FIGURE 1

FIGURE 1

Mouse Eomes has multiple isoforms, including a mammalian-specific alternative splicing event. (A) Gene model with conservation track and sequence logos for variant region. All transcripts are Ensembl version 107 annotations - ∆VR transcript is ENSMUST00000111763; FL transcript is ENSMUST00000035020; ∆CTD transcript is ENSMUST00000150633. Annotated transcript sizes are indicated, as well as amino acid conservation of the VR between placental mammals, other tetrapods and teleosts, and the variation within the terminal VR arginine codon. The VR is defined by the amino acids present in ENSMUSP00000035020 (encoded by ENSMUST00000035020) that are absent from ENSMUSP00000107393 (encoded by ENSMUST00000111763). The T-box is outlined in orange and the VR in pink. Asterisks indicate known phosphorylated amino acid residues. RT-PCR primer pairs are indicated as half arrows and colour-coded as follows: black–to establish connectivity between the annotated start codon and ∆CTD isoform 3′ UTR; green–to assess total Eomes through amplification of exon 2–4; blue–to amplify Eomes cDNA between exon 4 and the distal 3′ UTR; red–to assess alternate splicing at exon 6. (B) BLOSUM62 average distance evolutionary tree of the Tbr1 subfamily showing relationships between mouse and zebrafish genes. (C) Northern blot showing Eomes transcripts in different cell types using a probe against the T-box. Data for two independent trophoblast stem (TS) cell lines are shown. Mesendoderm is P19Cl6 cells after 4 days of DMSO induced differentiation. CTLLs are IL-2-dependent T-cell lymphocytes derived from ATCC TIB-214. (D) RT-PCR showing relative levels of FL and ∆VR isoforms (left), and nested PCR showing FL/∆VR ratio for long 3′ UTR transcripts (right). Day 4 differentiated embryoid bodies contain cells mimicking embryonic endoderm. CTLLs are IL-2-dependent T-cell lymphocytes derived from ATCC TIB-214. EL4 cells are a negative control for Eomes expression. Gapdh is a loading control. Locations of primer pairs used for RT-PCR are shown in panel A and the text colour-coded accordingly. Nested PCR to analyse exon 6 splicing in transcripts containing the long 3′UTR was performed using the blue primer pair in panel A, followed by the red primer pair.