p57Kip2 increases HSC numbers through an expansion of the catecholamine-secreting SA compartment. (A) Immunostaining for Th (red) with 4′,6-diamidino-2-phenylindole (DAPI) nuclear stain (blue) on cryosections from E11 p57Kip2 wild-type (WT) and knockout (KO) embryos. Dashed line shows outline of the aorta. Scale bars indicate 50 μm. (B) Percentage of Ngfr⁺ SA cells from E11 p57Kip2 WT and KO embryos in the different cell cycle stages. DAPI–stained Ngfr⁺ E11 AGM cells were analyzed by flow cytometry. Quantification of the catecholamines noradrenaline (C) and dopamine (D) by high-performance liquid chromatography in individual p57Kip2 WT and KO E11 and E12 AGMs. Concentration is measured in femtomole per embryo equivalent (fM/ee). Black lines denote the mean; n = 14. Donor chimerism in recipients of AGM cells from E11 or E12 p57Kip2 WT or KO embryos treated in utero with the α-adrenergic receptor (Adra1 and Adra2) blocker phentolamine (E) or the β-adrenergic receptor blocker propranolol (F). (G) Donor chimerism in recipients of AGM cells from E11 or E12 p57Kip2 WT embryos treated in utero with the specific β-adrenergic receptor blockers betaxolol (for Adrb1), ICI 118 551 (for Adrb2), and SR 59230A (for Adrb3). (H) Donor chimerism in recipients of AGM cells from E11 or E12 p57Kip2 KO embryos treated in utero with the Adrb2 blocker ICI 118 551. Data points represent chimerism in individual recipients of 1ee (1-3 separate experiments for each condition) determined by flow cytometry after 4 months, with the dashed line indicating 5% threshold and the solid line the mean. (I) Flow cytometry analysis of Adrb2 expression on pro-HSCs (VEC⁺CD41⁺CD43^-CD45^–), pre-HSC I (VEC⁺CD41⁺CD43⁺CD45^–), and pre-HSC II (VEC⁺CD41⁺CD43⁺CD45⁺) from E10.5 AGMs. *P < .05, **P < .01, two-tailed, unpaired t test. D, dorsal; SSC, side scatter; V, ventral; VEC, VE-Cadherin.