Generation and validation of murine Wac and zebrafish waca mutants.

(A) Floxed mouse Wac genetic locus and genotyping primers. Genotyping primers (magenta lines) reside external to the loxP sites (orange triangles) flanking exon 5 and nearby introns. (B) The recombined DNA band was Sanger-sequenced and results show the start codon (blue) and the novel stop codon introduced by the frameshift mutation resulting from Cre-mediated deletion of the locus used to generate the locus. (C) Example images of adult WT and Wac Het mice. (D) DNA gel of genotyping for WT and recombined (Het) Wac alleles. (E) waca KO zebrafish were generated by CRISPR/Cas9. KO target sites for sgRNAs are indicated by red arrowheads and predicted protein structures for KO mutations indicated below panel. (F) WT and waca KO zebrafish have grossly normal sizes but waca KO exhibited a shortened jaw (black triangle) at 5 dpf. (G) example waca genotyping results. Abbreviations: (aa) amino acid; (bp) base pair; (Chr) chromosome; (dpf) days post fertilization; (WT) wild type. Scale bars (C) = 1 cm and (F) = 200 µm.

Craniofacial changes in mutants.

(A, B) Dorsal views of the P0 calvaria stained with Alizarin red for bone. (A’, B’) The suture and fontanel areas in (A) and (B) are pseudo-colored green (anterior) and orange (posterior). (C, D) Dorsal views of P30 WT and Wac Het skull bones; arrow (D) points to a gap in the interfrontal suture. (F) Frontal bone and (P) Parietal bone; white dashed lines are widths measured in (G, H). (E, F) Quantification of P0 fontanel suture areas pseudo colored in A’ and B’; WT n = 4, Het n = 5. Quantification of the skull width across the frontal (G) and parietal (H) bones at P30 in WTs and Wac Hets; WT n = 4, Het n = 4. (I, J) The waca KO zebrafish show a shortened jaw structure, compared to WT. Lines denote lower jaw length. (K) Quantification of lower jaw length, data expressed as the mean ± SEM; n = 9 WT, 11 waca Het and 4 waca KO, **p < 0.01. (L, M) Cartilage staining of zebrafish using alcian blue, ventral view. (N) Measurements of the width of Meckel’s cartilage in waca KO zebrafish at 13 dpf (red line); WT n=10, KO n = 8. Data are expressed as the mean ± SEM. *p < 0.05 and ***p < 0.001. Scale bars (B’) = 1 mm, (D) = 4 mm, (J, M) = 200 µm.

Behavioral changes in mutants.

(A) 6-8 week WT and Wac Het mice were tested in the 3-chamber social/novel object test, WT n = 16 and Het n = 15, and the Y-maze (B), n = 13 both groups, with Wac Hets showing deficits in each. (C, D) Example heat maps during the 17-19-minute timeframe of the social cohesion test in zebrafish; waca KO males and females were more dispersed (E, F), see methods for test details. (G, H) Distance moved was greater in male waca KOs in the novel tank assay, n = 11, both groups, compared to females, n = 12 (WT) and 11 (KOs). Data are expressed as the mean ± SEM; *p < 0.05, **p < 0.01 and ***p < 0.001.

Murine Wac depletion leads to elevated seizure susceptibility and loss of GABAergic markers.

(A) Schema depicting the seizure induction by PTZ. Briefly, P30 mice were administered PTZ intraperitoneally and then assessed for the highest seizure severity score over the course of 20 minutes. The maximum seizure severity score over 20 minutes was quantified (B); n = 16 (WTs) and 22 (Hets). (C) Table of cortical interneuron cell counts in the somatosensory cortex at P30. (D-F) Immunofluorescent images of PV and cell density quantification in mice somatosensory cortex, n = 3, both groups. (G-I) Immunofluorescent images of LHX6 and cell density quantification in mice somatosensory cortex, n = 3, both groups. Data are expressed as the mean ± SEM, *p < 0.05 and ****p < 0.0001. Scale bar = 100µm (H).

Cell counts and biochemical assessment of key markers in P30 Wac WT and Het mice.

(A) WT and Wac Het somatosensory cortices were counted for major cell classes, including markers of neurons and glia, n = 3 each group, parentheses indicate Standard error of the mean. (B) P30 somatosensory cortex tissue was probed via western blotting for WAC and proteins known to be altered in other models or of brain markers. (C) Quantification of protein bands were normalized to GAPDH or the total protein if a phosphorylated protein. Data are expressed as the mean ± SEM, n = 4 all groups.* p < 0.05 and ** p < 0.01. Abbreviations: (WB) western blot and (kDa) kiloDaltons.

MRI reveals increased brain volume in mouse Wac Het males.

(A) There was a significant increase in whole brain volumes in HET compared to WT male mice. Female HET mice did not exhibit significant changes in brain volume relative to WT. Data are expressed as the mean ± SEM; *p < 0.05. (B) %change between cortical regions were larger in most HET mice areas, with distinct regions different between sexes. (Bolded bars indicate significant differences p<0.05, t-test).

Transcriptomic characterization by bulk RNA-seq.

(A) Volcano plot representing DE of the SVA-corrected DE model in (A). Labeled genes pass FDR < 0.1. (B) Box plots of RPKM expression values of significantly DE genes belonging to enriched GO terms analyzed. Triangles represent female, circles represent male samples.