|
Fig. 1
Expression of Overstabilized Plk4 in the Developing Zebrafish Induces Centriole Amplification and Subsequent Apoptosis
(A) Centriole duplication pathway in WT (left) and centriole amplification by expression of HS-inducible OS-Plk4 (right). The blue circle indicates a phosphate group. In the WT scenario, Plk4 is autophosphorylated and then degraded, leading to centriole duplication and four centrioles. OS-Plk4 lacks the phosphodegron domain; thus, it is not autophosphorylated and not degraded, leading to centriole amplification and multiple centrioles.
(B and C) A WT fish (B) and a transgenic (Tg)HS-OS-Plk4 fish (C). HS was applied at 24 hpf, and fish were imaged at 24 hphs/48 hpf and stained with acridine orange to label apoptotic cells. Left: bright field, right: acridine orange. Scale bars, 500 µm.
(D) Confocal scans, en face, of a mitotic WT neuroepithelial cell (left) with two poles, each containing two centrin spots (magenta) and a mitotic HS-OS-Plk4 neuroepithelial cell (right) with three poles: one featuring three centrin spots and two containing two centrin spots (magenta). Merged with cell outlines (phalloidin, blue) and chromatin (DAPI, green). Scale bars, 5 µm. HS was applied at 24 hpf, and cells were fixed at 16 hphs.
(E) EM image of a WT neuroepithelial cell showing two centrioles (magenta arrows). Dashed line marks the apical side. Scale bar, 1 µm. See Figure S1C for full series of sections. HS was applied at 24 hpf, and cells were fixed at 10 hphs.
(F) EM image of a Tg(HS-OS-Plk4) neuroepithelial cell showing six centrioles (magenta arrows). Dashed line marks the apical side. Scale bar, 1 µm. HS was applied at 24 hpf, and cells were fixed at 10 hphs.
(G) Time series of confocal scans of retinae of Tg(HS-OS-Plk4) fish. HS was applied at 24 hpf, and fish were stained for apoptotic marker active-caspase-3 (green) and DAPI (gray). Scale bar, 50 µm.
See also Figures S1B?S1D.