Extended Data Fig. 9: Markers, transcriptional and morphogenetic functions of TGFβ signalling.

a, Single-molecule FISH for Id2, Bmp4 and Nodal genes of the TGFβ pathway, in an E3.5 blastocyst counterstained with Hoechst. Images are representative of five independent blastocysts. Scales bars are 30 µm for Id2 and 50 µm for Nodal and Bmp4 b, CRISPR strategy for the generation of Nodal deletion in ES cells. c, Venn diagram of all genes regulated after exposure to BMP4, Nodal or both (synergy). RNA sequencing showed that BMP4 and Nodal regulated a similar number of genes (BMP4: 904 genes, Nodal: 926 genes, genes with P < 0.05 (DESeq negative binomial distribution), of which 30% overlapped (413 genes). The GO analysis is shown for each group. See also Supplementary Table 2 sheets 1–4. d, RNA-sequencing analysis of the WNT-related genes in trophospheres stimulated with activators of the TGFβ signalling pathway. All genes are significantly regulated (P = 0.03, DESeq negative binomial distribution). This included the ligand Wnt6 (1.6-fold, P = 0.006, DESeq negative binomial distribution), which is expressed primarily in the cells surrounding the blastocyst cavity¹⁰, the corresponding receptor Fzd7 (2.8-fold, P = 0.002, DESeq negative binomial distribution), the intracellular effector Tcf4 (also known as Tcf7l2; 1.7-fold, P = 0.001, DESeq negative binomial distribution), the negative-feedback regulator Axin1 (1.5-fold, P = 0.005, DESeq negative binomial distribution, top), and of the reported cooperative SMAD/β-catenin targets Msx2 (1.6-fold, P = 0.00003, DESeq negative binomial distribution)⁴⁵ and Ctgf (3.6-fold, P = 0.003, DESeq negative binomial distribution)⁴⁶ (bottom). See also Supplementary Table 2 sheets 1–4. e, RNA-sequencing analysis of the Tgfb-related gene Id2 in TS cells, ES cells, blastoids, trophospheres, blastocysts (left) and in trophospheres after stimulation with Nodal, BMP4 or both (see Methods) (right). n = 2 independent biological samples. The centre depicts the mean. Error bars denote the s.d. f, Number of transcripts measured by RNA-sequencing analysis for markers of epithelial development Cldn4, Krt8 and Krt18. n = 2 independent biological samples. The centre depicts the mean. Error bars denote the s.d. g, Yield of cystic structures for a combination of ES and TS cells as compared to TS cells alone (trophospheres) and trophospheres stimulated with BMP4 and Nodal. Horizontal bars denote the mean yield. Error bars are s.d. *P = 0.02, Student’s t-test. n = 3 independent microwell arrays (see Methods).