Prenatal β-catenin/Brn2/Tbr2 transcriptional cascade regulates adult social and stereotypic behaviors

Abstract

Social interaction is a fundamental behavior in all animal species, but the developmental timing of the social neural circuit formation and the cellular and molecular mechanisms governing its formation are poorly understood. We generated a mouse model with mutations in two Disheveled genes, Dvl1 and Dvl3, that displays adult social and repetitive behavioral abnormalities associated with transient embryonic brain enlargement during deep layer cortical neuron formation. These phenotypes were mediated by the embryonic expansion of basal neural progenitor cells (NPCs) via deregulation of a β-catenin/Brn2/Tbr2 transcriptional cascade. Transient pharmacological activation of the canonical Wnt pathway during this period of early corticogenesis rescued the β-catenin/Brn2/Tbr2 transcriptional cascade and the embryonic brain phenotypes. Remarkably, this embryonic treatment prevented adult behavioral deficits and partially rescued abnormal brain structure in Dvl mutant mice. Our findings define a mechanism that links fetal brain development and adult behavior, demonstrating a fetal origin for social and repetitive behavior deficits seen in disorders such as autism.

Figure 1. Dvl1^−/−3^+/− mice display social deficits and embryonic brain enlargement

WT, Dvl1^−/− and Dvl1^−/−3^+/− mice behavioral phenotypes were determined in: (a) Whisker trimming (n≥11) (*p<0.0001), (b) nest building (n≥5) (*p<0.04), (c) three chamber social approach (n≥9) (*p<0.01) and (d) marble burying task (n≥8) (*p<0.02). e, WT, Dvl1^−/− and Dvl1^−/−3^+/− embryonic heads (E12.5–E13.5) or brains (E14.5-P0) were dissected, weighed and quantified as a percentage of the average WT brain weight for the indicated time point (mean±SEM, n≥5, ANOVA<0.001; *p<0.04 for comparison of the Dvl1^−/−3^+/− brains with those from WT). f, Coronal sections of WT, Dvl1^−/− and Dvl1^−/−3^+/− were Nissl stained and representative images of the neocortex are shown at E14.5 (scale bar: 250; 100μm). Nissl stained images were quantified as percentage of the average width of WT total neocortex (g), ventricular zone (h) and cortical plate (i) for the indicated time points. Results are presented as mean±SEM (n≥4, ANOVA<0.02; *p<0.02 for comparison of the brains from Dvl1^−/−3^+/− and Dvl1^−/− mutants with those of WT).

Figure 2. Early expansion of basal progenitors in Dvl1^−/−3^+/− embryos

a, Coronal sections from WT, Dvl1^−/− and Dvl1^−/−3^+/− embryonic brains were immunostained with anti-Sox2 (Red) and anti-Tbr2 (Green) (scale bar: 100μm). Quantification of the single and double labeled cells was performed and results are presented as mean±SEM (n≥4). b, Sox2 single labeled cells (ANOVA<0.01; *p<0.01 for comparing the results of the Dvl1^−/−3^+/− with those of all the mouse groups); c, Tbr2 single labeled cells and d, Sox2⁺Tbr2⁺ double labeled cells. (ANOVA<0.02; *p<0.002 for comparing the results of the Dvl mutants with those of WT mouse groups).

Figure 3. Early differentiation of deep layer neurons in Dvl1^−/−3^+/− embryos

a, WT and Dvl mutant embryonic brain sections were immunostained with DAPI (Blue), anti-Ctip2 (Green) and anti-Cux1 (Red) (Scale bar: 200μm), Representative images of the staining are shown. b, Quantification of the percentage of Ctip2 labeled cells are presented as mean±SEM (n≥4, ANOVA<0.007; *p<0.01 for comparing the results of the Dvl1^−/−3^+/− with those of the WT mice). c, WT and Dvl embryonic brain sections were immunostained with DAPI (Blue), anti-NeuN (Green) and representative images of the staining are shown. (Scale bar: 200μm). d–e, Coronal sections of WT and Dvl embryonic brains at E18.5 were immunostained with DAPI (Blue), anti-FoxP2 (Green) (d) and anti-Brn2 (Red) (e). Representative images of the staining are shown. (Scale bar: 200μm). f–h, Quantification of the percentage of Brn2⁺ neuron in layer V (ANOVA<0.04) (f), layers II–III (ANOVA<0.003) (g) and VZ/SVZ (ANOVA<0.009) (h), results are presented as mean±SEM of n=3–5 per group (**p<0.01 and *p<0.05 for comparing the results of the Dvl mutants with those of the WT). Black, green, blue and red bars represent WT, Dvl3^+/−, Dvl1^−/− and Dvl1^−/−3^+/− genotypes, respectively.

Figure 4. β-catenin/Brn2/Tbr2 transcriptional cascade directly regulates NPCs proliferation

WT and Dvl1^−/−3^+/− brains at E16.5 were double-labeled with DAPI (blue) and anti-Brn2 (red) and representative images are presented (Scale bar: 100μm) (a) and in WT and Dvl1^−/−3^+/− NPCs (Scale bar: 100μm) (b). c, Brn2 labeled NPCs were quantified as the percentage of total cells counted (DAPI). Results are presented as mean±SEM (n≥3, *p<0.01 for comparing the percentage of Brn2⁺ cells in the Dvl1^−/−3^+/− NPCs with those of the WT NPCs). d, WT and Dvl1^−/−3^+/− NPCs were transfected with TOP-Flash and fire-fly renilla reporters and treated with either 5mM LiCl or 100ng/ml Wnt3A. Results are presented as mean±SEM (n≥3, ANOVA<0.001; *p<0.02 and **p<0.002 for comparing the luminescence ratio in the Dvl1^−/−3^+/− NPCs with those of the WT NPCs). e, WT and Dvl1^−/−3^+/− NPC treated with 5mM LiCl were immunostained for Brn2 and representative images are presented (Scale bar: 100μm). f, Schematic Image of the promoter region of Brn2, the predicted region for TCF binding is marked by red rectangle. g, At E14.5 WT and Dvl1^−/−3^+/− cortexes were isolated and β-catenin ChIP was preformed followed by qPCR analysis for the Axin2 intron1 and the predicted region on the Brn2 promoter. (mean±SEM, n≥4, *p<0.04 for comparing the enrichment in the Dvl1^−/−3^+/− cortical cells with those of the WT cortical cells). h, Schematic image of the promoter region of Tbr2, the red rectangle indicates the predicted regions for Brn2 binding. i, At E14.5 WT and Dvl1^−/−3^+/− cortexes were isolated and Brn2 ChIP was performed followed by qPCR analysis for the nestin intron2 and the predicted binding sites on the Tbr2 promoter. Results are presented as fold enrichment of the Brn2 signal above the IgG control (mean±SEM, n≥4, *p<0.04, **p<0.003, for comparing the enrichment in the Dvl1^−/−3^+/− cortical cells with those of the WT cortical cells). j, WT and Dvl1^−/−3^+/− NPCs grown as monolayer cultures were transfected with pCAG-Brn2. NPCs were immunostained for DAPI, Brn2 and Tbr2. Representative images are presented (Scale bar: 100μm). k, pCAG-Brn2 transfected WT and Dvl1^−/−3^+/− NPCs were immunostained for DAPI and DAPI⁺ nuclei were counted. Results are presented as mean±SEM (n≥4, ANOVA<0.04 *p<0.05 for comparing the results of the Dvl1^−/−3^+/− with those of the WT and the Brn2 transfected Dvl1^−/−3^+/− NPCs groups). l, pCAG-Brn2 transfected WT and Dvl1^−/−3^+/− NPCs were immunostained for DAPI, Brn2 and Tbr2. The percentage of Brn2⁺ and Tbr2⁺ cells was measured. Results are presented as mean±SEM (n≥4, AVOVA<0.04 *p<0.03, comparing the results of the GFP transfected Dvl1^−/−3^+/− with those of the WT and the Brn2 transfected Dvl1^−/−3^+/− NPCs). m, WT and Brn2^−/− brain coronal sections from E14.5 were immunostained with anti-Sox2 (Red) and anti-Tbr2 (Green) and representative images are presented (scale bar: 100μm). WT and Brn2^−/− brain coronal sections from E14.5 were immunostained with anti-Ctip2 (Green) and representative images are presented (scale bar: 100μm). n, The number of Sox2⁺, Tbr2⁺ and Sox2⁺Tbr2⁺ labeled cells were quantified and the results are presented as mean±SEM (n≥4, ANOVA<0.04; *p<0.001 for comparing the results of the Brn2 mutants with those of the WT embryos). o, Nest building activity in WT and Brn2^+/− cages was assessed and measurements of the nest depth are presented as mean±SEM (n≥4, *p<0.003 for comparing the results of the Brn2 mutants with those of the WT cages). p, WT and Brn2^+/− mice were assessed in the social approach task and scored, results are presented as mean±SEM (*p<0.002 for comparing the results of the time spent sniffing the mouse compared with the object). q, The marble burying task was scored and the results are presented as mean±SEM (*p<0.02 for comparing the results of the Brn2^+/− mutants with those of the WT mice).

Figure 5. Embryonic activation of Wnt canonical pathway regulates Dvl1^−/−3^+/− embryonic brain phenotype

a, WT and Dvl mutant brains at E14.5 following CHIR99021 treatment were Nissl stained and representative images are presented (scale bar: 100μm). Nissl stained images were quantified as a percentage of the average width of WT total neocortex (ANOVA<0.02) (b), ventricular zone (c) and cortical plate (ANOVA<0.001) (d). Results are presented as mean±SEM (n≥4, ANOVA<0.01; *p<0.02 for comparison of the brains from Dvl1^−/−3^+/− mock treated with those of WT mock treated embryos). e, Coronal sections of WT and Dvl mutant mock and CHIR99021 treated embryo brains at E14.5 were double label with anti-Sox2 (Red) and anti-Tbr2 (Green) and representative images are presented (scale bar: 100μm). Quantification presented as mean±SEM of Sox2 single labeled cells (f); Tbr2 single labeled cells (g) (n≥4, ANOVA<0.05 *p<0.03 for comparing the results of the WT mock and CHIR99021 treated embryos) and Sox2⁺Tbr2⁺ double labeled cells (h) (ANOVA<0.006; *p<0.01 for comparing the results of the Dvl1^−/− and Dvl1^−/−3^+/− mock treated with those of the other embryo genotypes). (i) WT and Dvl1^−/−3^+/− brains at E14.5 from mock or CHIR99021 treated embryos were immunostained with DAPI (Blue) and anti-Ctip2 (Green) and representative images are presented (Scale bar: 200μm). j, Quantification of Ctip2⁺ cells (mean±SEM) are presented as percentage of the average number of Ctip2⁺ cells from WT mock treated embryos (n≥4, ANOVA<0.02; *p<0.01 for comparing the results of the Dvl1^−/−3^+/− mock treated with those of the Dvl1^−/−3^+/− CHIR99021 treated and WT embryos).

Figure 6. Prenatal Wnt canonical pathway regulates adult social behavior

WT and Dvl mutant (Dvl1^−/− and/or Dvl1^−/−3^+/−) adult mice treated with CHIR99021 or mock treated only as embryos (E9.5–14.5) were tested in behaviors found to be abnormal in the Dvl mutants. a, Nest depth was measured after 24hr and the recorded depth are presented as mean±SEM (n≥3, ANOVA<0.002; *p<0.04 for comparing the results of the Dvl1^−/− and Dvl1^−/−3^+/− mock treated mice with those of the other mouse groups). b, The social approach task was recorded, scored and the results are presented as mean±SEM (*p<0.02 for comparing the results of the time spent sniffing the mouse compared with the object). c, The marble burying task was scored and the results are presented as mean±SEM (ANOVA<0.001 ;*p<0.03 for comparing the results of the Dvl1^−/−3^+/− mock treated and WT CHIR99021 treated mice with those of the other mouse). Total activity in the open field task (in meters distance traveled, d) the time spent in the center of the arena relative to their total activity (e) in the open field task are presented as mean±SEM. There were no significant genotypic differences in these tasks. WT and Dvl1^−/− mock and CHIR mice treated in utero between E9.5–14.5 (n≥9) were imaged using an MRI separately, with both a T2-weighted anatomical scan and a subsequent Diffusion Tensor Imaging (DTI) scan. (f) Total brain volume (mm³) for the four groups are presented as mean±SEM (*p<0.0004 for comparison of the results from Dvl1^−/− with those of WT brains). g, Voxel-wise analysis highlighting significant relative volume differences (false discovery rate, FDR) throughout the brain between the mock treated Dvl1^−/− and WT mice (h) Fractional anisotropy (FA) analysis of significant differences (FDR) throughout the brain between the mock treated Dvl1^−/− and WT mice. i, Voxel-wise analysis of significant relative volume differences (FDR) throughout the brain between the Dvl1^−/− CHIR and WT mock treated mice.