Wei et al., 2016 - Loss of ZNF32 augments the regeneration of nervous lateral line system through negative regulation of SOX2 transcription. Oncotarget   7(43):70420-70436 Full text @ Oncotarget

Fig. 1

ZfZNF32 expression pattern during early embryonic development. A. ZfZNF32 mRNA was distributed uniformly from the zygote stage to the larval stage according to Q-PCR. B. Agarose gel electrophoresis of the (A) PCR product. C. Whole-mount in situ hybridization revealed the dynamic expression of zfZNF32 during zebrafish development (from the 1 cell stage to 5 dpf), and zfZNF32 was mainly restricted to the nervous system and pectoral fin (lateral view and dorsal view), as indicated by arrows.

Fig. 2

Establishment of zfZNF32 knock-out zebrafish and lateral line system regeneration. A. Agarose gel electrophoresis showing the Cas9-mediated zfZNF-/- with bases deleted compared with WT. B. A total of 85 bp were deleted (in blue font) in the zfZNF-/-. C. The regenerative capacity of the lateral line system was increased in 4 dpf zfZNF-/- at 12 and 24 hrs after neomycin treatment. Hair cells in the lateral line were stained with DASPEI (red fluorescence). The L1 was chosen as the representative regenerating neuromast, as indicated by arrows, and shown in the lower right corner of each figure with 200×magnification.

Fig. 3

ZNF32 negatively regulates SOX2 expression in vivo and in vitro. A. The early nerve stem cell factors nestin, SOX2 and olig2 were detected by WISH at 32 hpf in zfZNF-/- and WT. mRNA expression is indicated by arrows. B. WISH revealed SOX2 expression in zfZNF-/- and WT at bud, 24 hpf and 48 hpf stages. SOX2 mRNA expression is indicated by arrows. C. The regenerative capacity of the lateral line system was decreased in 4 dpf zfZNF-/- microinjected with SOX2 MO compared with control MO at 24 hrs after neomycin treatment. Hair cells in the lateral line were stained with DASPEI (red fluorescence). The L1 was chosen as the representative regenerating neuromast, as indicated by arrows, and shown in the lower right corner of each figure with 200×magnification. Relative gene expression of ZNF32 and SOX2 in BE(2)-C cell lines was detected at the mRNA level by Q-PCR D. and at the protein level by western blotting E. Untreated BE(2)-C cells served as controls. BE(2)-C lv-NC and BE(2)-C lv-ZNF32 cells were transiently transfected with pSG5-Vec and pSG5-ZNF32. Q-PCR and western blotting were performed to detect the relative expression of ZNF32 and SOX2 at the mRNA F. and protein level G. All of the quantitative values are presented as the means±S.D. *P< 0.05, **P< 0.01. NS, not significant.

Acknowledgments:
ZFIN wishes to thank the journal Oncotarget for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ Oncotarget