Dissecting the autism-associated 16p11.2 locus identifies multiple drivers in neuroanatomical phenotypes and unveils a male-specific role for the major vault protein

Abstract

Background: Using mouse genetic studies and systematic assessments of brain neuroanatomical phenotypes, we set out to identify which of the 30 genes causes brain defects at the autism-associated 16p11.2 locus.

Results: We show that multiple genes mapping to this region interact to regulate brain anatomy, with female mice exhibiting far fewer brain neuroanatomical phenotypes. In male mice, among the 13 genes associated with neuroanatomical defects (Mvp, Ppp4c, Zg16, Taok2, Slx1b, Maz, Fam57b, Bola2, Tbx6, Qprt, Spn, Hirip3, and Doc2a), Mvp is the top driver implicated in phenotypes pertaining to brain, cortex, hippocampus, ventricles, and corpus callosum sizes. The major vault protein (MVP), the main component of the vault organelle, is a conserved protein found in eukaryotic cells, yet its function is not understood. Here, we find MVP expression highly specific to the limbic system and show that Mvp regulates neuronal morphology, postnatally and specifically in males. We also recapitulate a previously reported genetic interaction and show that Mvp^+/-;Mapk3^+/- mice exhibit behavioral deficits, notably decreased anxiety-like traits detected in the elevated plus maze and open field paradigms.

Conclusions: Our study highlights multiple gene drivers in neuroanatomical phenotypes, interacting with each other through complex relationships. It also provides the first evidence for the involvement of the major vault protein in the regulation of brain size and neuroanatomy, specifically in male mice.

Keywords: Autism spectrum disorders; Brain anatomy; Major vault protein; Mouse genetic studies; Sex differences.

Fig. 1

Mouse neuroanatomical studies for the identification of NAP genes at the 16p11.2 autism-associated locus. A Schematic representation of the human 16p11.2 region showing gene content and order with genes that underwent mouse neuroanatomical studies in red. B At bregma + 0.98 mm and bregma − 1.34 mm, 22 and 45 parameters were measured, respectively. C These 67 parameters (listed in Additional file 2: Table S2) are grouped into five categories (brain size, ventricle, cortex, commissure, and subcortex) on the two coronal sections. D Bar plots of the percentage change of male Del/+ relative to male WT mice for each of the parameters measured. The colored region indicates the presence of a significant parameter at the 0.05 level. White indicates a p-value > 0.05. E Bar plots of percentage change of female Del/+ relative to female WT. F Left: representative brain image stained with Nissl-luxol of adult male WT and Del/+ mice showing the cortical layers of the somatosensory cortex at bregma − 1.34 mm. Right: histograms showing the thickness of the upper and lower layers of the somatosensory cortex in male Del/+ mice. G Histograms showing the thickness of the upper and lower layers of the cortex in female Del/+ mice. H Venn diagram illustrating NeuroAnatomical Phenotype (NAP) genes (genes whose mutations yield neuroanatomical phenotypes) in male mice positioned in each category. Phenotypic directionality is color-coded. Blue corresponds to a reduction in the size of the brain regions affected, red to increase, and yellow occurring of both. I, J Bar plots showing the number of NAPs per section (bregma + 0.98 mm in orange and bregma − 1.34 mm in blue) for each gene assessed for male (I) and female (J), respectively. Genes are listed on the x-axis and sorted according to the number of NAPs

Fig. 2

MVP expression is highly specific to the limbic system. A, B Mvp qRT-PCR expression in the liver (LIV), cerebellum (CRB), hippocampus (HP), and cortex (CTX), in male (A) and female (B) WT mice. Normalization was done using the Gnas/Hprt ratio. Plots are shown as mean ± SEM. Immunofluorescence of MVP (green) in CRB (C), ventral HP (D), and deep layers of the cingulate gyrus (Cg) (E). Arrowheads point to MVP-positive cells. AV, arbor vitae; gr/mole, granular/molecular layer of CRB; or/py, oriens/pyramidal layer of HP. F–O Selection of MVP immuno-histo-fluorescence (IHF) images on the coronal brain sections, each of 30 µm in thickness, performed throughout the entire brain from bregma + 1.41 mm to bregma − 7.67 mm. MVP presence was revealed using an anti-vault antibody (N2-B15) and secondary antibodies fused to Alexa488 fluorochrome (green). Dashed lines delineate regions with MVP-positive cells (white), MVP-positive tract (yellow), and MVP-negative cells (gray). It is noteworthy to mention that the experiment was repeated on a new set of brain samples on sagittal orientation, ranging from lateral + 0.24 mm to lateral + 0.84mm. Quantitative analysis both on the coronal and sagittal sections was done, and MVP expression was scored as mild (+), moderate (++), or strong (+++) across all the positive brain regions. The complete analysis is available in Additional file 2: Tables S3-S4. Pir, piriform cortex; MS, medial septum; DBB, diagonal band of Broca; TS, triangular septal nucleus; ZI, zona incerta; RM, retromammillary nucleus; py/mol, pyramidal/molecular layer of the hippocampus (HP); DG, dentate gyrus of the hippocampus; 4V, fourth ventricle; CP, choroid plexus; MVe, medial vestibular nucleus; Sol, solitary nucleus; AP, area postrema; 10N, vagus nucleus; Raphe, raphe magnus nucleus. P, Q IHF of MVP, NeuN neurons, and Hoechst on the brain sections from male (P) and female (Q) WT mice. The dashed square delineates the region presented at a higher magnification on individual channels for MVP, NeuN, and Hoechst, and merge of the three. In all structures, the MVP signal came from the cytoplasm

Fig. 3

Validation and phenotyping of Mvp-deficient mice. A Schematic representation of Mvp knockout model construction. The knockout mutants were obtained by a promoter gene trap design, resulting in the insertion of a β-galactosidase/neomycin phosphotransferase (β-GEO) cassette within the first intron of the Mvp gene. The polyA-tail (pA) serves to end the transcription and thus discard the transcription of Mvp gene. B Validation of Mvp knockout at the protein level using Western blot (WB) from liver extracts of Mvp^+/+ (n = 2), Mvp^+/− (n = 2), and Mvp^−/− (n = 3) mice. Actin was used as a loading control for normalization. For quantification, see Additional file 1: Fig. S6D. C Viability screen of Mvp knockout mice. Left: schematic representation of heterozygous-by-heterozygous breeding scheme, with expected Mendelian ratio of segregation of the offspring. Right: graph represents the ratio of each genotype of successfully genotyped mice obtained from heterozygous-by-heterozygous breeding (n = 617). D, E Graph showing the total brain area (1_TBA) measured at four time points, embryonic age 18.5 (E18.5), postnatal day 10 (P10), P45, and P120, in male (D) and female (E) Mvp^−/− mice (n = 5–7 in each group). F Half images of the coronal brain sections stained with Nissl-luxol at the four time points from male Mvp^+/+ and Mvp^−/− mice. Plots are shown as mean ± SEM. Two-tailed Student t test equal variance (D, E). *p < 0.05, **p < 0.01

Fig. 4

Involvement of the major vault protein in male-specific neuroanatomical phenotypes. A, B Left: schematic representation of the 24 brain regions assessed at bregma + 0.98 mm and − 1.34 mm in male Mvp^+/− (A) and male Mvp^−/− (B) mice at P120. Colored regions indicate the presence of at least one significant parameter within the brain region at the 0.05 level. White indicates a p-value > 0.05, and gray shows not enough data to calculate a p-value. Right: histograms of percentage change relative to Mvp^+/+ for each of the 42 parameters (see Additional file 2: Table S2). C, D Left: coronal sections of Cg (C) and somatosensory cortex S2 (D) from Mvp^+/+ and Mvp^−/− at P120. Right: cell count and average cell area measures within Cg and S2. E Left: schematic representation of the 22 brain regions quantified at lateral + 0.60 mm on the parasagittal section from Mvp^+/+ and Mvp^−/−. Colored regions indicate the presence of at least one significant parameter within the brain region at the 0.05 level. White indicates a p-value > 0.05, and gray shows not enough data to calculate a p-value. Right: histograms of percentage change relative to Mvp^+/+ (set as 0) for each of the measured parameters (listed in Additional file 2: Table S6). F Left: schematic representation of a hippocampal neuron in culture and the different measures taken. Middle: neurons stained with MAP2 (red) and SMI312 (green). Right: graphs showing measures of soma area, growth cone area, and axonal length from hippocampal neurons derived from multiple male Mvp^+/+ and Mvp^−/− embryos. G Graphs showing measures of soma area, growth cone area, and axonal length from hippocampal neurons derived from multiple female Mvp^+/+ and Mvp^−/− embryos (n > 3 embryos per group, Additional file 5: Table S9). Plots are shown as mean ± SEM (except for A, B, and E). Two-tailed Student t test equal variance (C, D, F, and G). *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 5

Behavioral analysis of Mvp and Mapk3 genes. Selection of behavioral paradigms among 17 tested in 12 Mvp^+/+;Mapk3^+/+ and 12 Mvp^+/−;Mapk3^+/− mice, from 11 to 25 weeks of age. Elevated plus maze (A). Open field (B). Fear conditioning (C). Tail suspension (D). PTZ test (E). Forced swim (F). G Summary table of core functions assessed in various cohorts. Green indicates significance, gray no difference, and white indicates not done (not d.). Arrows indicate the directionality of the effect, and the equal signs indicate no changes. A comprehensive description of behavioral tests, raw datasets, and additional findings are provided in Additional file 9, Additional file 6: Tables S10, Additional file 7: Tables S11, Additional file 8: Tables S12, and Additional file 1: Figs. S10-S12, respectively. The mean and standard error of the mean are shown in the graphs. Two-tailed Student t test equal variance (A, D–F) and two-way ANOVA with Sidak post hoc (B, C). *p < 0.05, **p < 0.01, ***p < 0.001