A nonsense mutation of zebrafish kl gene. (a) The αklotho protein has a signal peptide, two glycosidase domains and a transmembrane domain. A nonsense mutation caused a truncation in the first glycosidase domain. (b) The T-to-A mutation generated an MseI restriction enzyme site. (c) The size of the genomic PCR products was 240 bp. The PCR products amplified from mutant alleles were digested by MseI to generate two 120-bp fragments.

Zebrafish kl mutants become thinner and undergo premature death. (a–d) The kl^+/+ female (a), kl^+/+ male (b), kl^−/− female (c) and kl^−/− male (d) zebrafish at 4 mpf. The kl^−/− fish showed no morphological defects at this stage. (e–l) Lateral and dorsal views of head in kl^+/+ female (e,i), kl^+/+ male (f,j), kl^−/− female (g,k) and kl^−/− male (h,l) at 5 mpf. Note that the eyeball is protruding to the outside in the kl^−/− female. (m–p) The kl^+/+ female (m), kl^+/+ male (n), kl^−/− female (o) and kl^−/− male (p) zebrafish at 6 mpf. The kl^−/− fish became skinny. (q) The kl^+/+ fish (n = 32) survived over 9 mpf. All of the kl^−/− fish (n = 28) underwent premature death before 9 mpf.

Swimming performance in kl mutants declines by 4 mpf. The critical swimming speed (U_crit) of 3 mpf and 4 mpf zebrafish were measured using a swimmill. The kl^+/+ female (n = 3), kl^+/+ male (n = 4), kl^−/− female (n = 4) and kl^−/− male (n = 3) were assayed at 3 mpf. The kl^+/+ female (n = 13), kl^+/+ male (n = 9), kl^−/− female (n = 6) and kl^−/− male (n = 3) were assayed at 4 mpf. Open black (kl^+/+) and red (kl^−/−) circles indicate U_crit of individual samples. Closed black (kl^+/+) and red (kl^−/−) circles with error bars indicate mean ± sem.