(A) Targeting strategy to generate the Kdm2b conditional allele. Exons 16–19 were targeted with loxp sites and a neomycin (Neo) cassette flanked by Frt sites. To remove the Neo cassette and generate the Kdm2b^lox allele, mice were crossed with the actin-Flpe strain. Tissue-specific deletion was achieved by crossing with appropriate Cre strains. Red arrows depict the position of PCR primers for the detection of Kdm2b^lox (P1:P2) and the recombined allele Kdm2b^Δ (P3:P4). (B) Upper left: representative PCR to detect the Kdm2b^lox allele in tail-tip DNA samples from WT, heterozygous, and homozygous mice. Bottom left: Kdm2b^fl/fl MEFs infected with adenoviruses that express GFP or Cre recombinase. PCR of genomic DNA shows excision of the floxed allele upon Cre expression. Asterisk indicates internal PCR control. Right: Western blot showing downregulation of long and short isoforms (arrowheads) of KDM2B from MEF whole-cell lysates. NS, nonspecific. (C and D) Gross images of WT and Kdm2b-null embryos at (C) E9.5 and (D) E10.5. Neural tube defects and excencephaly (white arrows), craniofacial defects (red arrow), and lack of yolk sac vasculature were observed in KO embryos. The red dotted line traces the aorta/AGM. Scale bars: 0.5 mm (C); 1 mm (D). (E) Gross images of E11.5 WT and Tie2-Cre Kdm2b^fl/fl embryos. Red dotted line traces the aorta/AGM, and the black arrow points to the heart. Scale bar: 2 mm. (F) Flow cytometric analysis of dissected AGM showing the frequency of double-positive VE-cadherin/CD45 cells. Graph shows the mean ± SEM. n = 3. *P < 0.05. (G) Immunofluorescent staining of aortas from E10.5 embryos for KDM2B (red; nuclear) and c-Kit (green; cell surface). White arrow shows budding hemogenic endothelium and yellow arrow a circulating “stem-like” cell. Nuclei were stained with DAPI (blue). Scale bars: 10 μm.