Prdm6 Is Essential for Cardiovascular Development In Vivo
Figure 3
Regular differentiation, recruitment and proliferation of Prdm6-deficient smooth muscle cells.
(A) ES cell lines were differentiated in vitro into SMA-positive (stained in red) smooth muscle-lineage cells (nuclei are stained in blue by DAPI). The numbers and frequencies of smooth muscle-lineage cells obtained from wild type control and two independent Prdm6-deficient (del/del) ES cell lines (#1 and #2) are given in the table. (B) Smooth muscle cells are normally recruited to aortic vessels of Prdm6del/del embryos. E12.5 embryos, either Prdm6wt/wt or Prdm6del/del, were immunohistochemically stained against SMA. Scale bars represent 10 µm. (C) Normal proliferation of SMCs in the yolk sac vasculature of Prdm6del/del embryos. Pregnant Prdm6wt/del mice from matings with Prdm6wt/del male mice were injected with BrdU at E11.5 and euthanized, and embryos with the wild type control genotype (wt/wt or wt/del) or Prdm6-deficient genotype (del/del) were sectioned and co-stained using antibodies to SMA (green fluorescence within the cytoplasm), BrdU (red fluorescent nuclei) and DAPI (blue, nuclear). Left: overlays of SMA and BrdU. Right: corresponding overlays of SMA and DAPI. Arrows indicate BrdU-positive SMCs. Representative yolk sac vessels are depicted. SMCs of the aortic arch arteries exhibited equivalent staining (not shown). Scale bars represent 20 µm. (D) Quantitative analysis of immunocytochemical staining, as shown in (C). Total SMCs (SMA + DAPI double-positive cells) and proliferating BrdU-positive SMCs (SMA + BrdU double-positive cells) were counted. The ratio of the latter to the former was defined as the proliferative index (percentage of BrdU-positive SM cells) for yolk sac SMCs (ysSMCs) and aortic arch artery SMCs (aaaSMCs). Data were obtained by counting at least 150 SMCs per vessel type from three embryos per genotype and are depicted as the mean +/- SD.