Fig. 1

Zebrafish IFT gene structure and location of mutations. A: Gene structure of the zebrafish ift57, ift88, and ift172 genes is shown with exons as solid boxes. The 5′-UTR and 3′-UTR regions are shown as white boxes. ift57 and ift172 mutants resulted from retroviral insertional mutagenesis (Sun et al.,[2004]) and the insertion site is shown as an open arrowhead. The causative mutation for ift88 was identified in exon 11 (Tsujikawa and Malicki,[2004]), which is shown by a closed arrowhead. An arrow shows the location of the intragenic RFLP used for genotyping ift88 mutants. B: Generic diagram for genotyping ift57 and ift172 insertional mutants. PCR was performed with a three-primer combination of a forward primer (FP), a reverse primer (RP), and a viral-specific primer (MSL4 or NLTR3). Due to the size of the viral insert (>6 kb), the FP and RP product only amplified from the wild type allele. If a mutant allele was present, the FP and a viral-specific primer generated a PCR product. When analyzed by agarose gel electrophoresis, homozygous embryos produce a single band, whereas heterozygous embryos produced bands from both alleles. C: Typical examples of genotyping analysis for ift57 (top), ift88 (middle), and ift172 (bottom) embryos. Homozygous ift57 mutants (M) produced a single 210-bp band, whereas reactions from heterozygous embryos (H) produced the mutant band and a 448-bp wild type band (W). The RFLP in the ift88 gene can be digested by Bcl I on the wild type allele but fails to cut on the mutant allele. ift88 mutant embryos (M) were identified by the absence of a lower band following Bcl I digestion, as previously described (Tsujikawa and Malicki,[2004]). Following PCR and Bcl I digestion, heterozygous animals (H) retained both the cut and uncut products. Homozygous ift172 embryos produced a single band of 265 bp, whereas reactions from heterozygous embryos produced the mutant band and a 461-bp band. Asterisks (*) denote excess primer at the bottom of the gel. D: Western blot analysis of 48-hpf embryos. Extracts of wild type (wt) and mutant embryos (ift88^-/- or ift57^-/-) were immunoblotted for either IFT88 or IFT57 protein. Acetylated tubulin (AcTub) was used as a loading control.