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Fig. 5 Generation of slc12a3-deficient zebrafish. (A) WISH analysis using the slc12a3 probe in wild-type larvae at 4 dpf. (B) Targeted depletion of the slc12a3 gene. The CRISPR/Cas9 target site is located at exon 1. A genotype with an 8-bp deletion was used to establish the slc12a3 knockout line (highlighted in red). (C) Schematic diagram of the rearing timeline. Larvae were reared in egg water from 0 to 5 dpf and in system water after 5 dpf. The fish at 2 mpf were harvested for Na+ and Cl− measurements. The fish at 3 mpf were harvested for morphological observation. (D,E) Na+ and Cl− concentrations were measured in control and slc12a3-deficient male and female fish at 2 mpf, respectively. (F–I) The general morphological observations of control and slc12a3-deficient male and female at 3 mpf, respectively. (F,G) Male and female control fish at 3 mpf. (H,I) Male and female slc12a3-deficient fish at 3 mpf. (J,K) Body weight and body length of control and slc12a3-deficient male and female at 3 mpf.