Kupffer’s vesicle in the zebrafish embryo. A. A dorsal view of Kupffer’s vesicle (KV) in a live zebrafish embryo at 8-somite stage (8 ss) of development. This is a brightfield image taken using a Zeiss Discovery V12 stereomicroscope. B. A schematic diagram of KV shows cell shapes at middle focal plane, and cilia (red and blue) projecting into the lumen to drive fluid flow within the KV after remodeling at 8 ss. A = Anterior; P = Posterior, L = Left; R = Right; D = Dorsal; V = Ventral. Anterior KV cells are represented in blue and posterior KV cells are red. Arrow = strong leftward flow.

Two transgenic zebrafish strains–Tg(dusp6:memGFP) and Tg(sox17:GFP-CAAX)–provide bright labeling of dorsal forerunner cells (DFCs) that give rise to Kupffer’s vesicle (KV) cells. A and B. Embryo diagrams (green represents DFC/KV cells) and inversed fluorescence images of membrane-localized GFP expression in DFCs in Tg(dusp6:memGFP) transgenic zebrafish at 80% epiboly stage (80% E) (A) and the tailbud stage (B) when migratory DFCs form a rosette structure. C and D. GFP expression in Tg(sox17:GFP-CAAX) transgenic zebrafish marks KV cell membranes during KV lumen formation at the 2 somite stage (2 ss) (C) and in the mature organ at 8 ss (D) after KV remodeling. A = Anterior; P = Posterior, L = Left; R = Right; D = Dorsal; V = Ventral. Anterior cells = blue, Posterior cells = red.

A. Double transgenic Tg(sox17:Cre^ERT2); Tg(ubi:Zebrabow) zebrafish are incrossed to obtain embryos. B. Time course of mosaic labeling of KV cells. Brief treatment of double transgenic Tg(sox17:Cre^ERT2); Tg(ubi:Zebrabow) embryos with 4-OHT from the dome stage (4 h post-fertilization) to the shield stage (6 hpf) generates low levels of Cre activity that changes expression of default RFP to expression of CFP or YFP in a subset of KV cells. C. Structure of the ubi:zebrabow and sox17:Cre^ERT2 transgenes and the possible recombination outcomes of the Zebrabow transgene by Cre recombinase activity in KV cell lineages. Cre can mediate the deletion of sequences flanked by loxP sites (orange triangles) or variant lox2272 sites (blue triangles), leaving behind single loxP or lox2272 sites that are not cross-compatible with each other. D. Mosaic labeled YFP⁺ KV cells (pseudo-colored green) at the middle plane of KV at tailbud stage and 8 somite stage (8 ss). Scale bars = 20 μm. E. 3D reconstructions of single KV cells (green) using Imaris software at tailbud and 8 ss. Dashed line indicates KV lumen surface. Scale bars = 10 μm.

Schematic representing embryo immobilization technique used for live imaging with an inverted confocal microscope. A live embryo is covered with liquid low-melting point (LMP) agarose in a MatTek dish and then positioned such that DFC/KV cells are close to the glass bottom. Once solidified, the agarose is covered by embryo medium.

A. A region of interest (ROI) bounding box (yellow box) around an entire 3D image of mosaic-labeled KV cells. Scale bar = 30 μm. B. The ROI bounding box can be re-sized to include only a single cell. Scale bar = 20 μm. C. The software 3D renders only the cell included in the ROI. Scale bar = 30 μm.

A. 3D renderings of cells in the anterior or posterior region of KV at the 2 somite stage (2 ss) or 8 somite stage (8 ss). Cell height (h), length (l), and width (w) measurements are shown at 2 ss. Dashed line indicates KV lumen surface. Scale bars = 10 μm. B-C. Box and whisker plots showing quantification of length to width ratio (LWR) that describes the shape of KV anterior or posterior cells (B), and quantification of cell volumes (C). n = 27 anterior cells at 2 ss; n = 25 posterior cells at 2 ss; n = 21 anterior cells at 8 ss; n = 22 posterior cells at 8 ss. Anterior and posterior KV cells have similar shapes and volumes at 2 ss, and then undergo asymmetric morphological changes that result in different cell shapes and volumes at 8 ss. n = number of cells analyzed. NS = not significant; ****P < 0.001. These images and results are modified from Dasgupta et al. (2018).