Absence of sws1 Opsin Expression History in Zebrafish Rod Photoreceptors. (A) Lineage tracing strategy in zebrafish. Lineage tracing in zebrafish was accomplished using a feedforward system that has been previously designated as “Kaloop” (Distel et al., 2009) (see Figure S6). Two constructs, Tg[sws1:KalTA4] and Tg[UAS:nfsB-mCherry-V2A-KalTA4] (ua3139 and ua3137, respectively), were independently inserted to generate the transgenic zebrafish. In ua3137, a fluorescent reporter protein mCherry is fused in-frame to a bacterial nitroreductase gene (nfsB), and the fusion protein is connected via the labile linker peptide V2A to a second copy of the KalTA4 transcription factor.

(B) Lineage tracing of sws1-expressing cells using sws1:KalTA4; UAS:nfsB-mCherry-V2A-KalTA4 zebrafish line. The UV-sensitive cones (cyan, 10C9.1) and rods (green, 4C12) of 4-dpf zebrafish were labeled using immunohistochemistry. Representative images from 1 of 10 left eyes. D, dorsal; V, ventral; N, nasal; T, temporal. Scale bar, 50 µm in low-magnification images and 20 µm in high-magnification image (bottom right).

Absence of sws2 Opsin Expression History in Zebrafish Rod Photoreceptors, related to Figure 6. (A) sws2 opsin lineage tracing strategy in zebrafish. Lineage tracing was performed using a Kaloop feedforward system. Two constructs Tg[sws2:KalTA4] and Tg[UAS:nfsB-mCherry-V2A-KalTA4] (ua3138 and ua3137, respectively) were independently inserted to generate the transgenic zebrafish. In ua3137, a fluorescent reporter protein mCherry is fused in-frame to a bacterial nitroreductase gene (nfsB), and the fusion protein is connected via the labile linker peptide V2A to a second copy of the KalTA4 transcription factor. (B) Lineage tracing of sws2- expressing cells using sws2:KalTA4; UAS:nfsB-mCherry-V2A-KalTA4 zebrafish line. The UV-sensitive cones (cyan, 10C9.1) and rods (green, 4C12) of 4 dpf zebrafish were labeled using immunohistochemistry. Representative images are shown for 1 of the 10 left eyes. A vast majority of lineage-traced cells (4,273 of 4,278; 99.88%) clearly lack immunolabeling by 4C12 (a rod marker), whereas 5 of the cells (0.12%) showed somewhat ambiguous (unclear detection) labeling. D, dorsal; V, ventral; N, nasal; T, temporal. Scale bar: 50 µm in low magnification images, and 20 µm in the high magnification image.

Validation of the Kaloop System, related to Figure 6. The expression of efna5a activates twhh1 promoter, which in turn would induce transgene under UAS. (A) The mü4380 zebrafish line was initially recovered from an enhancer trap screen (Distel et al., 2009). In mü4380, developmentally early (20-30 hpf) activation of the nearby endogenous efna5a gene induces twhh-driven production of KalTA4, which acts upon the UAS promoter to drive expression of the reporter fluorescent protein mCherry. This source of KalTA4 can also act upon the reporter element of the ua3137 construct. Cryosections of 13 dpf mü4380 or mü4380 x ua3137 larvae were imaged. The regions of magenta fluorescence detected only in the mü4380 x ua3125 larvae provide one line of validation for the lineage tracing function of the Kaloop construct. Cyan shows nuclear stain by ToPRO3. Scale bar: 40 µm. (B) Larval ua3139;ua3137 (Kaloop; see Figure 6A) and ua3016;c264 zebrafish were injected with KalTA4 mRNA as single-celled zygotes shortly after fertilization, and the level of global mCherry fluorescence is shown at three time points, most visible in the brain. Fluorescence is maintained over time in the Kaloop line but not the ua3016;c264 line, providing further validation that the Kaloop construct has lineagetracing functionality. In green is manually enhanced autofluorescence, to provide a landmark in each image. In the some cases, extra green fluorescence can be seen in the heart; this is a reporter gene built into the inserted construct which serves only to identify carriers of the [sws1:KalTA4] or [sws1:Gal4] transgene. In the c264, there is an additional red heart marker which can be seen in some images, which identifies carriers of the [UAS:nfsbmCherry] c264 transgene. dpf, days post fertilization; D, dorsal; V, ventral; T, temporal; N, nasal. Scale bar: 50 µm.