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Fig. 3 Cell types and patterns in Nodal explants at the middle gastrulation (A) Single-cell transcriptomes were collected from Nodal explants at 8 hpf. Cell-type annotations and their corresponding colors are shown at the right (clusters colored in gray indicate the cell types of Nodal explant at 10 hpf). (B) UMAP plot of single-cell transcriptomes (leftmost); cells are colored according to their cell-type annotations. UMAP colored according to expression of the indicated genes (selected marker genes of each cluster) (left) and WISH for the same genes in Nodal explants (middle) and embryos (right). (C) Normalized expression domain of ectoderm markers (tfap2c, otx2a, rx3, and foxb1a) (top) and mesoderm/endoderm markers (frzb, sox32, ntd5, and wnt8a) along the A-P axis of Nodal explants at 8 hpf. (D–F) HCR co-staining of key markers of ectoderm (D), mesoderm/endoderm (E) and ectoderm and mesoderm (F) in Nodal explant at 8 hpf. Sections of HCR co-staining samples (locations of the sections as white vertical dotted line and white arrows indicated) were imaged (E and F) to visualize the expression patterns of key markers on the cross sections of Nodal explant along the A-P axis. Normalized expression domains of the genes were shown at the bottom of each panel. (G–J) Simplified schematics of the main cell types and their patterns in the Nodal explants (G, I, and J) and embryos (H) at the same stages. The statistics of observed/total sampled Nodal explants were shown in bottom left of the panels (biological replicates). Each ISH or HCR experiment was performed for at least 2–3 independent replicates (technical replicates). Scale bars: 200 μm (B), 50 μm (D–F). See also Figure S3, Table S1, and Data S1.