 Title

Use of a carbonic anhydrase ca17a knockout to investigate mechanisms of ion uptake in zebrafish (Danio rerio)
 Authors
 Zimmer, A.M., Mandic, M., Yew, H.M., Kunert, E., Pan, Y.K., Ha, J., Kwong, R.W.M., Gilmour, K.M., Perry, S.F.
 Source
 Full text @ Am. J. Physiol. Regul. Integr. Comp. Physiol.
Genotyping and confirmation of ca17a−/− deletion in larval zebrafish (Danio rerio). A: nucleotide alignment of the sequenced ca17a+/+ and ca17a−/− alleles showing the CRISPR sgRNA target sequence in the shaded box and the positions of the 7 nucleotide (nt) deletion and C to T substitution mutations in the red box. B: multiplex PCR products from fin clips of F2 larval offspring of F1 ca17a+/− crosses. The general amplicon (477 base pairs; bp) band appears for all genotypes, the 147bp band is the ca17a+/+specific amplicon, and the 352bp band is the ca17a−/−specific amplicon. C: representative Western blot of Ca17a and βactin protein expression in ca17a+/+, ca17a+/−, and ca17a−/− whole larvae homogenates at 9 days postfertilization (dpf). D: immunofluorescent staining of Ca17a in the yolk sac of ca17a+/+, ca17a+/−, and ca17a−/− 4 dpf larval zebrafish. Scale bar = 50 μm. 
Survival of ca17a+/+, ca17a+/−, and ca17a−/− zebrafish (Danio rerio) larvae over development. A: percent survival of larvae at 5 and 30 dpf under flowthrough conditions. B: survival curves of larvae from 5 to 30 dpf under static conditions. There was a significant interaction of genotype and age on survival in A (twoway RM ANOVA; genotype × age, F = 57.6, P < 0.01; genotype, F = 57.6, P < 0.01; age, F = 2.0, P = 0.19; n = 5 tanks for ca17a+/+, n = 6 tanks for ca17a+/−, and n = 3 tanks for ca17a−/−). Bars with different letters are significantly different (P < 0.05) from one another. Survival curves in B were analyzed using Markov chain Monte Carlo sampler for multivariate generalized linear models (n = 8 tanks for ca17a+/+ and ca17a−/−, n = 7 tanks for ca17a+/−). *Significant difference from the wildtype ca17a+/+. Data are presented as means ± SE. dpf, days postfertilization. PHENOTYPE:

Na^{+} uptake rates and whole body Na^{+} content of ca17a+/+, ca17a+/−, and ca17a−/− zebrafish (Danio rerio) larvae over development. There was a significant effect of genotype on Na^{+} uptake (A) (ANOVA; 4 dpf: F = 133, P < 0.01, n = 8; 9 dpf: F = 60.6, P < 0.01, n = 8 for ca17a+/+ and ca17a+/−, n = 7 for ca17a−/−) and on Na^{+} content in 4 dpf (B) (ANOVA; F = 4.2, P = 0.03, n = 7 for ca17a+/+ and ca17a+/−, n = 6 for ca17a−/−) but not 9 dpf (ANOVA; F = 1.0, P = 0.35, n = 8 for ca17a+/+ and ca17a+/−, n = 5 for ca17a−/−) larvae. Bars with different letters are significantly different (P < 0.05) from one another across genotypes in 4dpf (uppercase letters) and 9dpf (lowercase letters) larvae. Data are presented as means ± SE. dpf, days postfertilization. 
Cl^{−} uptake rates and whole body Cl^{−} content of ca17a+/+, ca17a+/−, and ca17a−/− larvae (A and B), as well as sham Ca17a morpholinotreated larvae (C and D), and the effect of ethoxzolamide on Cl^{−} uptake (E) in zebrafish (Danio rerio) larvae. There was a significant effect of genotype on Cl^{−} uptake in 4 and 9dpf larvae (A) (ANOVA; 4 dpf: F = 55.1, P < 0.01, n = 16 for ca17a+/+ and ca17a+/−, n = 12 for ca17a−/−; 9 dpf: F = 21.5, P < 0.01, n = 13 for ca17a+/+, n = 12 for ca17a+/−, n = 11 for ca17a−/−) but not Cl^{−} content in 4 dpf (B) (ANOVA; F = 1.1, P = 0.36, n = 7 for ca17a+/+ and ca17a+/−, n = 6 for ca17a−/−) or 9 dpf (ANOVA; F = 1.9, P = 0.18, n = 8 for ca17a+/+ and ca17a+/−, n = 5 for ca17a−/−) larvae. There was an effect of Ca17a knockdown on Cl^{−} uptake in 4dpf zebrafish (C) (Welch’s twosample t test; t = 3.5, P < 0.01, n = 6), but not Cl^{−} content (D) (Welch’s twosample t test; t = −0.8, P = 0.5, n = 6). There was an effect of ethoxzolamide on Cl^{−} uptake in 4dpf zebrafish (E) [oneway nonparametric ANOVA (Kruskal–Wallis); χ2 = 12.1, P < 0.01, n = 6]. Values with different letters are significantly different (P < 0.05) from one another. Data are presented as means ± SE. dpf, days postfertilization. 
The effect of Ca17a knockdown on chloride (Cl^{−}) uptake in 4dpf zebrafish (Danio rerio) larvae reared in lowCl^{−} water and the effect of rearing in lowCl^{−} water on ca17a protein abundance in 4dpf larvae. There was an effect of ca17a knockdown and rearing water Cl^{−} concentration on Cl^{−} uptake (A) (twoway ANOVA; Cl^{−} concentration: F = 51.9, P < 0.01, morpholino: F = 110.1, P < 0.01, Cl^{−} concentration × morpholino: F = 13.6, P < 0.01, n = 7). There was an effect of rearing water Cl^{−} concentration on Ca17a protein abundance (B) (Welch’s twosample t test; t = −2.8, P = 0.02, n = 6). Different letters signify significant (P < 0.05) differences between sham and morpholino larvae within a rearing water Cl^{−} concentration and bars with an asterisk indicate significant effects of rearing water Cl^{−} concentration. Data are presented as means ± SE. 
Ionocyte density in ca17a+/+, ca17a+/−, and ca17a−/− zebrafish (Danio rerio) larvae at 4 dpf. Density of H+ATPaserich cells (A) and Na^{+} Cl^{−}cotransporter cells (B) significantly differed among genotypes (ANOVA; A: F = 7.7, P < 0.01, n = 14 for ca17a+/+, n = 19 for ca17a+/−, and n = 23 for ca17a−/−; B: F = 20.5, P < 0.01, n = 15 for ca17a+/+, n = 16 for ca17a+/−, and n = 26 for ca17a−/−, respectively). There was no effect of genotype on Na^{+}K^{+}ATPaserich cells (C) (ANOVA; F = 1.3, P = 0.29, n = 8 for ca17a+/+ and ca17a+/−, n = 11 for ca17a−/−). Bars with different letters are significantly different (P < 0.05) from one another across genotypes. Data are presented as means ± SE. PHENOTYPE:

Epithelial H^{+} flux in ca17a+/+, ca17a+/−, and ca17a−/− zebrafish (Danio rerio) larvae. The H^{+} flux was measured in the ventral yolk sac area of 4dpf larvae (A) and the pharyngeal arch area of 9dpf larvae (C). Scale bars = 100 μm. There was an effect of genotype on H^{+} flux at the yolk sac of 4 dpf larvae (B) (ANOVA; F = 4.7, P = 0.02, n = 7 for ca17a+/+ and ca17a+/−, n = 6 for ca17a−/−) but not H^{+} flux at the pharyngeal arch region of 9dpf larvae (D) (ANOVA; F = 0.8, P = 0.45, n = 8 for ca17a+/+ and ca17a+/−, n = 7 for ca17a−/−). Bars with different letters are significantly different (P < 0.05) from one another. Data are presented as means ± SE. dpf, days postfertilization. 
Gas transfer, respiratory exchange ratio (RER; /Ṁo_{2}) and ammonia excretion in ca17a+/+, ca17a+/−, and ca17a−/− zebrafish (Danio rerio) larvae at 4 dpf and 9 dpf. There was no effect of genotype on O_{2} consumption (A) (Ṁo_{2}; ANOVA; 4 dpf: F = 1.7, P = 0.19; 9 dpf: F = 0.7, P = 0.49), CO2 excretion (B) (Ṁco2; ANOVA; 4 dpf: F<0.1, P = 0.99; 9 dpf: F = 0.3, P = 0.72), or RER (C) (ANOVA; 4 dpf: F = 0.9, P = 0.42; 9 dpf: F = 2.1, P = 0.15) in 4 and 9 dpf larvae (4 dpf: n = 12 for ca17a+/+ and ca17a−/−, n = 10 for ca17a+/−; 9 dpf: n = 8 for ca17a+/+ and ca17a−/−, n = 7 for ca17a+/−). There was an effect of genotype on ammonia excretion in 4dpf (D) (ANOVA; F = 6.1, P < 0.01, n = 9 for ca17a+/+ and ca17a+/−, n = 8 for ca17a−/−) but not 9dpf larvae (ANOVA; F = 3.1, P = 0.07, n = 9 for ca17a+/+ and ca17a+/−, n = 6 for ca17a−/−). Bars with different letters are significantly different (P < 0.05) from one another across genotypes. Data are presented as means ± SE. dpf, days postfertilization. 