Satou-Kobayashi Y , Kim JD , Fukamizu A , Asashima M .

             somitogenesis
             nuclear envelope organization
             pattern specification process
             endoderm development
             mesoderm development
             axis specification
             cellular response to growth factor stimulus
             negative regulation of endoplasmic reticulum unfolded protein response

	Figure 1. Transcriptomic relationships among AC samples. (A) Overview of the sample preparation for a time-course RNA-seq. ACs were collected without activin A treatment (Pre_activin), and after activin A treatment for 1, 3, 6 and 9 h (Post 1h_activin, Post 3h_activin, Post 6h_activin, and Post 9h_activin). (B) Principal component analysis (PCA) of ACs. The first two principal components PC1 and PC2 represented 53.7% of the total variance. Each dot denotes a single biological replicate, and dashed circles represent three replicates for each individual sample. Black dot, Pre_activin; blue dot, Post 1h_activin; purple dot, Post 3h_activin; green dot, Post 6h_activin; red dot, Post 9h_activin. (C) Hierarchical clustering of the expression profiles between Pre_activin and Post_activin groups. Individual samples are shown in columns, and genes in rows. The upper axis shows the clusters of samples, and the left vertical axis shows clusters of genes. The heatmap represents relative expression (red, high; white, intermediate; blue, low expression). Rep1-3; the biological replicate 1–3. The heatmap was visualized using the CLC Genomic Workbench software 12.0 (QIAGEN).
	Figure 2. Differentially expressed genes (DEGs) between Pre_activin and each Post_activin sample. DEGs were identified in the comparison of gene expression between Pre_activin and each Post_activin sample based on log2 FC ≥ 2 or ≤  − 2 with FDR at p < 0.05. (A) Volcano plots representing DEGs in each comparison. Each dot represents individual DEG. Dotted vertical lines, log2 FC ≥ 2 or ≤  − 2; dotted horizontal line, the significance cut-off (p < 0.05). (B) Line graph showing the total number of upregulated and downregulated DEGs in each comparison. (C) Bar chart showing the ratio of common DEGs among different Post_activin samples and that of unique DEGs in each Post_activin sample. Common DEGs and unique DEGs are represented by solid or stripe pattern fill, respectively. (B,C) Magenta, upregulated DEGs; blue, downregulated DEGs.
	Figure 3. Common upregulated DEGs among Post_activin samples. (A) Venn diagrams showing overlap among upregulated DEGs. The numbers of common and unique upregulated DEGs are displayed in colored ellipses. Known activin responsive genes are listed as indicated. (B) Gene ontology (GO) enrichment analysis of 1861 common upregulated DEGs between Post 1 h, 3 h, 6 h, and 9h_activin. The top ten enriched GO terms in biological processes, and the -log10 (P-value) of the significant GO terms are displayed in order of significance from the bottom of the list. (C) Left Venn diagram showing the comparison of DMZ-enriched genes at stage 10.5 (Ding et al.), those at stage 11 (Kakebeen et al.) and common upregulated DEGs among all Post_activin samples. The representatives of overlapping genes among three studies are indicated. Right Venn diagram showing the comparison of VMZ-enriched genes at stage 10.5 (Ding et al.) and common upregulated DEGs among all Post_activin samples. The numbers of common and unique genes in independent studies are displayed in colored circles.
	Figure 4. Clustering of gene expression trajectories. (A) K-means clustering of 2000 variable genes following activin A treatment. Genes were classified into four clusters based on similar expression patterns. Individual samples are displayed on the vertical axis, and genes on the horizontal axis. The colors in heatmap indicates relative expression of each tile (red, high; white, intermediate; blue, low expression). The heatmap was visualized using the iDEP.93 online tools (http://bioinformatics.sdstate.edu/idep/). The number of genes and schematic diagrams representing specific expression patterns in each cluster are shown on the left. (B) Heatmaps showing the gene expression of five representatives in each cluster. The expression of genes observed in activin A-treated ACs (left), and that in whole gastrula embryos (stages 9, 10 and 12) (right). The transcriptomes of the whole embryos were obtained from “Session et al.” dataset. The color scale bar is shown at the top right corner of the figure. The heatmap represents relative gene expression (red, high; white, intermediate; blue, low expression). The heatmaps were visualized using the CLC Genomic Workbench software 12.0 (QIAGEN). (C) GO enrichment analysis of genes in different clusters and the genes of cluster C assigned in the mesoderm development GO term. (left) The top ten enriched GO terms in biological processes, and the -log10 (P-value) of the significant GO terms are displayed in order of significance from the bottom of the list. Results of GO analysis in each cluster are indicated by different colors: blue, cluster A; yellow, cluster B; purple, cluster C; green, cluster D. (right) The predicted protein–protein interaction (PPI) network for genes in cluster C. Nodes associated with mesoderm development represent as pink circles, and other nodes as white circles. Edges are as grey lines. The thickness of connected lines indicates the strength of the interaction.
	Figure 5. Upregulation of socs3 in ACs following activin A treatment. Semiquantitative RT-PCR of the expression of socs3 in ACs. Dissected ACs were cultured with or without of activin A. (A) Electrophoresis gel of RT-PCR products. #1–3 indicate the three biological replicates. The upper panel shows the expression of socs3 and the lower panel shows the expression of eef1a1, which was used as the loading control. Full size images of each gel are presented in Supplementary Figure S10. For (B), the densitometry for socs3 expression. Error bars indicate s.e.m. (n = 3). **p < 0.01, p-value was calculated using Student’s t-test after the one-way analysis of variance. N.S., not significant. (A,B) Cultivation times are indicated as follows: 0 h, 0 h (the absence of cultivation); 1 h, 1 h; 3 h, 3 h; 9 h, 9 h.

Altmann, The latent-TGFbeta-binding-protein-1 (LTBP-1) is expressed in the organizer and regulates nodal and activin signaling. 2002, Pubmed, Xenbase

Altmann, The latent-TGFbeta-binding-protein-1 (LTBP-1) is expressed in the organizer and regulates nodal and activin signaling. 2002, Pubmed , Xenbase
Amiri, Transcriptome and epigenome landscape of human cortical development modeled in organoids. 2018, Pubmed
Angerilli, The Xenopus animal cap transcriptome: building a mucociliary epithelium. 2018, Pubmed , Xenbase
Ariizumi, Control of the embryonic body plan by activin during amphibian development. 1995, Pubmed , Xenbase
Ariizumi, Dose and time-dependent mesoderm induction and outgrowth formation by activin A in Xenopus laevis. 1991, Pubmed , Xenbase
Ariizumi, In Vitro Induction of Xenopus Embryonic Organs Using Animal Cap Cells. 2017, Pubmed , Xenbase
Asashima, Mesodermal induction in early amphibian embryos by activin A (erythroid differentiation factor). 1990, Pubmed , Xenbase
Bajpai, Insights into gene expression profiles induced by Socs3 depletion in keratinocytes. 2017, Pubmed
Basso, Reverse engineering of regulatory networks in human B cells. 2005, Pubmed
Briggs, The dynamics of gene expression in vertebrate embryogenesis at single-cell resolution. 2018, Pubmed , Xenbase
Bright, Combinatorial transcription factor activities on open chromatin induce embryonic heterogeneity in vertebrates. 2021, Pubmed , Xenbase
Charney, A gene regulatory program controlling early Xenopus mesendoderm formation: Network conservation and motifs. 2017, Pubmed , Xenbase
Chen, Functional evaluation of ES cell-derived endodermal populations reveals differences between Nodal and Activin A-guided differentiation. 2013, Pubmed
Chiu, Genome-wide view of TGFβ/Foxh1 regulation of the early mesendoderm program. 2014, Pubmed , Xenbase
Cordenonsi, Links between tumor suppressors: p53 is required for TGF-beta gene responses by cooperating with Smads. 2003, Pubmed , Xenbase
De Robertis, The establishment of Spemann's organizer and patterning of the vertebrate embryo. 2000, Pubmed , Xenbase
Ding, Spemann organizer transcriptome induction by early beta-catenin, Wnt, Nodal, and Siamois signals in Xenopus laevis. 2017, Pubmed , Xenbase
Ding, Genome-wide analysis of dorsal and ventral transcriptomes of the Xenopus laevis gastrula. 2017, Pubmed , Xenbase
Dunican, xDnmt1 regulates transcriptional silencing in pre-MBT Xenopus embryos independently of its catalytic function. 2008, Pubmed , Xenbase
Erkenbrack, Divergence of ectodermal and mesodermal gene regulatory network linkages in early development of sea urchins. 2016, Pubmed
Fasterius, Analysis of public RNA-sequencing data reveals biological consequences of genetic heterogeneity in cell line populations. 2018, Pubmed
Forrai, Absence of suppressor of cytokine signalling 3 reduces self-renewal and promotes differentiation in murine embryonic stem cells. 2006, Pubmed
Gallinari, HDACs, histone deacetylation and gene transcription: from molecular biology to cancer therapeutics. 2007, Pubmed
Ge, iDEP: an integrated web application for differential expression and pathway analysis of RNA-Seq data. 2018, Pubmed
Gentsch, The Spatiotemporal Control of Zygotic Genome Activation. 2019, Pubmed , Xenbase
Green, Graded changes in dose of a Xenopus activin A homologue elicit stepwise transitions in embryonic cell fate. 1990, Pubmed , Xenbase
Gupta, Developmental enhancers are marked independently of zygotic Nodal signals in Xenopus. 2014, Pubmed , Xenbase
Harland, Formation and function of Spemann's organizer. 1997, Pubmed
Hayata, Dullard/Ctdnep1 regulates endochondral ossification via suppression of TGF-β signaling. 2015, Pubmed
Jukam, Zygotic Genome Activation in Vertebrates. 2017, Pubmed
Kakebeen, A temporally resolved transcriptome for developing "Keller" explants of the Xenopus laevis dorsal marginal zone. 2021, Pubmed , Xenbase
Kaufmann, Antagonistic actions of activin A and BMP-2/4 control dorsal lip-specific activation of the early response gene XFD-1' in Xenopus laevis embryos. 1996, Pubmed , Xenbase
Koide, Xenopus as a model system to study transcriptional regulatory networks. 2005, Pubmed , Xenbase
Kubo, Development of definitive endoderm from embryonic stem cells in culture. 2004, Pubmed
Kuliyev, Expression of Xenopus suppressor of cytokine signaling 3 (xSOCS3) is induced by epithelial wounding. 2005, Pubmed , Xenbase
Lee, Inhibition of FGF signaling converts dorsal mesoderm to ventral mesoderm in early Xenopus embryos. 2011, Pubmed , Xenbase
Li, Murine embryonic stem cell differentiation is promoted by SOCS-3 and inhibited by the zinc finger transcription factor Klf4. 2005, Pubmed
Mamada, Involvement of an inner nuclear membrane protein, Nemp1, in Xenopus neural development through an interaction with the chromatin protein BAF. 2009, Pubmed , Xenbase
Margolin, ARACNE: an algorithm for the reconstruction of gene regulatory networks in a mammalian cellular context. 2006, Pubmed
Miyazaki, mNanog possesses dorsal mesoderm-inducing ability by modulating both BMP and Activin/nodal signaling in Xenopus ectodermal cells. 2012, Pubmed , Xenbase
Morley, A gene regulatory network directed by zebrafish No tail accounts for its roles in mesoderm formation. 2009, Pubmed
Mughal, Reference gene identification and validation for quantitative real-time PCR studies in developing Xenopus laevis. 2018, Pubmed , Xenbase
Ohkuro, Calreticulin and integrin alpha dissociation induces anti-inflammatory programming in animal models of inflammatory bowel disease. 2018, Pubmed
Osada, Activin/nodal responsiveness and asymmetric expression of a Xenopus nodal-related gene converge on a FAST-regulated module in intron 1. 2000, Pubmed , Xenbase
Osada, XMAN1, an inner nuclear membrane protein, antagonizes BMP signaling by interacting with Smad1 in Xenopus embryos. 2003, Pubmed , Xenbase
Pan, The integral inner nuclear membrane protein MAN1 physically interacts with the R-Smad proteins to repress signaling by the transforming growth factor-{beta} superfamily of cytokines. 2005, Pubmed
Papin, Gradual refinement of activin-induced thresholds requires protein synthesis. 2000, Pubmed , Xenbase
Peng, Xenopus laevis: Practical uses in cell and molecular biology. Solutions and protocols. 1991, Pubmed , Xenbase
Rosa, Mix.1, a homeobox mRNA inducible by mesoderm inducers, is expressed mostly in the presumptive endodermal cells of Xenopus embryos. 1989, Pubmed , Xenbase
Ryan, The Xenopus eomesodermin promoter and its concentration-dependent response to activin. 2000, Pubmed , Xenbase
Ryan, Eomesodermin, a key early gene in Xenopus mesoderm differentiation. 1996, Pubmed , Xenbase
Sander, The opposing homeobox genes Goosecoid and Vent1/2 self-regulate Xenopus patterning. 2007, Pubmed , Xenbase
Satow, Dullard promotes degradation and dephosphorylation of BMP receptors and is required for neural induction. 2006, Pubmed , Xenbase
Session, Genome evolution in the allotetraploid frog Xenopus laevis. 2016, Pubmed , Xenbase
Shannon, Cytoscape: a software environment for integrated models of biomolecular interaction networks. 2003, Pubmed
Shimizu, A quantitative analysis of signal transduction from activin receptor to nucleus and its relevance to morphogen gradient interpretation. 1999, Pubmed , Xenbase
Sladitschek, A gene regulatory network controls the balance between mesendoderm and ectoderm at pluripotency exit. 2019, Pubmed
Smith, Identification of a potent Xenopus mesoderm-inducing factor as a homologue of activin A. 1990, Pubmed , Xenbase
Sudou, Dynamic in vivo binding of transcription factors to cis-regulatory modules of cer and gsc in the stepwise formation of the Spemann-Mangold organizer. 2012, Pubmed , Xenbase
Tada, Characterization of mesendoderm: a diverging point of the definitive endoderm and mesoderm in embryonic stem cell differentiation culture. 2005, Pubmed
Taira, The LIM domain-containing homeo box gene Xlim-1 is expressed specifically in the organizer region of Xenopus gastrula embryos. 1992, Pubmed , Xenbase
Takebayashi-Suzuki, Interplay between the tumor suppressor p53 and TGF beta signaling shapes embryonic body axes in Xenopus. 2003, Pubmed , Xenbase
Tan, RNA sequencing reveals a diverse and dynamic repertoire of the Xenopus tropicalis transcriptome over development. 2013, Pubmed , Xenbase
Umair, Dusp1 modulates activin/smad2 mediated germ layer specification via FGF signal inhibition in Xenopus embryos. 2020, Pubmed , Xenbase
Urrutia, Drosophila Dullard functions as a Mad phosphatase to terminate BMP signaling. 2016, Pubmed
van den Brink, Single-cell and spatial transcriptomics reveal somitogenesis in gastruloids. 2020, Pubmed
Wallingford, p53 activity is essential for normal development in Xenopus. 1997, Pubmed , Xenbase
Wang, DNA methylation dynamics during epigenetic reprogramming of medaka embryo. 2019, Pubmed
Watabe, Molecular mechanisms of Spemann's organizer formation: conserved growth factor synergy between Xenopus and mouse. 1995, Pubmed , Xenbase
Watanabe, Regulation of the Lim-1 gene is mediated through conserved FAST-1/FoxH1 sites in the first intron. 2002, Pubmed , Xenbase
Willems, Patterning of mouse embryonic stem cell-derived pan-mesoderm by Activin A/Nodal and Bmp4 signaling requires Fibroblast Growth Factor activity. 2008, Pubmed
Zhuang, Comparison of multi-tissue aging between human and mouse. 2019, Pubmed