Chapter 2 20 patterning28, disease modeling29,30, identification of new genes affecting glomerular function and tubulogenesis16,31,32and drug testing33. In the current study, we investigated the pathological and functional characteristics of the first zebrafish mutant model of nephropathic cystinosis. We elucidated the main pathophysiological defects causing the diseased phenotype, which can be used for targeting novel therapeutic approaches. Results Zebrafish ctns gene The zebrafish ctns(ENSDARG00000008890) is a 10 exon gene in chromosome 11. It corresponds to the coding 10 out of 12 exons of the human CTNS(ENSG00000040531,17p13.2)34. The zebrafish Ctns protein (UniProt F1QM07, 384 aa) has a 60.2% amino-acid identity and 78.5% similarity to the human cystine transporter cystinosin (UniProt O60931, 367 aa), with 75.6% identity and 88.8% similarity in the regions of the seven transmembrane domains (Fig. 1a). The genetic zebrafish mutant line (ctns‒/‒) used in the current study is homozygous for the nonsense mutation c.706C>T (at the 10th base position of exon 8 of the zebrafish ctns gene) leading to a premature stop codon (TAA) and truncated protein at glutamine 236 (p.Q236X). The translation product is thus devoid of the last four transmembrane domains and both lysosomal targeting motifs at the 5th cytosolic loop and the C terminal tail, which is expected to render the protein non-functional (Fig. 1a, b). Up to date, no paralogue gene to ctns has been reported in zebrafish. Morphology of ctns‒/‒ zebrafish larvae We initially evaluated the morphological phenotype of morphant and ctns‒/‒ larvae at 4 days post fertilization (4 dpf) (N=191 and 334, respectively) in comparison to wildtype (wt) larvae (N=152) according to the grading system of Hanke et al., 201316. Here, oedema was graded in four stages. Stage I: no signs of oedema; stage II: mild oedema; stage III: intermediate oedema; and stage IV: severe total body oedema. Seven percent of living morphant larvae showed stage IV oedema and extreme body curvature, while 16% and 24% showed milder oedema (stages III and II, respectively). The morphological changes were less severe in the genetic ctns‒/‒ larvae, as they showed milder pericardial oedema and body curvature (stage II) in 14% of larvae. More severe forms of oedema (Stages III-IV) were very rare (3 and 1%, respectively), while the majority (82%) were not deformed (Fig. 2a and Supplementary Fig. S1 online).
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