Expression of CD45 by mouse and human IACs.

<p>Transverse sections of AGM region were made from ICR mouse embryos at 9.5 and 10.5 dpc and from human embryos at 32 day-old, according to the Carnegie classification, stained with antibodies and observed by confocal microscopy. Arrowheads indicate IACs. (<b>A</b>) Mouse IACs in the omphalomesenteric artery (OMA) at 9.5 dpc expressed c-Kit, but not CD45. CD45 (green) and c-Kit (red). Magnified view of IACs is shown at right upper panel in Merge panel. Original magnification is 40x. (<b>B-D</b>) Mouse IACs in the dorsal aorta (DA) (B), OMA (C) and umbilical artery (UA) (D) at 10.5 dpc expressed c-Kit, and some expressed CD45. CD45 (green) and c-Kit (red). Original magnification is 40x. (<b>E</b>) All human IACs in the DA expressed CD34, and some expressed CD45. CD34 (green), CD45 (red) and TOTO-3 (blue). NT (Neural Tube); Ao (Aorta); Mn (Mesonephros). Original magnification is 20x.</p

Gene expression analysis in CD31⁺/CD34⁺/c-Kit⁺ AGM cells separated by CD45 expression.

<p>(<b>A</b>) Single cell suspensions of the caudal portion of embryos containing the AGM region at 10.5 dpc were prepared and analyzed by flow cytometry. Cells expressing CD31 and CD34, IAC markers, were first gated. The profile shows expression of c-Kit (x-axis) and CD45 (y-axis) in CD31⁺/CD34⁺ AGM cells (left). Based on intensity of CD45 expression, CD31⁺/CD34⁺/c-Kit⁺ AGM cells were separated into three fractions, CD45-negative (under 10² of CD45-fluorescence, same as negative control), -low positive (from 10².⁵ to 10³.⁵ of CD45-fluorescence), and -high positive (approximately over 10⁴ of CD45-fluorescence). Isotype control and compensation samples of flow cytometric analysis are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035763#pone.0035763.s004" target="_blank">Figure S4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035763#pone.0035763.s001" target="_blank">S5</a>. (<b>B</b>) The percentage of CD45-negative, -low positive, and -high positive c-Kit⁺/CD31⁺/CD34⁺ AGM cells was calculated both at 9.5 dpc (white bars) and 10.5 dpc (black bars). (<b>C-H</b>) Gene expression of <i>CD45</i> (C), <i>Runx1</i> (D), <i>c-Myb</i> (E), <i>Evi-1</i> (F), <i>SCL</i> (G) and <i>Gata2</i> (H) was analyzed in sorted CD45-negative, -low positive and -high positive c-Kit⁺/CD31⁺/CD34⁺ AGM cells. Expression levels of <i>CD45</i> mRNA are up-regulated as c-Kit⁺/CD31⁺/CD34⁺ cells express CD45 surface protein. Expression levels of <i>Runx1</i>, <i>c-Myb</i>, <i>Evi-1</i>, <i>SCL</i> and <i>Gata2</i> were highest in CD45-low positive c-Kit⁺/CD31⁺/CD34⁺ cells, whereas that of <i>Evi-1</i> was highest in CD45-negative c-Kit⁺/CD31⁺/CD34⁺ cells. RQ represents relative quantity of template in the original sample.</p

Confocal images of IACs expressing CD31/CD34/c-Kit in the AGM region.

<p>Transverse sections of AGM region from ICR mouse embryos at 9.0 and 10.5 dpc were stained with antibodies and observed by confocal microscopy. (<b>A</b>) IACs were observed in the omphalomesenteric artery (OMA) at 9.0 dpc (left; magnified view of IACs in upper right panel) and in the OMA, dorsal aorta (DA) and umbilical artery (UA) at 10.5 dpc (right). CD31 (red), c-Kit (green), and TOTO-3 (blue). Arrows indicate IACs. Original magnification is 20x. (<b>B-D</b>) IACs were observed in the DA (B), OMA (C) and UA (D) at 10.5 dpc. Left panel shows staining for CD31 (red), c-Kit (green), and TOTO-3 (blue), and right panel shows staining for CD34 (red), c-Kit (green), and TOTO-3 (blue) staining. Images were taken at 40x and zoom was used to show a detail at right lower panel. Another IAC in the DA is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035763#pone.0035763.s001" target="_blank">Figure S1</a>. (<b>E</b>) IACs expressing Ki-67, a marker of proliferation, were observed in the DA (left), OMA (middle) and UA (right). Ki-67 (red), c-Kit (green), and TOTO-3 (blue). Images were taken at 40x and zoom was used to show a detail.</p