Mosaic labeling of KV cells.

(A) A dorsal view of the tailbud in a live zebrafish embryo at the 8-somite stage (8 ss) of development. Kupffer’s vesicle (KV) is positioned at the end of the notochord. The inset shows GFP-labeled KV cells surrounding the fluid-filled KV lumen in a Tg(sox17:GFP-CAAX) transgenic embryo at 8 ss. This is the middle plane of the KV. Scale = 10 μm. (B) Schematic of cell shape changes during KV remodeling. KV-ant cells (blue) and KV-post cells (red) have similar shapes at 2 ss, but then undergo regional cell shape changes such that KV-ant cells are elongated and KV-post cells are wide and thin by 8 ss. These cell shape changes result in asymmetric positioning of motile cilia that generate fluid flows for left-right patterning. (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. (D) 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 to the shield stage generates low levels of Cre activity that changes expression of default RFP to expression of YFP in a subset of KV cells. (E) Mosaic labeled YFP⁺ KV cells at the middle plane of KV at 8 ss. (F) 3D reconstructed KV cells (green) and KV lumen (magenta) at 8 ss. Scale = 10 μm. (G–H) Morphometric parameters of 3D rendered KV-ant (G) and KV-post (H) cells: length = axis spanning from apical to basal side of the cell, height = axis spanning from dorsal to ventral side of the cell, and width = axis connecting lateral sides of the cell. Scale = 5 μm.

3D morphometric analysis of single cells reveals asymmetric cell volume changes during asymmetric KV cell shape changes.

(A) A lateral view of a mosaic labeled KV in a Tg(sox17:Cre^ERT2); Tg(ubi:Zebrabow) at 2 ss. The embryo diagram represents the orientation of the image. The notochord and KV are outlined in blue. Yellow lines mark the KV lumen. Examples of 3D reconstructed KV-ant and KV-post cells along the middle plane of KV are shown. Scale = 10 μm. (B) Representative snapshots of 3D rendered KV-ant and KV-post cells at different stages of KV development between 2 ss and 8 ss. The parameters including height (H), length (L) and width (W) were used to quantify cell morphology. Yellow lines indicate the KV luminal surface. Scale = 10 μm. (C–E) Quantification of height (C), length (D) and width (E) of individual KV-ant and KV-post cells during development. (F) A length-to-width ratio (LWR) was used to describe KV cell shapes. KV-ant and KV-post cells change shape between 4 ss and 6 ss. (G) Volume measurements of individual KV cells at different stages of development. Similar to cell shapes, KV-ant and KV-post cells change volume between 4 ss and 6 ss. All measurements presented in C-G were made on the same group of reconstructed cells. The number of KV-ant and KV-post cells analyzed is indicated in the graph in C. N = number of embryos analyzed at each stage. Graphs show the mean + SD. Results were pooled from three independent experiments. *p<0.01 and ns = not significant (p>5% with Welch’s T-Test).

Single KV cells were sampled from all positions along the middle plane of KV for morphometric analysis.

(A,B) Representative mosaic labeled KVs at pre (2 ss) and post-remodeling (8 ss) stages. YFP⁺ cells along the middle plane of the organ are numbered. KV lumen and cell boundaries are outlined. Yellow lines divide the KV lumen into anterior and posterior halves. (A’,B’) Pooled images of KV lumen boundaries and cell numbers show no bias in the distribution of mosaic labeled cells. Scale = 20 μm.

Ion channel activity mediates asymmetric KV cell volume changes, KV lumen expansion and KV cell shape changes.

(A) 3D reconstructed KV-ant and KV-post cells in mosaic labeled Tg(sox17:Cre^ERT2); Tg(ubi:Zebrabow) control embryos (treated with vehicle DMSO) showed asymmetric cell volume changes and asymmetric cell shape changes (length-to-width ratio) between 2 ss and 8 ss. (B) Inhibiting the Na⁺/K⁺-ATPase with ouabain treatments reduced KV lumen expansion and disrupted asymmetric cell volume changes. KV cells in ouabain treated embryos did not undergo asymmetric shape changes. (C–D) Interfering with Cftr function using the small molecule inhibitor CFTRinh-172 (C) or cftr MO (D) also blocked KV lumenogenesis and disrupted asymmetric cell volume changes and shape changes of KV cells. (E) Quantification of 3D reconstructed KV lumen volumes (insets depict lumen in YZ axis) in control and treated live embryos at 8 ss. For all quantitative analyses, the mean + SD is shown. The number of KV-ant and KV-post cells analyzed is indicated in the graphs in A-D. N = number of embryos analyzed. Results were pooled from two independent trials. Scale = 20 μm, *p<0.01 and ns = not significant (p>5% with Welch’s T-Test).

Ouabain treatments between 4–6 ss do not block KV lumen expansion.

(A–B) Mosaic-labeled KV in a control embryo (A) and an embryo treated with ouabain from 4 to 6 ss (B). (C) Measurements of maximum KV lumen area indicated that ouabain treatments between 4–6 ss were not effective at blocking KV lumen expansion. N = number of embryos analyzed. Shown is the mean + SD. ns = not significant (p>5% with Welch’s T-Test).

Vertex model simulations for cell shapes during KV remodeling.

(A–D) Vertex model simulations with N = 10 KV-ant and KV-post cells. Upper and lower panels respectively show force-balanced states at 2 ss and 8 ss. All shown simulations start from the same initial cell positions, but the mechanical parameters differ. The full simulation box is cropped in order to focus on the KV. For the example of panel A, Figure 4—figure supplement 1B shows the respective full state. (A) Both KV and external cells are solid-like (interfacial tensions ${\mathrm{\Lambda }}^{\mathrm{K}\mathrm{V}-\mathrm{K}\mathrm{V}}=-50$ and ${\mathrm{\Lambda }}^{\mathrm{e}\mathrm{x}\mathrm{t}-\mathrm{e}\mathrm{x}\mathrm{t}}=150$), and the lumen cross-sectional area expands according to experimental measurements between 2 and 8 ss. (B) KV cells are solid-like ($Λ Fig. 5 Interfering with Junction plakoglobin inhibits KV lumen expansion.(A) Immunostaining with Jup antibodies shows Jup enrichment at lateral membranes of KV cells marked by membrane-targeted GFP expression in Tg(sox17:GFP-CAAX) embryos. Embryos injected with jupa MO-1 showed reduced Jup protein levels. Boxes indicate enlarged regions shown as individual channels. Arrows point out representative lateral membranes. Scale = 20 μm. (B) Immunoblotting confirmed reduction in Jup protein level (arrowhead) in jupa MO-1 injected embryos relative to wild-type (WT) and control MO injected embryos. The graph shows normalized Jupa band intensities. Shown is the mean + SD for three independent experiments. (C) At 8 ss, control embryos showed an inflated KV lumen (red arrow) that was labeled using ZO-1 antibody staining. Embryos injected with jupa MO-1 to knockdown Jup expression in all cells (global knockdown) or specifically in DFC/KV cells (DFCjupa MO-1) appeared normal at 8 ss except that the KV lumen failed to expand. Interfering with Jup by injecting JUP-naxos mRNA also inhibited KV lumen expansion. Scale = 10 μm. (D) Quantification of KV lumen area in control and treated embryos at 8 ss. Co-injecting jupa MO-1 with jupa mRNA significantly rescued lumenogenesis defects. Shown are mean + SD for three independent experiments. (E) The number of ciliated KV cells was not different among the treatment groups. Shown is the mean + SD for results pooled from three independent experiments. (F) Representative images of at 8 ss in control and jupa MO injected embryos treated with vehicle (DMSO) or CFTRact-09. The graph shows KV lumen area (outlined by yellow line) in control and treated embryos. Scale = 10 μm. Shown is the mean + SD for two independent experiments.(G) Representative images of KV lumens of contro MO and jupa MO embryos injected with rhodamine-dextran. Scale = 20 μm. The graph shows percentage of embryos retaining and losing the fluorescent dye between 6 ss and 8 ss from two independent trials. N = number of embryos analyzed. *p<0.01 and ns = not significant (p>5% with Welch’s T-Test). EXPRESSION / LABELING: Gene: EGFP Fish: sny101Tg sny101Tg + MO2-jupa + MO4-tp53 Knockdown Reagents: MO2-jupa MO4-tp53 Anatomical Term: Kupffer's vesicle membrane Stage: 1-4 somites Expression / Labeling details PHENOTYPE: Fish: AB AB + MO1-jupa + MO4-tp53 AB + MO2-jupa + MO4-tp53 sny101Tg + MO2-jupa + MO4-tp53 Condition: chemical treatment by environment: CFTR potentiator Knockdown Reagents: MO1-jupa MO2-jupa MO4-tp53 Observed In: Kupffer's vesicle anatomical space Kupffer's vesicle tight junction Stage: 5-9 somites Phenotype details Fig. 5-S1 jupa is maternally supplied and enriched in precursor dorsal forerunner cells (DFCs) and KV.(A–E) RNA in situ hybridizations of jupa during early zebrafish development. (A) Antisense jupa probe revealed maternal jupa mRNA in 2 cell stage embryo. (B) A control jupa sense probe showed no staining. (C–D) jupa mRNA was detected in DFCs (black arrow) and KV cells (red square) during epiboly and early somite stages. (E) Enlarged region depicting jupa staining in KV (black arrow). Fig. 5-S2 Interfering with Junction plakoglobin inhibits KV lumenogenesis but not ciliated cell number.(A) Immunostaining with Jup antibodies showed that embryos injected with jupa MO-2 had reduced Jup protein levels in KV relative to control embryos (image of control MO embryo reproduced from Figure 5A for comparison). Boxes indicate enlarged regions shown as individual channels. Lateral membranes of KV cells are marked by membrane-targeted GFP expression in Tg(sox17:GFP-CAAX) embryos. Arrows point out representative lateral membranes. Scale = 20 μm. (B) Representative KV lumens labeled using ZO-1 antibody staining at 8 ss from different treatments. Scale = 10 μm. (C) KV cilia labeled using acetylated-tubulin antibody staining at 8 ss from different treatments. Scale = 10 μm. Fig. 5-S3 Jupa knockdown results in reduced E-cadherin levels at lateral membranes of KV cells.Immunostaining with E-cadherin (E-cad) antibodies showed E-cad enrichment at lateral membranes of KV cells marked by membrane-targeted GFP expression in Tg(sox17:GFP-CAAX) embryos. Embryos injected with jupa MO-1 showed a moderate reduction of E-cad protein enrichment at KV membranes. Arrows point to representative lateral membranes in individual channels. The graph represents normalized E-cad intensities from two experiments. N = number of embryos analyzed. Scale = 5 μm. *p<0.05 (Welch’s T-Test). Fig. 6 Asymmetric cell shape changes in KV are separable from lumen expansion.(A) Mosaic labeled KV cells in control MO injected embryos showed asymmetric changes in cell volumes and cell shapes between at 2 ss and 8 ss. (B–C) Perturbing cell-cell junction integrity in KV by interfering with jupa (B) or lgl2 (C) expression inhibited KV lumen expansion, but asymmetric cell volume changes occurred that were similar to controls. In addition, asymmetric KV cell shape changes occurred normally in jupa and lgl2 MO embryos. (D) Quantification of 3D reconstructed KV lumen volumes (insets depict KV lumen in YZ axis) in control and treated live embryos at 8 ss. For quantitative analyses, the mean + SD is shown. The number of KV-ant and KV-post cells analyzed is indicated in the graphs in A-C. N = number of embryos analyzed. Data for control MO and jupa MO experiments are pooled from three independent experiments and lgl2 MO data are pooled from two experiments. Scale = 20 μm. *p<0.01 and ns = not significant (p>5% with Welch’s T-Test). EXPRESSION / LABELING: Genes: RFP YFP Fish: a131Tg; sny120Tg a131Tg; sny120Tg + MO1-llgl2 + MO4-tp53 a131Tg; sny120Tg + MO2-jupa + MO4-tp53 Condition: chemical treatment by environment: afimoxifene Knockdown Reagents: MO1-llgl2 MO2-jupa MO4-tp53 Anatomical Terms: Kupffer's vesicle Kupffer's vesicle cell Stage Range: 1-4 somites to 5-9 somites Expression / Labeling details PHENOTYPE: Fish: a131Tg; sny120Tg + MO1-llgl2 + MO4-tp53 a131Tg; sny120Tg + MO2-jupa + MO4-tp53 Condition: chemical treatment by environment: afimoxifene Knockdown Reagents: MO1-llgl2 MO2-jupa MO4-tp53 Observed In: Kupffer's vesicle anatomical space Stage: 5-9 somites Phenotype details 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 @ Elife $