Fig 1.
Developmental stages selected for Cap Analysis of Gene Expression (CAGE) study.
(A) Representative images of chicken embryos used for RNA preparation. (B) Temporal distribution of these selected stages (red dots) during chicken embryogenesis. X-axis: 46 Hamburger and Hamilton (HH) stages represented in numerical order. Y-axis: log scale of incubation time needed to reach these stages (based on reported estimates), e.g., Y value of 2.0 = 100 hours. Numerical values for this plot can be found in S1 Data. (C) Principal coordinates analysis (PCoA) of all 16 developmental samples. Four groups, representing early, mid, mid-late, and late developmental phases, can be recognized. (D) PCoA of all samples. Primary cells are clearly grouped away from each other and from developmental samples. Raw data for PCoA can be accessed at http://fantom.gsc.riken.jp/5/suppl/Lizio_et_al_2017/.
Fig 2.
Chicken-ZENBU views of transcription start site (TSS) peaks and expression levels.
(A) A robust single TSS peak (arrow) is correctly mapped to the known Ensembl and RefSeq 5′ end of GAPDH. Right panel: zoomed-in view of left panel. (B) Ambiguity in ACTB gene annotation can be resolved using Cap Analysis of Gene Expression (CAGE). (C) The TSS representative of RPL32 gene does not confirm either available annotation, suggesting the incorrectness of both gene models. (D) Bar graph of expression values shows NANOG pluripotency gene present at early stages, then down-regulated at later stages. (E) Late stage–specific expression of GFAP gene (Hamburger and Hamilton stage 41 [HH41] and HH45). Samples in the bar graphs are sorted by developmental stage.
Fig 3.
Expression profiles of stage- and cell type–specific transcription start sites (TSSs).
Cap Analysis of Gene Expression (CAGE) TSSs associated with pluripotency and germ layer–specific genes show distinct expression patterns during development. Pluripotency-related genes (NANOG, POU5F3, MYC, EOMES) show early stage–specific expression. Ectoderm-, mesoderm-, and endoderm-related genes show opposite expression patterns, being activated at later stages of development. X-axis represents developmental stages; y-axis represents tag per million (TPM) expression values on a logarithmic scale. Numerical values for this plot can be found in S1 Data.
Fig 4.
Expression in tags per million (TPM) across all 26 samples for the top 30 housekeeping genes with lowest variance is compared to expression of well-known housekeeping genes GAPDH and ACTB. Genes are listed by variance (low to high). Numerical values for this plot can be found in S1 Data.
Fig 5.
Shared and unique transcription factor binding site motifs in the vicinity of transcription start sites (TSSs) enriched in sample subgroups.
(A) The top 50 enriched motifs in each subgroup are shown. Embryo group contains early, middle, middle-late, and late subgroups. Primary cells group contains mesenchymal stem cells (MSCs), aortic smooth muscle cells (SMCs), and hepatocytes subgroups. Other tissues group contains leg and wing buds and extraembryonic subgroups. (B) Venn diagram showing overlaps of those top 50 motifs for embryo, primary cells, and other tissues against housekeeping groups. The number of shared motifs between groups is low, suggesting stage- or cell type–specific regulatory programs.
Fig 6.
Induction of endogenous Brachyury expression mediated by Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-on.
(A) Chicken-ZENBU view of a 6-kb window on chromosome 3, showing the transcription start site (TSS) location for the Brachyury gene. Small rectangle represents the region shown in (B). (B) Sequences of 4 single guide RNAs (sgRNAs; red underline) located within 300 bp upstream of the Brachyury TSS. Blue: protospacer-adjacent motif (PAM). (C) Electroporated embryos were grown to Hamburger and Hamilton stage 10 (HH10), and sgRNA-expressing territories were assessed by co-electroporated GFP signal. Embryos were then processed for Brachyury in situ hybridization (shown here). (D) Magnified view of mid-region of the embryo, showing ectopic Brachyury+ cells in somites and neural tube.