Disrupting ythdf2 in zebrafish is associated with head and brain size phenotypes. (A) Copy‐number‐estimate plot (QuickMer2) using sequencing data from the APP proband harboring a de novo 109‐kb duplication on chromosome 1 compared with their parents harboring two diploid copies. (B) IGV plot showing discordant reads in the APP proband supporting a tandem duplication. (C) An illustration of the tandem duplication on chromosome 1 in an APP proband encompassing GMEB1 and all but the last exon of YTHDF2. (D) Cartoon depicting the CRISPR‐based knockdown (KD) and overexpression using in vitro transcribed mRNAs (mRNA) experimental paradigms by injection of nucleic acid into single‐cell zebrafish embryos. (E) Morphometric measurements were produced using VAST platform images and automated feature extraction via FishInspector (Teixidó et al. 2019) of body length, distance between the eyes, telencephalon width, and head‐trunk angle. (F) Features were quantified in 3 dpf larvae by comparing ythdf2 knockdown (KD, n = 37) versus scrambled gRNA controls (Cont., n = 34) and YTHDF2 overexpression (mRNA, n = 55) versus injection controls (Cont., n = 55) (top). Similar comparisons were made for gmeb1 KD (n = 33) versus scrambled gRNA controls (n = 34) and GMEB1 mRNA (n = 26) vs. injection controls (n = 26) (bottom). (G) Knockdown and mRNA injected zebrafish harboring a pan neuronal marker (HuC:eGFP) reveal brain size differences at 3 dpf. Representative control, knockdown, and mRNA injected zebrafish images of transgenic larvae. Scale bar is 100 μm. (H) ythdf2 knockdown embryos show significantly decreased midbrain volume (Wilcoxon t test, p value = 0.006). YTHDF2 mRNA‐injected embryos show both significantly increased midbrain (Wilcoxon t test, p value = 0.014) and forebrain (Wilcoxon t test, p value = 0.001). All p values are adjusted for multiple‐testing using Bonferroni correction and only significant comparisons depicted as: ≤ 0.05*, ≤ 0.01**, ≤ 0.001***.
|
