(A) Whole mount in situ hybridization was used to detect the spatial expression of aqp8ab mRNA. (A, part a) 14 hpf, lateral view, no staining. (A, part b) 24 hpf, lateral view, no staining. (A, part c) 36 hpf, lateral view, no staining. (A, part d) 48 hpf, lateral view, no staining. (A, part e) 72 hpf, overview of whole body, intestine (arrow). (A, part f) 4 dpf, overview of whole body, intestine (arrow). (A, part f’) 4 dpf, ventral view, intestine. (A, part g) 5 dpf, overview of whole body, intestine (arrow). (A, part g’) 5 dpf, lateral view, intestine. (A, part h) 6 dpf, overview of whole body, intestine (arrow). (B) Whole mount in situ hybridization analysis was used to detect ifabp mRNA expression in different stages of zebrafish embryonic development. (B, part a) 4 dpf, ventral view of whole body, intestine (arrow). (B, part b) 5 dpf, lateral view, intestine (arrow). (B, part b’) 5 dpf, lateral view, intestine. (B, part c) 6 dpf, overview of whole body, intestine (arrow). (B, part c’) 6 dpf, lateral view, intestine. (B, part d) 6 dpf, ventral view, intestine.

(A–C) Corresponding transverse sections through the three different regions of intestine at 4 dpf depicted in left image of the top row. (A) The aqp8ab mRNA was mainly expressed in epithelial cells lining the intestinal cavity. The developing intestine adjacent to the yolk was seen at 4 dpf (red dotted lines). (B) The aqp8ab mRNA expression was more intense in the layer lining the intestinal cavity. The intestinal tract contained a small lumen (arrow). (C) The aqp8ab mRNA expression was still detected in the layer lining the intestinal cavity. (D–F) Corresponding histological cross-sections through the different regions of intestine at 5 dpf depicted in right image of the top row. Cross-sections analysis showed that aqp8ab mRNA expression was more intense in the layer lining the intestinal lumen. n, notochord; i, intestine; s, somite; y, yolk; ib, intestinal blub.

(A) Schematic diagram showing aqp8ab sgRNA targeting site (indicated by arrowhead) in the third exon of aqp8ab gene. Starting codon (ATG) site is indicated by arrow. The sgRNA targeting sequence is highlighted in brown and the PAM in purple. (B) Mutation pattern of mRNAs-injected embryos. Numbers in the brackets show the number of nucleotides that were deleted (–) or inserted (+). Inserted nucleotide is in red, changed nucleotide is in blue. WT, wild-type. (C) Three heritable mutants were identified by screening. F0 offspring were out-crossed with wild-type fish to produce F1, and the DNA extracted from tail fins of F1 adults were used for the identification of heritable mutants by sequencing. (D) Schematic diagram showing the predicted proteins encoded by the three mutated alleles. The mutants are reading frameshift mutations that result in truncated proteins.

(A) Microscopy analysis of embryos development at 3–6 dpf in control group and aqp8ab mutants. Red arrows indicate pericardial edema. (B) Whole mount in situ hybridization detection of the expression of ifabp at 3–6 dpf after aqp8ab deletion. Red arrows indicate intestinal bifida.

(A) Schematic diagram showing transverse sections through the trunk at four different regions. (B) At 4 dpf, the control group showed a single intestinal tract, in which a lumen was already formed (stars). The aqp8ab mutants displayed double different intestinal tracts with no lumen. (C) At 5 dpf, the diameter of intestinal tract in the control group grew bigger. The aqp8ab mutants showed double different intestinal tract with no lumen.

At 4–5 dpf, the control group had a single intestinal tract, the aqp8ab mutants displayed double different intestinal tracts. However, aqp8ab mRNA injection could rescue the phenotype caused by aqp8ab mutation. In the rescue experiment, left embryos shown here are lateral view with anterior to the left, and right embryos shown here are ventral view with anterior to the left.