Lumen and cilia formation in Kupffer's vesicle. (A-C)Images of a time-lapse multi-photon confocal movie of a zebrafish embryo expressing Tg(β-actin:HRAS-EGFP;sox17:GFP). Single focal planes at the interior of the DFC cluster are shown at the bud stage and subsequent time points, as indicated. Anterior is to the top. At the bud stage, bottle-shaped DFCs are arranged around two focal points (A, arrowheads). Shortly after, these focal points have coalesced into a single focal point (B, arrowhead) from which a single lumen forms (C). (D-I) Confocal images of Tg(sox17:GFP)-expressing embryos immunolabelled with an anti-ZO-1 antibody. GFP, green; anti-ZO-1, red. Single focal planes at the interior of the DFC cluster (D-F) and 3D renderings of the anti-ZO-1 labelling (G-I) are shown. Arrowheads mark equivalent structures in single focal planes and renderings. At the bud stage, multiple, widely spread ZO-1-rich accumulations are found in the interior of the DFC cluster (D,G). At the 1-somite stage, ZO-1 clusters are more connected and condensed, and small lumina are observed (E,H). At the 2-somite stage, a single, large lumen delineated by ZO-1 is observed inside the cluster (F,I). (J-O) Confocal images of Tg(sox17:GFP)-expressing embryos (green) double labelled with anti-ZO-1 (blue) and anti-acetylated α-tubulin (red) antibodies. Single focal planes are shown for merged (J-L) and single (M-O) channels. At the 1-somite stage, DFCs are positioned around multiple small lumina delineated by ZO-1, in which short, tubulin-rich cilia are observed (J,M). Between the 2- and 4-somite stages, a single, expanding lumen is observed (K,L), which contains cilia that are increasing in length (N,O). Scale bars: 30 μm.
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