FIGURE SUMMARY
Title

In vivo imaging of hedgehog pathway activation with a nuclear fluorescent reporter

Authors
Mich, J.K., Payumo, A.Y., Rack, P.G., Chen, J.K.
Source
Full text @ PLoS One
Fig. 1

Generation of zebrafish with nuclear Hh pathway reporters.

(A) Design of the Gli-dependent nuclear mCherry reporter constructs. (B–C) Tg(8xGliBS:mCherry-NLS) and Tg(8xGliBS:mCherry-NLS-Odc1) larvae at 84 hpf. Fluorescence and brightfield images of representative zebrafish are shown, and the telencephalic nuclei (tn), optic tectum (ot), cerebellum (c), and hindbrain (hb) are labeled. Embryo orientations: lateral view and anterior left. Scale bars: 200 μm.
Fig. 2

Tg(8xGliBS:mCherry-NLS-Odc1) zebrafish exhibit enhanced reporter turnover.

(A–B) Brightfield and fluorescence images of 10-somite (~14 hpf) Tg(8xGliBS:mCherry-NLS) and Tg(8xGliBS:mCherry-NLS-Odc1) embryos. (C–F) The transgenic lines at 30 hpf after the addition of cyclopamine or vehicle control at the 10-somite stage. The embryos were concurrently treated with phenylthiourea (0.003%, w/v) to block pigmentation. Brightness-enhanced fluorescence images are also shown to more clearly depict mCherry-positive cells in cyclopamine-treated embryos. The zebrafish embryos in (A) and (B) are the same animals shown in (D) and (F), respectively. Embryo orientations: A–B, dorsal view and anterior left; C–F, lateral view and anterior left. Scale bars: A–B, 100 μm; C–F, 200 μm.
Fig. 3

Tg(8xGliBS:mCherry-NLS-Odc1) zebrafish report Hh pathway activity with high sensitivity and fidelity.

(A–D) Videomicroscopy frames of a Tg(8xGliBS:mCherry-NLS-Odc1) embryo during early somitogenesis. (E) Somitic region of a Tg(8xGliBS:mCherry-NLS-Odc1) zebrafish at 24 hpf, revealing cellular differences in nuclear mCherry fluorescence. Muscle pioneer cells (mp) and superficial slow-twitch muscle fibers (sstm) are labeled. (F–H) Tg(8xGliBS:mCherry-NLS-Odc1) zebrafish treated with cyclopamine or shha mRNA at the one-cell stage. Fluorescence and brightfield images of representative 24-hpf embryos are shown. (I–M) Tg(8xGliBS:mCherry-NLS-Odc1) zebrafish in wildtype or the indicated mutant backgrounds. Fluorescence and brightfield images of representative 36-hpf embryos are shown. The fluorescence micrographs were acquired with longer exposure times than those in panels F–H to enable the detection of low-level reporter activity. Brightfield micrographs show the distinctive you-type phenotypes observed within the same clutch of embryos. Embryo orientations: A–D, dorsal view and anterior up; E–M, lateral view and anterior left. Scale bars: A–E, 20 μm; F–M, 200 μm.
Fig. 4

Visualization of Hh signaling during adult fin regeneration.

Caudal fin of a Tg(-2.4shha:GFP-ABC;8xGliBS:mCherry-NLS) zebrafish three days after amputation. Brightfield (A) and fluorescence micrographs (B–C) are shown, with the dashed boxes corresponding to the magnified views below. Fin orientations: lateral views, anterior left. Scale bars: A–C, 2 mm; B2–C2, 200 μm.
Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ PLoS One
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