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Fig. 1
Pathogenicity analysis in vivo by morpholino gene knockdown and CRISPR/Cas9 knockout in zebrafish. Morpholino knockdown of ckap2l, dennd3, ncl1, nup98, and tbata resulted in a HSCR-like phenotype when compared to control (a–j). Kaede-expressing enteric neurons were absent in the distal intestine at 5 dpf. The number of embryos with phenotype out of the total number of embryos observed is shown. Co-injection of p53 morpholino reproduced the phenotype except ckap2l, indicating the loss of enteric neurons in dennd3, ncl1, nup98, and tbata knockdown was not the result of p53-induced apoptosis (k–o). The results were verified by CRISPR/Cas9 knockout of ckap2l, dennd3a and b, ncl1, nup98, and tbata, in which the HSCR-like phenotype was reproduced (p–t). Dotted lines outline the intestines. Asterisks indicate the position of the anus. Arrows indicate the position where the aganglionic region begins. Scale bar = 200 μm. MO morpholino, nt nucleotide