in situ hybridization analysis confirms that the differential GFP expression between pt101 and pt102 occurs at the mRNA level. A. The mRNA expression patterns of the GFP transgene in pt101 and pt102 and of the zebrafish tyrp1 gene in the wildtype at different developmental stages confirm that the GFP expression in pt101 is similar to that of the endogenous tyrp1 gene; whereas, in pt102, the GFP mRNA expression is dramatically reduced in the RPE (arrow). B. Three control hybridizations verified that the in situ signals shown in A are specific: GFP anti-sense probe for wildtype fish, GFP sense probe for pt101, and tyrp1 sense probe for wildtype fish. All embryos were treated with PTU to block melanin formation.

The Fugu tyrp1 promoter directs GFP expression in RPE cells in pt101 and pt102. A—F. The lateral views of the eyes of pt101, pt102, and wildtype under a fluorescence stereomicroscope at 72 hpf. A—C are images taken in bright field and D—F are their respectively corresponding images in GFP fluorescence field. Arrows indicate the stronger GFP appearance at the boundary regions of the eyes. Arrowheads indicate what is likely to be choroidal melanophores adjacent to the RPE. G—I. In pt101, GFP signal in the eye (G, arrow) colocalizes with RPE specific marker zpr2 (H, arrow) at 5 dpf. I is the merged image of G and H. The arrowhead in I indicates the choroidal melanophores. J—L. In pt102, the weak GFP expression (J, arrow) in the eye also localizes to the zpr2-stained RPE (K). L is the merged image of J and K.

GFP expression profiles in pt101 and pt102 between 12 hpf and 48 hpf. The embryos are treated with PTU to block melanin pigmentation so that GFP signal can be visualized. Pictures of wildtype embryos were also taken as controls for bright field and fluorescence field imaging. Arrow1 and arrow2 indicate the weak GFP expression in the eye in pt101 at early developmental stages; no GFP signal can be detected in wildtype and pt102 in the same areas at the same developmental stages. Arrow 3 and arrow4 indicates the early expression of GFP in the NCDM that are located behind the ear (black arrowheads). All fluorescence images were taken at the same photographing setting. Insets 1, 2, 3, and 4 are the close-up images of the regions indicated by arrows 1, 2, 3, and 4, respectively. White arrowheads in the insets indicate the GFP fluorescence in the eye or the NCDM.

GFP expression in the RPE in pt101 is much stronger than that in pt102. A. A picture of a histological section of the zebrafish retina at 3 dpf shows the geometric contour of the RPE. The diagram on top illustrates the areas where the average pixel values of fluorescence signal were measured: Areas b represent the boundary region of the eye (about 7.2 μm in diameter); areas i represent the interior of the eye (about 18 μm in diameter). The microscopic focus plane for photographing is indicated with the dashed line. For detailed explanation of how relative GFP signal is determined, see Methods and Materials. B. The relative GFP intensity in the RPE increases during development in both pt101 and pt102. However, at 36 hpf, 48 hpf, and 72 hpf, the GFP signal in pt101 is about 27-, 20-, and 37-fold higher than that in pt102, respectively.

The Fugu and zebrafish tyrp1 genes are conserved. A. The zebrafish TYRP1 protein (zTYRP1) is highly similar to the Fugu TYRP1 protein (fTYRP1). The identical amino acid residues are highlighted in the darker shade and the amino acid residues that have similar chemical characteristics are indicated with the lighter shade. The two proteins are 73% identical at the amino acid level. B. The CONREAL algorithm identifies a number of potential conserved elements in the regulatory regions of the Fugu and zebrafish tyrp1 genes. The thick blue horizontal bar represents the span of the 6 kb DNA sequence that is immediately upstream of the zebrafish tryp1 gene. The thick red horizontal bar represents the span of the Fugu tyrp1 promoter. The black lines between the blue and red bars link the regions that show conservation of potential cis-elements, which are also indicated in the histogram in the box above.

The Southern Blot analysis demonstrates that the transgenic integration sites in pt101 and pt102 are different. A. A portion of the transgene construct pTol2-ftyrp1:GFP is indicated to illustrate the critical features of the transgene: ftyrp1 pro. for Fugu tyrp1 promoter, GFP for the coding region of the green fluorescent protein, SV40 polyA for SV40 mRNA polyadenylation signal, Tol2 for Tol2 transposon elements. The diagram is not drawn to the scale. EcoR I and Xho I site and the Digoxigenin-probe recognition site are indicated. B. The Southern blot: Lane 1, Markers. The molecular sizes (in base pairs) of the markers are indicated on the left. Lane 2, pTol2-ftyrp1:GFP as a positive control. Lane 3, pt101 genomic DNA. Lane 4, pt102 genomic DNA. Lane 5, pT2KXIGΔin as a negative control. Lane 6, AB wildtype genomic DNA as a negative control.

GFP positive melanophores are detected and isolated from the ftyrp1:GFP transgenic zebrafish via flow cytometry. A–C. Histograms of the spectra of fluorescence intensities of the dissociated cells of wildtype (A), pt101 (B) and pt102 (C) illustrate that GFP cells can be detected by the flow cytometer. An arbitrary fluorescence intensity threshold is indicated with arrows; above the threshold, no wildtype cells are detected. The weak fluorescence below the threshold is mainly contributed by background noise. In pt101 and pt102, GFP cells display high fluorescence intensities (B and C, brackets). D–F. The confocal immunohistochemical analysis reveals that GFP cells isolated from pt101 are composed of RPE cells (arrowheads) and the NCDM (arrows). The NCDM are positive for GFP only; whereas RPE cells are positive for both GFP and the RPE marker zpr2. Of the 242 GFP cells analyzed, 12% are zpr2 positive. G–I. On the contrary, none of the 152 analyzed GFP cells that were isolated from pt102 at 5 dpf displayed apparent zpr2 signal. J–L. Immunostaining of the isolated pt102 GFP cells with anti-GFP antibodies (red) confirms that the green fluorescence is indeed derived from GFP expression. M–O. The integrity of the isolated pt102 GFP positive cells is largely maintained, as demonstrated by the DIC image (O) that shows a continuous cell membrane boundary and a distinct nuclear structure. The presence of the nuclei was also confirmed by the nuclear staining with TO-PRO-1 (N).

Partial recovery of melanin reveals that the Fugu tyrp1 promoter drives GFP expression in the neural crest-derived melanophores but neither in xanthophores nor in iridophores in pt101. A–C. GFP is only expressed in the skin cells that contain melanin at 6 dpf (B, green, arrows) but not in yellow appearing xanthophores (arrowheads). C is the merged image of A and B. The bright field image was taken with transmission light. D–F. GFP is not expressed in silvery iridophores (arrowheads) but in melanophores (arrows) at 6 dpf. F is the merged image of D and E. The bright field image was taken with incident light to visualize iridophores. G–I. GFP (H) colocalizes to all melanin-containing NCDM (G) that are extensively distributed in the flat tail region at 72 hpf. The arrows indicate the same distribution patterns of GFP signal and melanin in two examples of the NCDM. I is the merged image of G and H. J–O. To determine if weak expression of GFP occurs in xanthophores and iridophores in the transgenic fish, we took fluorescence images of 6 dpf wildtype (J–L) and pt101 (M–O) larvae at a longer exposure time that allowed the recording of the weak autofluorescence in the wildtype. Compared to fluorescence signal in wildtype fish, no elevation of green fluorescence was observed in pt101 for both xanthophores (arrows) and iridophores (arrowheads), supporting the notion that no detectable GFP is being expressed in these two cell populations. J and M are the bright field images with incident light illumination. K and N are the fluorescence images. L and O are the merged images of the bright field and fluorescence images. The scale bars represent 70 μm.