Fig. 3

Zebrafish mutants of ASD genes display brain size phenotypes

(A) Whole-brain volume mapping pipeline (STAR Methods).

(B) Eight-region zebrafish brain atlas derived from Thyme et al. (2019) and Randlett et al. (2015).^22,42

(C) Hierarchical clustering of regional brain volume measurements comparing mutant and background-matched wild-type fish. Each rectangle in the clustergram represents the signed -log₁₀-transformed p values from linear mixed models (LMM) (red, increased in mutant; blue, decreased in mutant). Regions with significant volume differences by LMM (p < 0.05) are outlined in yellow. p-values for homozygous and heterozygous mutants are shown in Figure S3A. For raw volume quantifications by region for all fish, see Table S3.

(D) tERK-immunostained brains of dyrk1aa^Δ77/Δ77dyrk1ab^Δ8/Δ8 and wild-type larvae at 6 dpf. Note the decrease in forebrain volume in mutants (arrowheads). Dorsal views. FB, forebrain; OT, optic tectum; CB, cerebellum. Scale bar, 50 μm.

(E) Regional brain volume differences in dyrk1aa^Δ77/Δ77dyrk1ab^Δ8/Δ8 (n = 18) and wild-type (n = 18) relative to the standard zebrafish reference brain ⁴² (dotted line). ^∗∗∗∗p < 0.0001, ^∗∗∗p < 0.001, ^∗p < 0.05 (one-way ANOVA).

(F) Voxel-wise Z score values representing brain volume differences in dyrk1aa^Δ77/Δ77dyrk1ab^Δ8/Δ8 versus background-matched wild-type larvae. Images shown from left to right represent sequential slices: axial views, top row, dorsal to ventral; sagittal views, bottom row, lateral to medial. Scale bar, Z score (red/yellow, increased in mutant; cyan/purple, decreased in mutant).

(G) Voxel-wise Z score values representing brain volume differences for the following lines: scn1lab^Δ44/Δ44, chd8^Δ5/Δ5, tbr1a^Δ64/Δ64tbr1b^Δ10/Δ10, kdm5ba^Δ17/Δ17kdm5bb^Δ14/Δ14, and cul3a^Δ7/Δ7cul3b^Δ20/+. Axial views, top row; sagittal views, bottom row. The horizontal green line in the sagittal view indicates the slice shown in the axial view. FB, forebrain; MHB, midbrain-hindbrain boundary. Scale bar, Z score (red/yellow, increased in mutant; cyan/purple, decreased in mutant). For the number of animals used in imaging experiments, see STAR Methods (average n = 23–26 per genotype in two to four independent clutches).

(H) Genotype-level p values for each brain region combined for all homozygous mutants and cul3a^Δ7/Δ7cul3b^Δ20/+, excluding dyrk1a, using Fisher’s method. Scale represents the -log₁₀-transformed combined p value. The forebrain (telencephalon) shows the most significant difference in volume (p = 8.32 × 10⁻⁶, Fisher’s combined probability test).