Generation of slc12a10.2-deficient zebrafish. (A) WISH analysis using the slc12a10.2 probe in wild-type larvae at 4 dpf. (B) Targeted depletion of the slc12a10.2 gene. The CRISPR/Cas9 target site is located at exon 1. A genotype with an 11-bp deletion was used to establish the slc12a10.2 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 6 dpf were harvested for Na+ and Cl− measurements. (D,E) Na+ and Cl− concentrations were measured in control and slc12a10.2-deficient larvae at 6 dpf. **P < 0.01. (F,G) Sodium green staining in the gills of control and slc12a10.2-deficient larvae at 6 dpf (ventral views).

The lethality of the slc12a10.2-deficient zebrafish is rescued by exogenous Cl− supplement. (A) 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 survival ratio of fish reared under this condition was analyzed until 20 dpf. (B) Comparison of the survival ratios of prl+/+, prl−/−, slc12a10.2+/+, and slc12a10.2−/− larvae (n = 25 for each genotype). (C–H) The general morphological observations of control larvae and slc12a10.2-deficient larvae at 6, 8, and 10 dpf. (C,E,G) Control larvae. (D,F,H) slc12a10.2-deficient larvae. (I) Schematic diagram of the rearing timeline. Larvae were reared in egg water from 0 to 5 dpf and in brackish or artificial water after 5 dpf. The survival ratios of fish reared under different conditions were analyzed until 20 dpf. (J–M) The survival ratios of control and slc12a10.2-deficient larvae in system water + sea salt, system water + Mix 1, system water + Mix 2, and system water + Mix 3.

Expressions of prl and PRL targets in control and slc12a10.2-deficient larvae at 4 dpf. (A) Schematic diagram of the rearing timeline. Larvae were harvested at 4 dpf for WISH and qPCR analysis. (B,C) WISH analysis using the probe of prl in control and slc12a10.2-deficient larvae at 4 dpf. (D,E) WISH analysis using the probe of atp1a1a.5 in control and slc12a10.2-deficient larvae at 4 dpf. (F,G) WISH analysis using the probe of slc9a3.2 in control and slc12a10.2-deficient larvae at 4 dpf. (H,I) WISH analysis using the probe of slc12a3 in control and slc12a10.2-deficient larvae at 4 dpf. Red arrows indicate the expressions of the nominated genes. (J) Expression levels of prl, prlra, prlrb, atp1a1a.5, slc9a3.2, and slc12a3 in control larvae and slc12a10.2-deficient larvae at 4 dpf were examined with qPCR. *P < 0.05. **P < 0.01.

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.

Expression patterns of prl and PRL targets in the slc12a3-deficient larvae at 4 dpf. (A) Schematic diagram of the rearing timeline. Larvae were harvested at 4 dpf for WISH analysis. (B,C) WISH analysis using the probe of prl in control and slc12a3-deficient larvae at 4 dpf. (D,E) WISH analysis using the probe of slc12a10.2 in control and slc12a3-deficient larvae at 4 dpf. (F,G) WISH analysis using the probe of atp1a1a.5 in control and slc12a3-deficient larvae at 4 dpf. (H,I) WISH analysis using the probe of slc9a3.2 in control and slc12a3-deficient larvae at 4 dpf. Red arrows indicate the expressions of the nominated genes. (J) Expression levels of prl, prlra, prlrb, atp1a1a.5, slc9a3.2, and slc12a10.2 in control larvae and slc12a3-deficient larvae at 4 dpf were examined with qPCR assay. *P < 0.05. **P < 0.01.