 27, Issue 5, Pages e6 (April 2019)
DOI: /j.celrep
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2. Cell Reports 2019 27, 1487-1502.e6DOI: (10.1016/j.celrep.2019.04.026)
Copyright © 2019 The Authors Terms and Conditions

3. Figure 1
Canonical Wnt Signaling Is Required for Dendritic Development of Layer II Pyramidal Neurons
(A) Timeline of the experiment and plasmids used. Coronal slices of P0 brains electroporated at E18 with tomato (TOM) and TOPdGFP (GFP) showing low level of canonical Wnt activity in migrating neurons (arrows) and high canonical Wnt activity in layer II neurons (arrowheads). CP, cortical plate; IZ, intermediate zone; L2, layer II; VZ, ventricular zone.
(B) Quantification of TOPdGFP+ cells within TOM+ population. Histograms represent mean + SEM of n = 5 brains (P0 migrating), 5 brains (P0 layer II), 5 brains (P4), 5 brains (P10), 4 brains (P21), and 3 brains (P45) from at least two independent experiments (one-way ANOVA followed by Bonferroni post-test).
(C) Timeline of the experiment and plasmids used. Coronal slices of P21 brains electroporated at E18 with control plasmid (GFP) (top panel) and dnTCF4 (expressed from E21) (bottom panel) showing cells iontophoretically injected with Lucifer yellow (LY) and their Neurolucida reconstructions.
(D) Quantification of total length and number of branchpoints in apical and basal dendrites of dnTCF4-expressing neurons as a percentage of control cells at P21. Histograms represent mean ± SEM of n = 4 and 5 brains for control and dnTCF4, respectively, from at least two independent experiments (Student’s t test). Absolute values (mean ± SEM): AD length, 1704 ± 150.5 μm for control and ± 92.64 μm for dnTCF4; AD branch points, ± for control and ± for dnTCF4; BD length, ± 45.29 μm for control and ± 28.43 μm for dnTCF4; BD branch points, ± for control and ± for dnTCF4. ADs, apical dendrites; BDs, basal dendrites. (E) Quantification of the total branch number for each branch order of control and dnTCF4-expressing neurons at P21. Data represent mean ± SEM of n = 7 and 3 brains for control and dnTCF4, respectively, from at least two independent experiments (two-way ANOVA followed by Bonferroni post-test).
∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < Scale bars, 200 μm (A, top panel), 50 μm (A, bottom panel, and C), and 20 μm (A, right panel).
See also Figure S1.
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4. Figure 2
Wnt LOF Has an Irreversible Effect on Dendritic Development and Acts Only during a Specific Time Window
(A) Timeline of the experiment. Neurolucida reconstructions of control cells and neurons expressing dnTCF4 from E21 to P15 analyzed at P90.
(B) Quantification of total length and number of branchpoints in apical and basal dendrites of control and dnTCF4-expressing neurons at P90. Histograms represent mean ± SEM of n = 5 and 4 brains for control and dnTCF4, respectively, from at least two independent experiments (Student’s t test).
(C) Timeline of the experiment. Neurolucida reconstructions of control cells and neurons expressing dnTCF4 from E21 to P7 analyzed at P21.
(D) Quantification of total length and number of branchpoints in apical and basal dendrites of control and dnTCF4-expressing neurons at P21, with dnTCF4 expression restricted from E21 to P7. Histograms represent mean ± SEM of n = 11 and 5 brains for control and dnTCF4, respectively, from at least two independent experiments (Student’s t test).
(E) Timeline of the experiment. Neurolucida reconstructions of control cells and neurons expressing dnTCF4 from P7 to P15 analyzed at P21.
(F) Same as (D), with dnTCF4 expression restricted from P7 to P15. Histograms represent mean ± SEM of n = 11 and 5 brains for control and dnTCF4, respectively, from at least two independent experiments (Student’s t test).
(G) Timeline of the experiment. Neurolucida reconstructions of control cells and neurons expressing dnTCF4 from P15 to P21 analyzed at P21.
(H) Same as (D), with dnTCF4 expression restricted from P15 to P21. Histograms represent mean ± SEM of n = 11 and 10 brains for control and dnTCF4, respectively, from at least two independent experiments (Student’s t test).
Controls in (C)–(H) are the same. ∗p < 0.05, ∗∗∗p < 0.001, and ∗∗∗∗p < Scale bars, 20 μm (A) and 50 μm (C, E, and G).
See also Figures S2 and S3.
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5. Figure 3
During Late Dendritogenesis, Wnt LOF Reduces Spine and Synapse Densities
(A) Timeline of the experiment. Confocal images of representative apical dendritic segments of P30 control cells and neurons expressing dnTCF4 from E21 to P7. Quantification of number of spines per micrometer. Histograms represent mean ± SEM of n = 6 and 4 brains for control and dnTCF4, respectively (control, 1,810 spines; dnTCF4, 721 spines), from two independent experiments (Student’s t test).
(B) Quantification of spine head width and length. Histograms represent mean ± SEM of n = 18 and 12 cells for control and dnTCF4, respectively (control, 299 spines; dnTCF4, 145 spines), from two independent experiments (Student’s t test).
(C) Timeline of the experiment. Confocal images of representative dendritic segments of P30 control cells and neurons expressing dnTCF4 from P21 to P30.
(D) Quantification of number of spines per micrometer. Histograms represent mean ± SEM of n = 6 brains/condition (control, 1,810 spines for apical dendrite and 1,110 spines for basal dendrites; dnTCF4, 1,356 spines for apical dendrite and 1,061 spines for basal dendrites) from two independent experiments (Student’s t test).
(E) Percentage of total spines on apical and basal dendrite classified as filopodia, thin, stubby, or mushroom. Histograms represent mean + SEM of n = 18 cells/condition (control, 371 spines for apical dendrite and 281 spines for basal dendrite; dnTCF4, 387 spines for apical dendrite and 393 spines for basal dendrite) from two independent experiments (two-way ANOVA followed by Bonferroni post-test).
(F) Timeline of the experiment. Confocal images of representative dendritic segments of P60 control cells and neurons expressing dnTCF4 from P21 to P30. Quantification of number of spines per micrometer. Histograms represent mean ± SEM of n = 5 brains/condition (control, 2,351 spines; dnTCF4, 970 spines) from two independent experiments (Student’s t test).
(G) Timeline of the experiment and plasmids used. Left panel: confocal images of representative dendritic segments of control cells and dnTCF4 neurons expressing PSD95.FingR-GFP at P30. Quantification of number of PSD95 puncta per micrometer. Histograms represent mean ± SEM of n = 6 brains/condition (control, 1,817 puncta; dnTCF4, 1,018 puncta) from two independent experiments (Student’s t test). Right panel: confocal images of representative dendritic segments of control cells and dnTCF4 neurons expressing GPHN.FingR-GFP at P30. Quantification of number of gephyrin puncta per micrometer. Histograms represent mean ± SEM of n = 6 brains/condition (control, 747 puncta; dnTCF4, 366 puncta) from two independent experiments (Student’s t test).
Controls in (A) and (D) are the same. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < Scale bars, 5 μm (A, C, F, and G).
See also Figure S4.
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6. Figure 4
Wnt Acts on Early Dendritic Development through Regulation of NT3 Transcription
(A) In situ hybridization for NT3 on coronal slices of P0, P3, P9, and P13 brains. neg, negative control.
(B) Timeline of the experiment. Neurolucida reconstructions of control cells and neurons expressing shNT3 from E21 to P7 analyzed at P7.
(C) Plasmid used. Quantification of total length and number of branchpoints in apical and basal dendrites of control and shNT3-expressing neurons at P7. Histograms represent mean ± SEM of n = 10 and 6 brains for control and shNT3, respectively, from at least two independent experiments (Student’s t test).
(D) Timeline of the experiment. Quantification of NT3 mRNA expression of dnTCF4-expressing neurons relative to control cells at P7. Histograms represent mean + SEM of n = 2 litters/condition (12 brains/condition in total) (Student’s t test).
(E) NT3 promoter and beginning of exon 1 sequences (3,710 nucleotides, National Center for Biotechnology Information [NCBI] GenBank: S ) contain eight potential TCF-LEF binding sites (red). +1 is the mRNA transcription start site. +271 AUG is NT3 protein start codon.
(F) Timeline of the experiment and plasmid used. Neurolucida reconstructions of control, dnTCF4-expressing neurons, and cells that co-express dnTCF4 and NT3 (dnTCF4+NT3) at P7.
(G) Quantification of total length and number of branchpoints in apical and basal dendrites of control, dnTCF4-expressing neurons, and cells that co-express dnTCF4 and NT3 (dnTCF4+NT3) at P7. Histograms represent mean ± SEM of n = 10, 4, and 10 brains for control, dnTCF4, and dnTCF4+NT3, respectively, from at least two independent experiments (one-way ANOVA followed by Bonferroni post-test).
∗p < 0.05, ∗∗p < 0.01, and ∗∗∗∗p < Scale bar, 200 μm (A) and 20 μm (B and E).
See also Figure S5.
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7. Figure 5
During Late Dendritogenesis, Spine Density Is Rescued by NT3 Overexpression
(A) In situ hybridization for NT3 on coronal slices of P21, P25, and P31 brains.
(B) Timeline of the experiment. Confocal images of representative dendritic segments of dnTCF4-expressing neurons and cells that co-express dnTCF4 and NT3 (dnTCF4+NT3) at P30.
(C) Quantification of number of spines per micrometer. Histograms represent mean ± SEM of n = 6 brains/condition (control, 1,810 spines for apical dendrite and 1,110 spines for basal dendrites; dnTCF4, 1,356 spines for apical dendrite and 1,061 spines for basal dendrites; dnTCF4+NT3, 1,839 spines for apical dendrite and 1,660 spines for basal dendrites) from two independent experiments (one-way ANOVA followed by Bonferroni post-test).
(D) Percentage of total spines on apical and basal dendrite classified as filopodia, thin, stubby, or mushroom. Histograms represent mean + SEM of n = 18 cells/condition (control, 371 spines for apical dendrite and 281 spines for basal dendrite; dnTCF4, 387 spines for apical dendrite and 393 spines for basal dendrite; dnTCF4+NT3, 404 spines for apical dendrite and 428 spines for basal dendrite), from two independent experiments (two-way ANOVA followed by Bonferroni post-test).
∗p < 0.05 and ∗∗∗∗p < Scale bar, 200 μm (A) and 5 μm (B).
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8. Figure 6
Wnt LOF during Early Development Reduces Activity-Dependent Transcription of Early Immediate Genes
(A) Timeline of the experiment and plasmid used.
(B) Coronal slices of P21 brains electroporated at E18 with either TOM/SAREdGFP or dnTCF4/SAREdGFP, with dnTCF4 expression from E21 to P7, showing decreased SARE expression in dnTCF4 cells. Quantification of SARE intensity/TOM intensity. Histograms represent mean ± SEM of n = 4 brains/condition (control, 943 cells; dnTCF4, 584 cells) from at least two independent experiments (Student’s t test).
(C) Coronal slices of P45 brains electroporated as in (B), showing decreased SARE expression in dnTCF4 cells. Quantification of SARE intensity/TOM intensity. Histograms represent mean ± SEM of n = 6 and 5 brains for control (715 cells) and dnTCF4 (443 cells), respectively, from at least two independent experiments (Student’s t test).
(D) Timeline of the experiment.
(E) Example traces of firing properties of control and dnTCF4 electroporated cells in response to depolarizing current injection at P21. Graph represents frequencies of action potentials in response to different depolarizing current injections. Data represent mean ± SEM of n = 12 and 9 cells for control and dnTCF4, respectively. No significant difference was measured between the two conditions (two-way ANOVA).
(F) Same as (E) at P45. Data represent mean ± SEM of n = 7 cells/condition. No significant difference was measured between control and dnTCF4 cells (two-way ANOVA).
∗p < 0.05 and ∗∗p < Scale bar, 50 μm (B and C).
See also Figure S6.
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9. Figure 7
Wnt LOF during Early Development Leads to Functional Deficits at Adult Age
(A) Timeline of behavioral experiments.
(B) Open field test showing higher distance traveled by dnTCF4 animals compared with control.
(C) Quantification of distance traveled every 150 s. n = 25 and 17 animals for control and dnTCF4, respectively (repeated-measures two-way ANOVA followed by Bonferroni post-test).
(D) Proportion of time spent immobile and mobile. n = 25 and 17 animals for control and dnTCF4, respectively (Student’s t test).
(E) Proportion of time spent in the internal and external zones. n = 25 and 17 animals for control and dnTCF4, respectively (Student’s t test).
(F) Barnes maze test showing spatial memory deficit in dnTCF4 animals.
(G) Timeline of Barnes maze test experiments.
(H) Schematic of Barnes maze and representative track plots of control and dnTCF4 animals in trial 4.
(I) Quantification of latency to find the escape cage. n = 24 and 18 animals for control and dnTCF4, respectively.
(J) Quantification of distance traveled in trial 1. n = 24 and 18 animals for control and dnTCF4, respectively (Student’s t test).
(K) Quantification of path efficiency. n = 24 and 18 animals for control and dnTCF4, respectively.
(L) Quantification of number of errors normalized on latency and percentage of time spent in the target quadrant as average of trial 2 and 3. n = 24 and 18 animals for control and dnTCF4, respectively (Student’s t test).
(M) Quantification of number of errors normalized on latency and percent of time spent in the target quadrant in trial 4. n = 24 and 18 animals for control and dnTCF4, respectively (Student’s t test).
∗p < 0.05 and ∗∗∗p <
See also Figure S7.
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