p57Kip2 is highly expressed in the SNS. Immunohistochemistry on cryosections from E11 wild-type embryos. (A) Immunostaining for p57Kip2 (red) and CD34 (green) with 4′,6-diamidino-2-phenylindole (DAPI) nuclear stain (blue). P57Kip2 expression is highlighted in endothelial cells (arrowheads), sub-endothelial mesenchyme (asterisk), and SA cells (arrows). (B) Immunostaining for p57Kip2 (red) and Ngfr (green) with DAPI nuclear stain (blue). P57Kip2 expression is highlighted in sub-endothelial mesenchyme (asterisk) and SA cells (arrows). Dashed line shows outline of the aorta. (C) Immunostaining for Pdgfrß (red) with DAPI nuclear stain (blue). Exclusion of Pdgfrß expression from SA cells is highlighted (arrows). Scale bars indicate 50 μm. (D) Sorting strategy for AGM subpopulations; HSPCs, CD34⁺CD45⁺; endothelial cells (EC), CD34⁺CD45^–; SA cells, Ngfr⁺Pdgfrß^–; mesenchymal cells (ME), Ngfr-Pdgfrß+. Gating was based on fluorescence minus one controls as shown in supplemental Figure 1. (E, F) p57Kip2 mRNA expression (relative to β-actin) by quantitative PCR in subpopulations sorted from E11 and E12 AGMs. n ≥ 3. Histogram represents mean ± SEM. (G) Immunostaining for p57Kip2 (red) and ckit (green) with DAPI nuclear stain (blue) on cryosections from E11 wild-type embryos. Intra-aortic clusters are highlighted by asterisks. D, dorsal; V, ventral.