Figure 2.

NOVA2 Downregulation Affects Neurite Outgrowth In Vitro and Leads to Decreased Number of Inter-tecta Axonal Tracts in Zebrafish In Vivo

(A) NOVA2 downregulation using siRNA affects neurite outgrowth. Neuro2A cells were transfected with different combinations of NOVA2 constructs (wild-type WT, Val261Glyfs^∗135 alias Mut1 or p.Tyr231^∗) and siRNAs together with the GFP-reporter Venus. 48 h after treatment, cells were stained against GFP and tubulin and counterstained with DAPI. Histograms represent the percentage of transfected cells with either multiple similar sized processes (gray), a single neurite (purple), or round undifferentiated cells without processes (yellow). Data are represented as mean ± SEM. Two-way ANOVA with Tukey’s multiple comparison test. n = 3 independent experiments. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗∗p < 0.0001, gray asterisks for differences in cells with multiple processes and yellow asterisks for undifferentiated cells.

(B) Top panel: Representative images of dorsal views of control, wild-type, and Mut1 NOVA2 mRNA-injected larva at 4 days post-fertilization stained with anti-acetylated tubulin (AcTub). Bottom panel: In vivo complementation assay. Representative images of dorsal views of morpholino (MO)-injected, MO+WT RNA-injected, and MO+Mut1 RNA-injected larva at 4 days post-fertilization stained with anti-acetylated tubulin (AcTub).

(C) Boxplots of inter-tecta axonal tracts’ count after acetylated Tubulin staining of 4 dpf control larva and larva injected with 6 ng of nova1 morpholino (MO), 6 ng of nova1 MO+25 pg of WT NOVA2 mRNA, 6 ng of nova1 MO+25 pg of Mut1 NOVA2 mRNA, 50 pg of WT NOVA2 mRNA, 50 pg of Mut1 NOVA2 mRNA. A t test was performed between pairs of conditions. p value < 0.001 are indicated by ^∗∗∗. n.s.: non-significant. n: number of larvae per condition.