Tfap2e morpholino KD model in zfl. (A?B) Chemiluminescence bands and box plot graph of western blots of zfl on Tfap2e morpholino (MO) KD and controls from 1?4?days post fertilisation (dpf). Each dot on the box plot graph represents an independent experiment with 50?µg zfl protein of an independent injection. Band intensities were plotted on a logarithmic scale. Note the increasing genuine Tfap2e expression between 2 dpf and 3 dpf and the significantly suppressed expression on TB and e3i3 MO KD at 3 dpf. The housekeeping control Gapdh shows a consistent expression over all timepoints. The uncropped western blot membrane pictures are provided in online supplemental data 11. (C) Agarose gel electrophoresis after rt-PCR on tfap2e at 3 dpf. Tfap2e KD with e3i3 MO leads to an alternative, 50?bp shorter transcript (arrow) with precise exclusion of exon 3. This was further confirmed by Sanger sequencing (online supplemental data 7). (D?E) Representative brightfield microscopy images of zfl and box plot graph on the rate of unaffected zfl on Tfap2e KD. Each dot in the box plot graph indicates an independent injection experiment. Wild type (wt) strain zfl on tfap2e MO KD at 3 dpf present with microcephaly, scoliosis and hypopigmentation. The KD results in a significant reduction of phenotypically unaffected zfl. Co-injection of wt human RNA or zebrafish RNA (zfRNA) in morphants does not rescue the phenotype. Moreover, overexpression of TFAP2E by wt TFAP2E human polyA mRNA (hRNA) injection into Ctrl MO zfl leads to expression of the morphant phenotype in some of these controls. (F) Kaplan-Meier survival curve of tfap2e MO KD zfl. Both morphants show a poorer survival rate with a rapidly decreasing survival after 3 dpf especially for the TB MO KD, congruent with the timepoint of physiological Tfap2e upregulation. n=4; p<0.0001 (Mantel-Cox test). Ns, not significant; *p<0.05 **; p<0.01; ***p<0.001; ****p<0.0001 (B, E: one-way analysis of variance (ANOVA) with Tukey?s multiple comparison). Number of investigated zfl n as indicated. Scale bar: 500?µm. KD, knockdown; TB, translation blocking; TFAP2E, transcription factor activator protein 2; zfl, zebrafish larvae.
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