Extended Data Fig. 3: Lineage delineation by transcriptome.

This figure is related to Fig. 2. a–c, Quality control of single-cell RNA-sequencing data. a, Sequence quality was evaluated by FastQC. b, Total reads, mapped reads and mapping ratios of 557 single cells. c, Saturation curve of sequencing. d, e, Integrated analysis of embryonic single-cell data from different source. We used the analytical strategies developed previously¹⁵ to analyse single-cell RNA-seq data (255 single cells) from 6–9-d.p.f. embryos in the study and single-cell RNA-seq data (216 single cells) from three previous reports^16,17,18 (later blastocysts or 6–7-d.p.f. blastocysts). The three datasets have previously been analysed¹⁵. d, PCA based on 12 lineage markers (NANOG, SOX2, KLF17 and TDGF1 for EPI; PDGFRA, GATA6, GATA4 and SOX17 for PrE; GATA3, GATA2, KRT18 and TEAD3 for TrB) showed clear separation between EPI, TrB or PrE could be attained for nearly all samples including our single cells from 6–9-d.p.f. blastocysts, which indicates that lineage delamination occurs at 6 d.p.f. The result is consistent with previous findings¹⁵. e, t-SNE analyses using 4,333 viable genes across all samples. The samples from previous studies were defined into four types: intermediate cells, EPI, PrE and TrB¹⁵. The combined Seurat revealed that most of cells independent of cell resource mixed well. Although most of samples were clustered into EPI, PrE or TrB, similar to the results using 12 lineage genes (d), some cells from 6-d.p.f. embryos remained in an intermediate state with overlapping expression of POU5F1, GATA6, PDGFRA and GATA3. Compared with cells from 6-d.p.f. embryos, cells from 7–9-d.p.f. embryos displayed a clearer separation. These data showed that cell fates of 7–9-d.p.f. embryos became more fixed. f–j, Lineage delineation by transcriptome. Analysis of genes corresponding to EPI, PrE and TrB from 7–9-d.p.f. embryos to understand the regulators involved in the segregation process. f, Heat map of lineage-specific genes of EPI, TrB and PrE from 7–9-d.p.f. embryos (Supplementary Table 1). Their representative transcriptional factors and KEGG pathways are shown, respectively. GO terms and KEGG pathways showed EPI-specific genes associated with signalling pathways regulating stem-cell pluripotency including PI3K–AKT, p53, RAP1 and MAPK. PrE-expressing genes related to TGFβ, PPAR and Ras signalling pathways. TrB-specific genes contributed to Hippo, HIF, PPAR and thyroid receptor signalling pathways. Notably, the PI3K–AKT signalling pathway was enriched in EPIs, PrEs and TrBs. To explain the difference, we examined gene expressions of the PI3K–AKT signalling pathway components in three cell types and found that cell types specifically expressed different genes of PI3K–AKT pathway (Supplementary Table 1.4). g, WGCNA dendrogram indicating different gene modules in all single cell samples from 7–9-d.p.f embryos. Three major branches corresponded to PrE (brown module), TrB (blue module) and EPI (turquoise module). h–j, Hub-gene-network analysis of transcriptional factors specific for PrE (brown module), TrB (blue module) and EPIs (turquoise module). The size of dots represents hubness. h, Hub-gene network of the EPI-specific gene module. In addition to well-known transcription factors (NANOG, PRDM14, SOX2, OCT4 (also known as POU5F1), ZSCAN10 and KLF17), new candidate factors may associate with EPI differentiation, such as VENTX, BCL11A, PBX1 and ARGFX. i, Hub-gene network of the PrE-specific gene module. High correlations of some transcription factors with PrE differentiation included GATA4, SOX17, GATA6 and HNF1B. j, Hub-gene network of the TrB-specific gene module. TrB-specific transcription factors, such as MYBL2, TFAP2A, DLX6 and GCM1. k, Comparison of lineage-specific total genes overlapping between a previous study¹⁸ and this study. In the previous study¹⁸, by analysing 5–7-d.p.f. embryos and combining the lineage-specific results, 439, 820 and 222 genes—which significantly maintained TrB-, EPI- and PrE-specific genes, respectively—were identified. Comparison analysis showed that although we identified more EPI-, PrE- and TrB-specific genes by our resource data, core lineage transcription factors (NANOG, POU5F1 and SOX2 for EPI; GATA6, SOX17 and GATA4 for PrE; GATA2 and GATA3 for TrB) are maintained cross different samples. The difference in gene expressions may be contributed by different development stages of embryos. Difference of gene expressions, including transcription factors, is summarized in Supplementary Table 1.6.