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Fig. S1. Division patterns and fates of dorsal surface epithelial cells. The division patterns of dorsal surface epithelial cells and the fate of daughter cells were analysed. Fourteen cells from three embryos were monitored from high to shield stage (3.3 to 6 hpf). We observed both ‘planar’ divisions, generating two surface epithelial cells, and ‘delaminating’ divisions, generating a surface cell and deep cell. Seven principal patterns were observed: a planar division giving rise to two EVL cells (A); a planar division giving rise to one EVL cell and one DFC precursor (B); a planar division giving rise to two DFC precursors (C); a planar division producing one EVL cell, while the second daughter cell divides to give rise to two DFC precursors (D); a delaminating division producing an EVL cell and a deep cell (E); a delaminating division producing a DFC precursor and one deep cell (F); and a delaminating division that gives rise to a deep cell, while the second daughter cell divides in a planar fashion to produce two DFC precursors (G). The frequency of each of these division/cell fate patterns among the 14 cells is summarised in the table (H).
Fig. S2. Morphological changes of the dorsal forerunner cell cluster at the end of epiboly. (A-D′) Three-dimensional renderings of the dorsal forerunner cell (DFC) cluster in a Tg(sox17:GFP) embryo as dorsal views (A-D; animal pole to the top) and sagittal views (A′-D′; enveloping surface layer to the left). (A,A′) DFC cluster at 75% epiboly stage. Cluster size is shown along the following axes: x (medio-lateral, yellow dotted lines in A), y (animal-vegetal, red dotted lines in A′), and z (surface-depth, green dotted line in A′). At this stage, the cluster is composed of approximately 36 cells. Note that the DFC cluster is still in contact with the enveloping layer (EVL) (white arrow in A′). (B,B′) After 60 minutes, the cluster has narrowed along the x and y axes (B and B′), and extended along the z axis (B′). At this stage, the number of DFCs had increased to 41, but remained approximately constant during the remainder of the recording. Note that the dorsal blastoderm (not labelled) has started to move between the DFC cluster and EVL (animal concavity in B′). (C,C′) After 120 minutes, the cluster has begun to extend along the x axis while further narrowing along the y (C) and extending along the z (C′) axes. Note that the DFC cluster is still partially attached to the EVL (white arrow in C′). (D,D′) After 180 minutes, the DFC cluster exhibits a spheroid shape with wider x, shorter y and thicker z dimensions (D and D′). Note that the DFC cluster is now completely detached from the EVL (white arrow in D′). (E) Quantification of the changes in DFC cluster size along the Y (yellow), Y (red) and Z (green) axes. Scale bar: 20 µm.
Fig. S3. Mesenchymal-to-epithelial transition of the dorsal forerunner cell cluster. Three-dimensional renderings (A-C) and single focal planes through the centre of the DFC cluster (A′-C′) in a Tg(Sox17:GFP) embryo. At 80% epiboly (A,A′), migratory DFCs show numerous cell protrusions without clear directionality (arrowheads in A). At bud stage, cell protrusions start diminishing as the cluster rounds up (arrowheads in B) and organises into a more compact structure (B′). At the 2-somite stage, the DFC cluster has transformed into a vesicle (C) with a lumen at its centre (C′). Scale bar: 20 µm.
Fig. S4. Quantitative changes in lumen volume, cilia number and cilia length during dorsal forerunner cell epithelialisation. Quantification of lumen volume, cilia number and cilia length measured from bud to 4-somite stages. Lumen volume begins to rapidly increase at the 2-somite stage (A). By contrast, cilia number reaches its maximum already at the 1-somite stage (B), whereas cilia length continuously increases between bud and 4-somite stages (C). Values for each stage are averages ± s.d. (vertical bars) of five embryos.
Movie 1. Dorsal forerunner cells are derived from the enveloping cell layer. Multi-photon confocal time-lapse movie of a Tg(β-actin:HRAS-EGFP) embryo starting at sphere stage (4 hpf). Stacks of multiple focal planes (1 µm apart) were recorded at each time point. Single focal planes at the surface level of the embryo were selected from the 4D data set and assembled into a movie. Dorsal view with animal pole to the top. In addition, a sagittal view at the position of the white line is simultaneously shown. A subset of surface epithelial cells disappears from the plane as trailing enveloping cell layer (EVL) cells move over them. Note the division of some marginal EVL cells prior to conversion to DFCs. Duration, 87 minutes.
Movie 2. Dorsal forerunner cell formation. Multi-photon confocal time-lapse movie of a Tg(β-actin:HRAS-EGFP) embryo starting at sphere stage (4 hpf). Single focal planes at the deep cell level of the embryo were selected from the 4D data set and assembled into a movie. Dorsal view with animal pole to the top. DFCs arise from surface epithelial cells, as trailing EVL cells move over them. Note that deep cells do not contribute to the DFC cluster. Duration, 87 minutes.
Movie 3. Surface epithelial cell behaviour on the lateral side of the embryo. Multi-photon confocal time-lapse movie of the lateral side of a Tg(β-actin:HRAS-EGFP) embryo starting at dome stage (4.2 hpf). Single focal planes at the surface level of the embryo were selected from the 4D data set and assembled into a movie. Animal pole is to the top. Surface epithelial cells divide and advance vegetally, but no ingressions at the margin are observed. Duration, 160 minutes.
Movie 4. Cell division and ingression of dorsal surface epithelial cells. Multi-photon confocal time-lapse movie of a Tg(β-actin:HRAS-EGFP) embryo starting at the high stage (3.3 hpf). Single focal planes at the surface level of the embryo were selected from the 4D data set and assembled into a movie. Three marginal cells can be observed to undergo a delaminating division (marked by asterisks). In one of these divisions (double asterisks), the newly formed surface daughter cell subsequently divides in a planar orientation, and the two new daughter cells are displaced below the surface epithelium to become DFCs. In the additional delaminating divisions (marked by a single asterisk), the newly formed surface daughter cells are then displaced below the surface epithelium without an additional round of division. Duration, 167 minutes.
Movie 5. Dorsal forerunner cell cluster morphogenesis. Multi-photon confocal time-lapse movie of a Tg(sox17:GFP) embryo. 3D rendering of the DFC cluster starting at 75% epiboly. Dorsal view with animal pole to the top. Initially, DFCs are in a broad, loose arrangement. The cluster progressively condenses while moving to the vegetal pole, acquiring a compact, spherical shape. Duration, 102 minutes.
Movie 6. Detachment of bottle-shaped DFCs from the EVL during late epiboly. Multi-photon confocal time-lapse movie of a Tg(β-actin:HRAS-EGFP) embryo starting at 90% epiboly (9 hpf). A single focal plane of the 4D data set was selected at each time point at the level of the narrow sides of a subset of bottle-shaped DFCs. A sagittal section at the position of the white line is simultaneously shown (see Fig. 2E-F′ for tracking of individual cells). At the beginning of the movie, several bottle-shaped DFCs are in contact with the overlying EVL via a single focal point. As the movie progresses, these bottle-shaped DFCs gradually detach from the EVL and orient their narrow sides to the interior to form a rosette structure. Duration, 102 minutes.
Movie 7. Kupffer’s vesicle (KV) lumen formation. Multi-photon confocal time-lapse movie of a embryo co-expressing Tg(sox17:GFP) and Tg(β-actin:HRAS-EGFP). Vegetal/posterior view with anterior to the top. Single focal planes through the interior of the DFC cluster starting at 100% epiboly are shown. DFCs initially form multiple rosette-like structures that progressively coalesce into a single rosette, at the centre of which a single lumen forms. Duration, 170 minutes.
Movie 8. Apical fusion of vacuole-like structures during Kupffer’s vesicle lumen expansion. Multi-photon confocal time-lapse movie of a Tg(sox17:GFP) embryo. Single focal planes of the DFC cluster starting at the 2-somite stage are shown. Vegetal/posterior view with anterior to the top. Several intracellular vacuole-like structures fuse with the apical surface of lumen-forming dorsal forerunner cells. Duration, 106 minutes.
Movie 9. Conversion of EVL to DFCs is regulated by Nodal signalling in a non-cell-autonomous manner. Confocal time-lapse movie of a Tg(β-actin:HRAS-EGFP) embryo co-injected with 50 pg of cyclops mRNA and rhodamine-dextran (identifying cyclops-expressing cells) into a single marginal blastomere at the 64-cell stage. The animal pole is to the top. 3D projections of the data set starting at dome stage are shown (4.2 hpf). Note that predominantly the rhodamine-negative cells become covered by rhodamine-positive surface cells. The same principal observation was made in all three embryos that were imaged. Duration, 129 minutes.
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