Fig. 2

Lack of hematopoietic stem cells (HSC) in Runx1 ^P2TAG/P2TAG embryos that harbors an aberrant RNA splice junction in the P1-Runx1 transcript. a Representative dot plots of fetal liver cells from Runx1 ^+/+ and Runx1 ^P2TAG/P2TAG 11.5 dpc embryos. Lin-negative (Lin⁻) cells were analyzed for ScaI and c-Kit expression. b Results of three independent colony forming assays of fetal liver cells. No colonies formed from Runx1 ^P2TAG/P2TAG cells. c Immunohistochemical analysis of c-Kit and CD31 expression in the dorsal aorta of 11.5 dpc embryos. In control Runx1 ^+/P2TAG samples, round c-Kit positive cells were seen budding from CD31 positive endothelial cells, whereas those cells were undetectable in Runx1 ^P2TAG/P2TAG embryos. Right graph shows a summary of the numbers of c-Kit expressing cells in the dorsal aorta of three embryos. Mean ± SD. d Immunoblot showing expression of Runx1 and Runx3 proteins in fetal liver from 11.5 dpc Runx1 ^+/+ and Runx1 ^P2TAG/P2TAG embryos. e RT-PCR analysis of P1- and P2-Runx1 transcripts in whole 11.5 dpc Runx1 ^+/+ , Runx1 ^+/P2TAG and Runx1 ^P2TAG/P2TAG embryos. Left gel image represents PCR products from a mixture of three primers, whose positions are illustrated on the right. I, II and III represent corresponding exons. f RT-PCR analysis focusing on the P1-Runx1 transcript using two primers, P1F and R. PCR product corresponding to the P1-Runx1 transcript is indicated. All eight P1-Runx1 transcripts from the Runx1 ^P2TAG/P2TAG embryos examined contained an aberrant splice junction between exon I and II, which resulted from upstream TAG sequences. AG in red font indicates a splice acceptor signal and dashed line indicates a splice junction. Aberrant splicing cause a frame shift and deduced amino acids are shown as a single letter