Fig. 1.

Fig. 1.

Pharyngeal mesoderm contains two vascular progenitor subpopulations. (A) In situ hybridization analysis of the expression of ZsYellow and nkx2.5. Numbers indicate PAA clusters. Lateral views with anterior on the left. (B,C) The expression patterns of the PAA progenitor marker nkx2.5 (B), and angioblast markers scl and etv2 (C) in Tg(nkx2.5:ZsYellow) embryos were detected by fluorescent in situ hybridization. Embryos were first subjected to fluorescent in situ hybridization and then to immunostaining with anti-ZsYellow antibody. Scale bar: 20 μm in B; 50 μm in C. (D) Colocalization analysis of nkx2.5 and etv2 by double fluorescent in situ hybridization. Scale bar: 20 μm. (E-G) The Kaede proteins in PAA cluster 3 and the subsequent posterior pharyngeal mesoderm on the right side of the Tg(nkx2.5:kaede) embryos were photoconverted at 22 hpf (E). Cells on the left side of the pharyngeal mesoderm remained unphotoconverted as an internal control (inset). Embryos were subsequently imaged in the green and red channels at 36 hpf (F) and 60 hpf (G). Scale bars: 50 μm. (H-K) Localized photoconversion of PAA cluster 5 (H) or Kaede⁺ cells between cluster 4 and 5 (J) at 36 hpf. Red, green and merged images of the same embryos at 54 hpf are shown in I and K. Scale bars: 50 μm. The photoconversion experiments in E-K were repeated three times independently, and three to five embryos per group were used each time. All the embryos analyzed showed similar distributions of photoconverted cells. The Kaede⁺ cells in PAA cluster 5 and between PAA clusters 4 and 5 gave rise to PAA5 and ventral aorta, respectively, indicating that the pharyngeal mesoderm contains two vascular progenitor subpopulations.