IACs express Twist1 at midgestation in mice. (A) Flow cytometric analysis of HSPCs over the course of development. Single cell suspensions of the caudal portion of embryos containing the p-Sp/AGM region at 9.5 dpc (upper left) and 10.5 dpc (upper right) were prepared. Cells expressing both CD31 and CD34 were gated first, and then among those cells, c-Kit⁺ cells were analyzed. FL cells at 14.5 dpc (lower left) were also prepared. Lineage⁻Sca1⁺ cells were gated first, and then among them, c-Kit⁺ and CD45⁺ cells were analyzed. BM cells at 2 to 3 months old (lower right) were prepared. Lineage⁻ cells were gated, and c-Kit⁺ and Sca1⁺ cells were analyzed among Lineage⁻ cells. Images are representative of 3 independent flow cytometric analyses (n = 3). (B) Twist1 expression in HSPCs. RNA was extracted from sorted HSPCs at 9.5-dpc and 10.5-dpc AGM region, 14.5-dpc FL, and 2- to 3-month-old BM shown in panel A, and Twist expression assessed using a TaqMan probe. Comparative expression levels were calculated using the 2^−ΔΔCT method. The 14.5-dpc FL HSPC sample served as a reference. Data shown are means ± SD of technical triplicate samples. *P < .05. (C) Confocal images of IACs expressing Twist1, CD31, and CD34 in AGM. AGM region sections were prepared from C57BL/6 mouse embryos at 10.5 dpc, stained with indicated antibodies, and observed by confocal microscopy. Left panels show Twist1 (green), CD31 (red), and TOTO-3 (blue) staining. Right panels show Twist1 (green), CD34 (red), and TOTO-3 (blue) staining. Magnified views of respective dashed boxes are shown adjacent at right. Bars represent 20 μm for all panels.