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Fig. 3
Wtx inactivation is not associated with developmental anomalies. A, Structure of the wtx gene locus on zebrafish chromosome 5. The coding sequence is indicated as a black box, grey boxes depict untranslated sequences. Binding sites of both TALEN pairs are indicated by arrowheads. Arrows (F, R) show primers used for amplification. B, Sequences of selected wtx mutations in F1 fish derived from TALEN‐1 injections close to the transcriptional start site and from TALEN‐1/TALEN‐2 co‐injection‐induced segmental deletions. Deletions are indicated by dashes. Underlined sequences correspond to the left arm and the right arm of TALENs. The total number of deleted nucleotides in mutant alleles is given in brackets. C, Predicted consequences of TALEN‐induced mutations for protein sequence. The mutations wtx del1 and wtx del2 are predicted to cause reading frame shifts resulting in premature translation termination codons at amino acid position 20 and 16, respectively. The mutation wtx del3 is predicted to delete 421 amino acids and therefore five of six conserved sequence blocks (orange, yellow, red, green, blue, purple) and thus nearly half of the protein. MBD, membrane binding domain; aa, amino acids. D, Localization of wild type and N‐terminally truncated Wtx. ZF4 cells were transiently transfected with the respective expression vector. HA‐epitope (green) was detected 48 hours post transfection (hpt). DAPI (blue) was used for nuclear staining. White arrow indicates membrane localization of wtx‐HA. Scale bars represent 10 μm. (right) Western blot against HA‐epitope of ZF4 cells transfected with the respective expression vector (48 hpt). E, Representative brightfield images of wild type and homozygous wtx del3 mutant zebrafish 48 hpf and F, 7 dpf. Mutant wtx alleles do not result in overt embryonic phenotypes. D, Wild type and homozygous wtx del3 mutant embryos, bearing Tg(wt1b:EGFP) transgene. Transgene expression (green) demarks the pronephros, consisting of glomeruli (G) and associated tubuli (T). E, At 7 dpf calcein staining (green) indicates craniofacial bone structures of wild type and wtx del3 mutant zebrafish larvae (head region). For orientation, E depicts eye, O demarks opercle