Fig. 2

sox32-positive cells are induced randomly within the first two cell tiers

(A) MIP of RNAscope for sox32 in a 5 hpf embryo. Nuclei marked with DAPI (gray). Dashed white line, embryo margin; arrowhead, dorsal. Inset: Z-reconstruction through the lateral regions of embryo.

(B) Digitization of embryo in (A) showing the centroids from segmentation of all nuclei in the embryo. sox32-positive cells are marked in red. Yellow line, embryo margin; arrowhead, dorsal. Inset: Positions of all nuclear centroids from inset in (A). YSL and cells beyond the margin, and thus eliminated from the analysis, are labeled in magenta. Blue dashed line, embryo margin.

(C) Reconstruction of embryo in (B) showing the position of sox32-positive cells (red). Arrowhead, dorsal.

(D) Distribution of 827 sox32-positive cells around the margin for 20 embryos collected from 4.25 to 5.25 hpf. Note that dorsal enrichment corresponds to DFCs. Dorsal, up.

(E) Relationship between sox32-positive cells (YSL and DFCs removed) and total cell number for embryos collected at 30 min intervals from a single clutch. Colors indicate embryonic stage.

(F) Donut graphs from the scRNA-seq dataset showing the percentage of cells in G1, G2/M or S phase for sox32-positive cells (above) and sox32-negative cells (below).

(G) Scatterplot from the scRNA-seq dataset showing sox32 expression levels in sox32-positive cells falling in the different phases of the cell cycle. Means ± SD are shown. Dotted line shows zero.

(H) MIP of RNAscope for sox32 (red) and tbx16 (cyan) combined with immunofluorescence (IF) for P-H3 (yellow) in a 5-hpf embryo. Nuclei are marked with DAPI (gray).

(I) Z-reconstruction through the lateral regions of the embryo shown in (H). Dashed white line, embryo margin. Arrowhead shows a cell positive for sox32, tbx16, and P-H3.

(J) Scatter dot plot showing percentage of cells staining for P-H3 among cells that are sox32⁺ and tbx16⁺, sox32^– and tbx16⁺, or sox32^–. Means ± SD are shown.

(K) Two representative embryos showing the distribution of sox32-positive cells around the margin. Average direction vector shown on each embryo. Length indicates direction bias and color indicates the total number of sox32-positive cells. Embryo pool shows vectors from the 20 embryos in (D) (only 16 vectors are shown, as the four youngest embryos have no progenitors). Dorsal, up.

(L) Distribution of average direction vectors for the 16 embryos in (K), grouped into 15-degree bins. Dorsal, up.

(M) Plot of direction bias against total number of sox32-positive endodermal progenitors for the 16 embryos in (K).

(N) Proposed model for the appearance of sox32-positive endodermal progenitors. A series of cells at the margin of the embryo proliferate through time (gray bifurcations). In a random manner, cells turn on sox32 (blue outlined cells), and expression is maintained (red cells).

See also Figure S2. Scale bars: 125 μm (A), 50 μm (A, inset), 200 μm (H), 28 μm (I). ^∗p < 0.05; ^∗∗p < 0.01.