Fig. S2

Related to Figure 1. Inhibitory and excitatory neurons in cntnap2ab mutants. Immunostainings of reporter gene expression in Tg(dlx6a-1.4kbdlx5a/dlx6a:GFP) and Tg(vglut:DsRed) in wild-type

(A-H) and cntnap2a^Δ121/Δ121cntnap2b^31i/31i (A′-H′) larvae at 4 dpf. (A-A′, E-E′): The deficit in GABAergic neurons and precursors (dlx5a6a:GFP+ cells) is evident in the ventral and dorsal telencephalon (tel), hypothalamus (hyp), and cerebellum (CB) (arrowheads in A-A′, E-E′). The reduction in GABAergic neurons by surface area of acetylated tubulin is significant in the forebrain and cerebellum (J).

I-K. dlx5a6a:GFP+ cells, vglut:DsRed and acetylated tubulin stainings were quantified by brain region at 4 dpf: forebrain (small brackets in D-D′, H-H′); hypothalamus (large brackets in D-D′, H-H′); cerebellum (arrowheads in E-E′, H-H′); and optic tectum (arrowheads in E-E′, H-H′). (forebrain and hypothalamus: wild-type, n=9; cntnap2ab, n=9; optic tectum and cerebellum: wild-type, n=7; cntnap2ab, n=8)

I. dlx5a6a:GFP+ cell number normalized to wild-type. (*p<0.001; **p<0.01; ***p<0.02; one-way ANOVA, Bonferroni corrected).

J. dlx5a6a:GFP+ cell number relative to acetylated tubulin surface area normalized to wild-type. Because there are significant reductions in GABAergic neurons relative to area in the forebrain and cerebellum, we concluded that the GABAergic deficits in these regions are not primarily attributable to the reduction in brain size. (*p<0.02; one-way ANOVA, Bonferroni corrected).

K. vglut:DsRed surface area relative to acetylated tubulin surface area normalized to wild-type. Unlike GABAergic neurons, there are no significant reductions in glutamatergic surface area relative to acetylated tubulin surface area across regions, suggesting these differences are due to the overall reduction in brain size as opposed to regional deficits (p=0.225, forebrain; p=0.444, hypothalamus; p=0.408, optic tectum; p=0.490, cerebellum; one-way ANOVA).

L-S. Analysis of the CNS of wild-type (L-S) and cntnap2a^Δ121/Δ121cntnap2b^31i/31i (cntnap2ab) (L′-S′) by in situ hybridization at 48 hpf: (L, L′): glutamate decarboxylase 1b (gad1b); (M, M′): vesicular glutamate transporter or solute carrier family 17, member 6b (vglut2a or slc17a6b); (N, N′): solute carrier family 6 (neurotransmitter transporter, glycine), member 5 (glyt2 or slc6a5); (O, O′): tyrosine hydroxylase (th1 or th); (P, P′): neuronal differentiation 1 (neurod or neurod1); (Q, Q′); transiently expressed axonal glycoprotein (tag1) or contactin 2 (cntn2); (R, R′): TUNEL staining at 28 hpf; (S, S′): phospho-histone H3 (H3P) (green) and acetylated tubulin (blue) immunostaining at 48 hpf. We observed that expression of gad1b was variable. No gross differences in the other neurotransmitter systems, markers of neuronal differentiation, apoptosis, or cell proliferation were observed.

(A-D, A′-D′, E-H, E′-H′) show all channels for the images in Figure 1C-F and 1C′-F′, respectively. (A-D, A′-D′): ventral views; (E-H, E′-H′, L-N, L′-N′; P-P′, R-R′, S-S′):): lateral views; (O-O′, Q-Q′): dorsal views. (tel, telencephalon; hyp, hypothalamus; OT, optic tectum; CB: cerebellum; HB, hindbrain; E, eye.)