Dixdc1KO Behavioral Assay Results and Sequence-disrupting SNVs in ASD, BD, Scz

1. Supplementary Table 1. Behavioral assay results in Dixdc1KO mice (CD-1 genetic background)

2. Supplementary Table 2. Sequence-disrupting SNVs (allele frequency <0.1% in ExAC) in DIXDC1 isoform 1 in ASD, BD, Scz. Singleton SNVs (unique SNVs not found in ExAC) are indicated by an *. Codon numbering is from the ATG for isoform 1. SNVs in the region common to isoform 1 and 2 are highlighted in purple.

3. Supplementary Table 2 (continued). Sequence-disrupting SNVs (allele frequency <0.1% in ExAC) in DIXDC1 isoform 1 in ASD, BD, Scz. Singleton SNVs (unique SNVs not found in ExAC) are indicated by an *. Codon numbering is from the ATG for isoform 1. SNVs in the region common to isoform 1 and 2 are highlighted in purple.

4. Supplementary Table 3. Sequence disrupting SNVs (allele frequency <0.1% in ExAC) in DIXDC1 isoform 2 in ASD, BD, Scz. Singleton SNVs (unique SNVs not found in ExAC) are indicated by an *. Codon numbering is from the ATG for isoform 2. SNVs in the region common to isoform 1 and 2 are highlighted in purple.

5. b c d a KO WT WT KO WT KO WT KO isoform 1 isoform 2 IgG IgG Tle4 Cux2 IP: - α Dixdc1 e WT KO g f WT KO WT KO Ctip2/Tbr1 Ctip2/Tbr1 h WT KO WT KO Prox1 Prox1 Supplementary Figure 1. (a) Immunoblot for Dixdc1 in neonatal WT and Dixdc1KO forebrain lysates immunoprecipitated (IP) with anti-Dixdc1 antibody. In WT (middle) both Dixdc1 isoform 1 (top band) and 2 (bottom band) are detected; in Dixdc1KO neither isoform is detected (right). No-antibody control (left ) performed in parallel with no antibody added to the IP. (b) Neonatal brains; left WT, right Dixdc1KO. Scale bar, 1mm. (c-d) in situ hybridization for Tle4 (c) and Cux2 (d), neonatal coronal brain sections. Scale bar, 1mm (e) Nissl staining of P30 coronal brain sections. Scale bar, 1mm. (f,g) Late embryonic (embryonic day 18.5) coronal brain sections immunostained with antibodies for CTIP2 (green) and Tbr1 (red) for neurons migrating to cortical L5/L6. Scale bar in (f), 500µm. Scale bar in (g), 150µm. (h) P0 coronal brain sections showing thalamic neurons immunolabeled with Prox1. Scale bar, 1mm.

6. Supplementary Figure 2. Depression-like behavior in the Tail Suspension Test. Dixdc1-/+ (HET) and -/- (KO) mice spend increased time immobile compared to +/+ (WT) littermates. *p≤0.05

7. c a b hippocampus cortex WT KO d e GFP/Gephyrin/VGAT WT in vitro KO f g GFP/Gephyrin/VGAT WT in vivo KO Supplementary Figure 3. Normal dendrite complexity and inhibitory synapse density in Dixdc1KO neurons (outbred mixed genetic background except as noted (blue) in c). (a) GFP expressingculturedhippocampal pyramidal neurons (DIV18). Scale bar=30µm. (b, c) Sholl analysis of cultured hippocampal (b) and cortical (c) pyramidal neurons. [blue = modestly reduced dendrite complexity of Dixdc1KO cortical neuronsin an isogenic C57Bl/6 background. (WT C57Bl/6 not significantly different than WT outbred, not shown.) All phenotypes reported in this article were significant in littermates from the mixed outbred (CD-1) genetic background – see Materials and Methods.] (d) Representative micrographs of cultured hippocampal neurons (DIV18) immunolabeled for GFP (dendrite), VGAT (inhibitory presynapse) and gephyrin (inhibitory postsynapse). Scale bar, 5µm. (e) Quantification of inhibitory (GABAergic) synapses (co-localized GFP, VGAT and gephyrin). (f) Representative micrographs (P30 cortex) immunolabeled for GFP (dendrite), VGAT (inhibitory presynapse) and gephyrin (inhibitory postsynapse). Scale bar, 5µm. (g) Quantification of inhibitory (GABAergic) synapses (co-localized GFP, VGAT and gephyrin).

8. * * ** Supplementary Figure 4. Withdrawal of lithium leads to behavioral relapse in the FST. Increased time immobile in Dixdc1KO mice is rescued by systemic injection of lithium (middle bars), compared to vehicle injected animals (left bars). Withdrawal of lithium for 14 days (right bars) leads to behavioral relapse. * p≤0.05; ** p≤0.01

9. b a isoform 1 isoform 2 Supplementary Figure 5. Increased burden of rare missense, nonsense and splice-disrupting SNVs in ASD from both the discovery (AASC) and replication (ASC+SSC) datasets in isoform 1 (a) and isoform 2 (b).