Alternative splicing signature of the three germ layers. (A) Diagrams of the eight types of AS analyzed in the study. Blue boxes, the constitutive exons; orange boxes, alternatively spliced exons/regions; solid lines, splice junctions. (B) Comparison of genes that are involved in gastrulation or germ layer specification and annotated to have different isoforms with those detected to be subjected to certain AS events using ASD. 1,346 out of 1,561 genes with annotated isoforms could be detected in our study. (C) The significantly different AS events in the three germ layers identified using ASD software (adjusted P-value<0.05). The number and percentage of the representative eight types of AS events are shown. (D-H) Validation of diverse AS events in genes that are functional at E7.5 using qPCR. In each case, the differential spliced exons detected by ASD and visualized using IGV are labeled by dotted boxes. The colored peaks represent the cover heights of the position (left panels). The AS events and the total expression level of each gene were analyzed using qPCR with isoform-specific primers or common primers (*P<0.05, **P<0.01, ***P<0.001) (right panels). Alternative last exons were labeled as 1a and 1b, respectively. The types of AS are shown in parentheses. ALE, alternative last exon; SE, skipped exon; MXE, mutually exclusive exon; e, exon; t, total; in, inclusion; ex, exclusion. Error bars represent s.e.m.; n=3.

Alternatively spliced genes are extensively involved in key developmental processes. (A) Analysis of the genes with significantly different AS events (adjusted P-value<0.05) shared by the three germ layers using Venn diagram. (B) GO term enrichment analysis of the 1,279 genes generating the 1648 AS events (P<0.01). End, endoderm; Mes, mesoderm; Epi, epiblast.

Splicing factors are differentially expressed and alternatively spliced across the germ layers. (A) The heat map of 39 significantly differentially expressed RNA splicing factors in the germ layers (at least one RPKM>5, Fold Change>2, FDR<0.001). (B) Venn diagram showing the targets of ESPRs (ESRP1 and ESRP2), ELAVL3 and PTBP2 in the 1,279 differential alternatively spliced genes. (C) Venn diagram showing the 73 alternatively spliced splicing factors. (D) Venn diagram showing the 11 differentially expressed and simultaneously alternatively spliced splicing factors. The name of the 11 splicing factors was shown. (E) The RPKM value and normalized signal intensity of Rbfox2 in the RNA-seq and Microarray data, respectively. Four probes were used in the Microarray analysis to detect Rbfox2. (F) The differential AS events of Rbfox2 between the three germ layers visualized using IGV software with RNA-seq mapped reads. The differential spliced exon detected by ASD is labeled by dotted boxes. The alternative first exons due to selective use of distal and proximal promoters are labeled as 1a and 1b, respectively. The colored peaks in each case represent the cover heights of the position. (G) Validation of the AS events and the total expression level of Rbfox2 using qPCR with isoform-specific primers or common primers (*P<0.05, **P<0.01, ***P<0.001). End, endoderm; Mes, mesoderm; Epi, epiblast. Error bars represent s.e.m.; n=3. See also Fig. S2.

AP usage is a prevalent machinery in controlling gene expression in post-gastrulation embryos. (A) GO term enrichment analysis of the 383 genes generating the 430 AP events (P<0.01). (B) Venn diagram of the significantly different AP usage events in the three germ layers (adjusted P-value<0.05). (C) Functional enrichment of GO terms for genes showing differential AP events between endoderm and mesoderm (End versus Mes), endoderm and epiblast (End versus Epi), and mesoderm and epiblast (Mes versus Epi), respectively.

Validation of the AP usage events in E7.5 mouse embryonic germ layers using qPCR. (A-J) Genes with significantly different AP events across the three germ layers identified by RNA-seq data analysis using IGV software. In each case, the positions of the predicted differential first exons generated by APs are labeled by dotted boxes. The colored peaks represent the cover heights of the position (left panels). The AP events and total expression level of each gene were analyzed using qPCR with exon-specific primers and common primers, respectively (*P<0.05, **P<0.01, ***P<0.001) (right panels). Alternative first exons in each gene were labeled as 1a, 1b, 1c and 1d, respectively. No coverage of Ash2l-1c and Ubtf-1c was detected by IGV. Error bars represent s.e.m.; n=3.