FIGURE 7

FIGURE 7

Measurements of aberrant neuron branching and apoptosis activation in zebrafish injected with CCNF gene variants S621G, S195R, R574Q, and wild type. (A) Blinded analysis of the number of aberrant branches in zebrafish expressing cyclin F S621G and S195R, respectively, had 2.01-fold (p-value = 0.006, n = 42) and 2.4-fold (p-value = 0.0011, n = 46) more aberrantly branched motor neurons compared to the wild type (one-way ANOVA). Region of interest (ROI) in zebrafish used for imaging. (B) Representative images of zebrafish expressing blue fluorescent protein in motor neurons used to analyse primary motor axon branching. Examples of aberrant branching indicated by red arrows. (C) Cyclin F S621G and S195R had displayed increased cleaved caspase-3 activity by 2.08-fold (n = 39, p-value = 0.0029 and 2.79-fold (n = 28, p-value = 0.0004) (one-way ANOVA adjusted with Kruskal–Wallis test). (D) Representative images of live zebrafish staining with AO. (E) AO positive cells in S621G and S195R were 1.4-fold (p-value = 0.0496, n = 37) and 1.45-fold (p-value = 0.0115, n = 57) higher, respectively, compared to the wild type (one-way ANOVA). (F) Representative images of zebrafish immunostained with cleaved caspase-3 primary antibody. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.