Fig 2: COUP-TFII binds to the -67/-34 bp Amhr2 region. DNA pulldown assays were performed using either nuclear extracts from MA-10 Leydig cells (A) or in vitro–produced COUP-TFII (B) along with various biotinylated double-strand oligonucleotides containing a high-affinity COUP-TFII DR1 sequence WT (HA-DR1 WT) or harboring a mutation that destroys the binding site (HA-DR1 Mut), the Amhr2 -67/-34 bp sequence either WT or with a mutation in the potential NRE (M1) or in the GC-rich sequence (M4), or the previously characterized SF1 distal and proximal elements. Beads alone (no oligonucleotide) were used as a negative control (Beads). Input in (A) corresponds to 5 µg of the nuclear extracts, and in (B) to 2.5% (v/v) of the product of the in vitro translation reaction used in the DNA pulldown assay (n = 3). Western blots were used to detect COUP-TFII. Input corresponds to 5 µg of the nuclear extracts used in the DNA pulldown assay (n = 3). In (B), two images of the same blot using different exposure times are shown.

Fig 3: Requirement of Tie2 for the maintenance of venous EC identity.(A) Quantitative expression analysis of venous markers including EphB4, APJ and COUP-TFII, and arterial markers including EphrinB2, NRP1, NOTCH1 and Dll4 in lung and retina tissues of Tek mutant and control mice (P7). (B, C) Immunostaining for Dll4 with retinas of Tek mutant and control mice (P9). Scale bar: 100 µm in B and C.DOI: http://dx.doi.org/10.7554/eLife.21032.014

Fig 4: Forkhead box transcription factors bind to the ACE2 proximal promoter region. (a) An EMSA was conducted with the R4 DNA probe. Nuclear extracts were from untransfected 832/13 cells or 832/13 cells transfected with COUP-TFII or PPAR? expression plasmids. Antibodies against COUP-TFII and PPAR? were included in the binding reactions as indicated. (b) The R6 region has similarity to FOXO1 and FOXA1 motifs as indicated by the BKL TRANSFAC program, whereas the mutation destroys the similarity. (c) An EMSA was done with the R6 probe and nuclear extracts from 832/13 cells that were untransfected or transfected with a FOXO1 expression plasmid (left panel). The right panel shows the effect on the band pattern when an antibody against FOXO1 is included. (d) An EMSA was done with the R6 probe and nuclear extracts from 832/13 cells that were untransfected or transfected with FOXA1, FOXA2, or FOXA3 expression plasmids (left panel). The right panel shows the effects on the band pattern when antibodies against the FOXA transcription factors are included in the binding reactions. (e) An EMSA was conducted with a nuclear extract from 832/13 cells. The probes were the human and mouse R6 regions as well as regions in the distal promoter region with a putative FOXA binding site. An antibody recognizing FOXA1 and FOXA2 was included in the binding reactions as indicated.

Fig 5: Inhibition of RA signaling pathway by BMS biases the differentiation of hiPSC toward Sinoatrial node-like cells. (A) BMS was introduced on day 5 at the concentrations indicated, the effect on the expression of NPPA, MYL7, COUPTFII, KCNJ5, and CX40 was analyzed by qPCR. (B) 5 µM BMS was introduced on day 5 after the differentiation, and the expression of SHOX2, TBX18, TBX3, HCN4, ISL1, CX30.2, CACNB1, CACNA1A, KCNN4, KCNK2, KCND2, and SCN3B was quantitated by qPCR on day 21. The expression was normalized to that of GAPDH. Data are presented as ‘Mean±SD’ from at least 3 independent experiments with duplicate on each sample, with ns denoting non-significant, *denoting p<0.05, **denoting p<0.01, and ***denoting p<0.001.