and the overexpression study of TSNARE1, CNTN4, SNAP91, and CLCN3, human wild-type capped mRNA for each gene was transcribed using the SP6 Message Machine Kit (Ambion). All RNAs were injected at the 1- to 4-cell stage at 200ng concentrations. Immunohistochemistry and phenotyping: For immunostaining purposes, all embryos were collected at 3dpf, dechorionated, and fixed in Dent’s solution (20% DMSO; 80% MeOH) overnight at 4°C. Embryos were rehydrated in a step-wise manner starting with 75% ethanol in 1XPBS, followed by 50%, and 25% ethanol solutions. Embryos were then bleached, post-fixed with 4% PFA, and permeabilized using proteinase-K. Embryos were then washed twice in IF buffer (1% BSA, 0.1% Tween-20 in 1XPBS) and incubated in primary antibodies for anti-α-acetylated tubulin (1:1000, Sigma-Aldrich, T7451) and anti-p-histone H3 (PH3; 1:500, Santa Cruz Biotechnology, sc-8656-R) in blocking solution overnight at room temperature (RT). Following two washes in IF buffer, embryos were placed in secondary antibody solution containing Alexa Fluor 594 goat anti-mouse IgG (1:1000) and Alexa Fluor 488 goat anti-rabbit IgG at 488(1:500; Invitrogen) in blocking solution for 2hrs at RT. Embryos were then washed and stored in IF buffer at 4°C until used for microscopy.
