H2afv expression successfully depleted by morpholino (MO) injection (A) RT-PCR using primers that detect both h2afva and h2afvb demonstrates high mRNA levels of expression across all stages of embryonic zebrafish development. (B) Whole mount immunofluorescence detects H2afv protein expression across stages of zebrafish embryonic development. Representative images from at least 5 embryos examined from 2 separate clutches are shown. (C) Western blot analysis demonstrates depletion of H2afv expression at 24 hpf in lysates from varying concentrations of h2afv MO injected embryos compared with control (uninjected) embryos. h2afv morphants (0.67 ng) were used for the rest of the study (marked*). Representative image of 2 different blots are shown. (D) Kaplan-Meier curve illustrates increased mortality of h2afv morphants compared with control (uninjected) embryos across 96 h post fertilization (hpf). MO injected at 3.3, 0.67 and 0.34 ng per embryo, n = number of clutches and number of total embryos analyzed per condition in parenthesis.
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