Comprehensive expression profile reveals dynamic changes in Xenopus gene expression depending on the duration of activin A treatment.
In amphibian embryo, the animal cap explants treated with activin A, which is a member of TGF-ß family protein, have a potent of Spemann-Mangold organizer-like activities, leading the induction of mesendodermal tissue.  We report the temporal RNA expression profile of the mesendodermal cell lineage induced by activin A to Xenopus animal cap explants, which have multipotency.  Depending on the duration of activin A treatment to animal cap, dynamic changes of gene expressions were observed. Overview of samples: As the sample for RNA-seq, animal cap explants from Xenopus blastulas (stage 8.5) were cultured in activin A solution (50 ng/mL) for 1, 3, 6, and 9 hours (Post 1h_, Post_3h_, Post 6h_, and Post 9h_activin), and activin A-untreated control (Pre_activin) were also prepared. Results: We first tested the principal component analysis (PCA) for gene expression at individual time points, and found that three biological replicates of individual time points made clusters together.  To compare the gene expression between activin A-treated and -untreated samples, we then visualized gene expression of 45099 annotated genes.  The heat map visualization showed the distinct clusters between individual time points, and also revealed the significant changes of gene expression levels depending on the duration of activin A treatment.  Analysis for differentially expressed genes (DEGs) (fold change >2 with false discovery rate at p<0.05) showed upregulated and downregulated genes by activin A treatment, in comparison with Pre_activin sample.  Gene number of upregulated (up), downregulated (down) genes, and treatment time of activin A were described as follows; 2573 (up), 111 (down), 1h; 4149 (up), 1901 (down), 3h; 6323 (up), 4704 (down), 6h; 8858 (up), 6008 (down), 9h.  From the data set of analysis for enriched Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, we assessed the molecular functions and signaling pathway of upregulated DEGs at each time point of activin A treatment, in comparison with Pre_activin sample. Conclusions: Regarding the temporal expression of organizer genes such as goosecoid (gsc), orthodenticle homeobox 2 (otx2) and chordin (chrd), our transcriptome data using mesendodermal cells derived from animal cap explants was correlated with previous findings using mesendodermal region from Xenopus embryos.  Our transcriptome data from animal cap assay may cover the transition of gene expression from undifferentiated cells to mesendodermal tissue during the normal early development. Overall design: Time-course RNA expression data from Xenopus animal cap treated with activin A. mRNA profiles of animal caps were generated by RNA sequencing using the NextSeq 500 (Illumina).