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

Temporal and spatial expression patterns of zebrafish orthologs. Whole mount in situ hybridized embryos hybridized with antisense riboprobes for dennd3a (a–d), dennd3b (e–h), ncl1 (i–l), nup98 (m–p), and tbata (q–t) at the indicated developmental stages. All columns show lateral views. Intestinal expression for all genes is apparent from 48 hpf onwards. Scale bar = 500 μm

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