Cystine content in homogenates of wild-type and ctns^−/− zebrafish. (A–F) Cystine content in whole body (A), kidney (B), testis (C), eyes (D), muscle (E) and brain (F) in wild-type and ctns^−/− 18-month-old male zebrafish. Cystine concentration is expressed as nmol/mg protein. Each dot represents one zebrafish, for a total of n = 3 wild-type and n = 3 ctns^−/− 18-month-old male zebrafish; unpaired t test with Welch’s correction, two tailed: * p < 0.05; ** p <0.01; ns: not significant.

Kidney histology in wild-type and ctns^−/− zebrafish. (A,B) Representative images of renal tubules of wild-type (A) and ctns^−/− (B) 18-month-old zebrafish. Details of proximal tubules with hyaline-like eosinophilic droplets (black arrowheads) and cytoplasmic vacuoles (*). H&E staining. The scale bars represent 50 µm. (C,D) Representative images of the PAS staining of renal tubules of wild-type (C) and ctns^−/− (D) 18-month-old zebrafish. Details of proximal tubules with hyaline-like droplets (black arrowheads) and cytoplasmic vacuoles (*). PAS staining. The scale bars represent 50 µm. PT: proximal tubule; DT: distal tubule.

Kidney cystine crystals in wild-type and ctns^−/− zebrafish PTECs. (A,B) Representative toluidine blue-stained images of renal proximal tubules in wild-type (A) and ctns^−/− (B) 18-month-old zebrafish. The scale bars represent 50 µm. (C,D) Figure 3C,D show a higher magnification of Figure 3A,B. Details of renal proximal tubules with cytoplasmic vacuoles ((D); *), rectangular and polymorphous vacuolar spaces ((D); black arrowheads). (E,F) Representative TEM images of the renal proximal tubule of wild-type (E) and ctns^−/− 18-month-old zebrafish (F). (F) Partial loss of proximal tubule brush borders (PB) and accumulation of the rectangular and polymorphous vacuoles (*) were observed in the renal proximal tubule of ctns^−/− zebrafish. The high-magnification view of the rectangle in the bottom right corner shows the polymorphous vacuoles and a large vacuole with straight membrane border segments (a straight line). The scale bars represent 5 µm.

Glomerular morphology in wild-type and in ctns^−/− zebrafish. (A,B) Representative images of the glomerulus of wild-type (A) and ctns^−/− (B) 18-month-old zebrafish. PAS staining. The scale bars represent 50 µm. (C,D) Representative TEM images of the glomerulus of wild-type (C) and ctns^−/− (D) zebrafish. The scale bars represent 1 µm. (E–G) The quantification of the surface areas of Bowman’s capsule (E), glomerular tuft (F), and Bowman’s space (G) of wild-type and ctns^−/− zebrafish in both genders. Each dot represents one zebrafish, for a total of n = 3 wild-type and n = 3 ctns^−/− 18-month-old zebrafish. Two-way ANOVA with Fisher’s least significant difference (PLSD) test: * p < 0.05; ns: not significant.

Increased apoptosis in PTECs with vacuoles in ctns^−/− zebrafish. (A,B) Representative images of the cleaved caspase-3 immunostaining of tubules of wild-type (A) and ctns^−/− (B) 18-month-old zebrafish. The scale bars represent 50 µm. (C,D) The bottom panels show a higher magnification of the boxed areas in the upper panels. Details of PTECs with cytoplasmic vacuoles (*). The scale bars represent 50 µm. (E,F) Representative images of the cleaved caspase-3 immunostaining of glomerulus of wild-type (E) and ctns^−/− (F) 18-month-old zebrafish. The scale bars represent 50 µm. (G) The quantification of the tubular caspase-3 staining positive area in wild-type and ctns^−/− zebrafish in both genders. Each dot represents one zebrafish, for a total of n = 3 wild-type and n = 3 ctns^−/− 18-month-old zebrafish. Two-way ANOVA with Fisher’s least significant difference (PLSD) test: ** p < 0.01; *** p < 0.001.

Hypopigmented spotted skin pattern in ctns^−/− zebrafish. (A,B) Representative pictures of wild-type (A) and ctns^−/− (B) 18-month-old zebrafish. The scale bars represent 0.5cm. The black arrowheads indicate the different striped skin patterns. (C,D) PAS-stained pictures of epidermis of wild-type (C) and ctns^−/− (D) 18-month-old zebrafish. The black arrowheads highlight the melanin layer. The interruption in the melanin layer is present in ctns^−/− (D) while it is absent in wild-type zebrafish (C). The scale bars represent 50 µm.

Spermatogenic cysts enriched in spermatozoa in ctns^−/−zebrafish. (A,B) Representative H&E stained pictures of testis of wild-type (A) and ctns^−/−(B) 18-month-old zebrafish. Dark blue and rounded staining represent the spermatozoa accumulated in the spermatogenic cysts (indicated with the asterisks). The scale bars represent 200 µm.

Egg production and percentage of fertilized eggs in wild-type and ctns^−/−zebrafish. (A) Egg production was measured as a total number of eggs in wild type and ctns^−/−zebrafish 18-month-old zebrafish. The egg production rate is 1.941 high in wild-type zebrafish compared with ctns^−/−18-month-old zebrafish (rate ratio = 1.941); 95% CI (lower bound = 1.165, higher bound = 3.234); Poisson regression analysis; p = 0.011. (B) The percentage of fertilized eggs was measured on the total number of eggs. The odds of the fertilized eggs are 3.504 high in wild-type zebrafish compared with ctns^−/−18-months-old zebrafish (odds ratio = 3.504); 95% CI (lower bound = 2.005, higher bound = 6.124); Logistic regression analysis; p < 0.001; n/a: not applicable.

Average swimming speed in wild-type and ctns^−/−zebrafish. The average speed is decreased in ctns^−/−zebrafish compared with wild-type in both genders. The average swimming speed is measured in pixel/second. Each dot represents one zebrafish, for a total of n = 6 wild-type and n = 6 ctns^−/−18-month-old zebrafish. Two-way ANOVA with Fisher’s least significant difference (PLSD) test: ** p < 0.01.

Eye histology in wild-type and ctns^−/−zebrafish. (A,B) Representative images of cornea of wild-type (A) and ctns^−/−(B) 18-month-old zebrafish. The stromal layer of the cornea (black arrowheads and black lines). H&E staining. The scale bars represent 50 µm. (C,D) Representative images of retina of wild-type (A) and ctns^−/−(B) zebrafish. H&E staining. The scale bars represent 50 µm. (E) Relative quantification of the thickness of the stromal layer of the cornea. Each dot represents one zebrafish, for a total of n = 3 wild-type and n = 3 ctns^−/−18-month-old zebrafish. Unpaired t test with Welch’s correction, two tailed: * p < 0.05.