Figure 2.

Strategies for transgenic expression of Nok in the RPE and for selection of desired transgenic zebrafish lines. A, The promoter of Fugu tyrosinase gene was used to direct pigment cell-specific expression of the full-length wild-type nok gene. Individual protein domains are indicated with gray boxes. A GFP coding region was inserted between the stop codon of the nok gene and the SV40 poly(A) signal-containing 3′-untranslated region. This GFP sequence serves as the target of a GFP antisense probe for in situ analyses. The Nok C-terminal region downstream of the m520 nonsense mutation site was used to make an affinity column to purify the anit-Nok^547–703 polyclonal antibodies. Anti-Nok^28–208 antibodies (generated in a previous study) (Wei and Malicki, 2002) recognize both the full-length Nok and the truncated Nok^m520. B, Western blot analyses showed that anti-Nok^547–703 antibodies recognized the full-length Nok protein from wild-type fish samples (WT) and the recombinant Nok-His and GST-Nok^547–703 fusion proteins expressed in Escherichia coli. Anti-Nok^547–703 antibodies do not recognize the truncated Nok^m520 mutant protein (m520 mutants). A few weaker bands indicate the existence of nonspecific reactions of the antibodies to some fish proteins. The positions of full-length and m520 mutant Nok bands were indicated with arrows on the right. C, An immunohistochemical analysis demonstrated that the anit-Nok^547–703 antibodies recognize full-length Nok in wild type at 72 hpf. Nok localizes to the apical regions of the RPE and the retina (arrows). The arrowheads indicate the nonspecific staining of cells of an unknown class, which appear to be randomly distributed in the brain and retina. In mutant retinas, only nonspecific staining was observed (arrowheads). Nuclear dye YO-PRO was used to visualize the overall shape of the eyes.