In situ hybridization of tmem88a and nkx2.5 in developing zebrafish embryos. (A-J2) tmem88a and nkx2.5 expression during early development in zebrafish. Whole-mount in situ hybridizations for tmem88a (A-E2) and nkx2.5 (F-J2) were performed on embryos ranging from 10.5 hpf to 24 hpf. (K-N) Double fluorescent in situ hybridizations for nkx2.5 (green) and tmem88a (red) at 13 hpf (eight-somite stage; K,L) and 19 hpf (M,N). The white dotted box in merged panels (K-M) outlines the region shown at 4× magnification to the right. A-J, K and M are lateral views with anterior to the left. A2-J2, L and N are dorsal views with anterior to the bottom. Scale bars: in J2, 200 μm for A-J2 in M, 200 μm for K,M; in N, 200 μm for L,N.

Genes upregulated in TMEM88 KD cells suggest activation of endothelial lineages. (A) Quantitative PCR analysis at day 5 and day 14 of selected genes involved in mediating endothelial cell development and differentiation. (B) Brightfield images (top) and immunofluorescence for vWF (bottom) of TMEM88 shRNA KD cells compared with control at day 5. vWF, green; DAPI, blue. Scale bars: 100 μm (brightfield); 200 μm (immunofluorescence). (C,D) Representative flow cytometry plot (C) and quantification (D) of mean fluorescence intensity of KDR and PDGFRα at day 5 for control versus TMEM88 KD populations. n=3-7 per group. Error bars represent s.e.m. ^*P<0.05.

TMEM88 isoform CRA_a is the isoform expressed in cardiomyocyte differentiation. (A) Schematic of TMEM88 isoforms. (B) PCR analysis of transcripts from cDNA (top) and RNA (bottom) at day 5 of cardiac differentiation showing that TMEM88 isoform CRA_a is the dominant isoform expressed in heart development.