Prdm6 affects angiogenic patterning.

<p>(<b>A</b>) Unusual clusters of densely growing vessel structures on yolk sacs of <i>Prdm6</i>-deficient embryos, as observed under a stereomicroscope. Scale bars correspond to 1 mm (left panel) or 100 µm (right panel). (<b>B</b>) Large vessels in the yolk sacs of E12.5 control and <i>Prdm6</i>-deficient (del/del) embryos under a stereomicroscope. Scale bars correspond to 500 µm. (<b>C</b>) Visualization of E10.5 (left panels) and E11.5 (right panels) yolk sac microvascular systems <i>via</i> immunofluorescent staining with an anti-CD31 primary antibody and a Cy3-conjugated secondary antibody. Scale bars correspond to 200 µm. (<b>D</b>) Quantitative morphometric analysis of the yolk sac vasculature as shown in (C). Avascular space and mean vessel diameters of yolk sacs at E10.5 – E 11.5 are shown as mean ± SEM, n=6. More details about this analysis are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081833#pone.0081833.s002" target="_blank">Figure S2</a>.</p

Selective disruption of <i>Prdm6</i> in vascular smooth muscle cells results in perinatal lethality.

<p>(<b>A</b>) <i>Prdm6</i>^wt/del;SM22-Cre mice were crossed with <i>Prdm6</i>^flox/flox mice, and the genotypes of the offspring were analyzed at three weeks of age. The <i>Prdm6</i>^flox/del;SM22-Cre genotype leads to deletion of Prdm6 in the SMC lineage. The frequencies of the resulting genotypes were calculated from a total of 28 offspring animals and compared to the expected Mendelian frequencies. (<b>B</b>) Newborn mice from the same crosses as in (A) were observed at day 1 and day 2 after birth and subsequently were genotyped. (<b>C</b>) Lungs from newborn <i>Prdm6</i>^flox/del control animals (viable) and SMC-conditional <i>Prdm6</i>^flox/del;SM22-Cre animals (deceasing) were embedded in paraffin, and sections were stained with hematoxylin and eosin. Scale bars correspond to 100 µm.</p

Deregulated expression of angiogenesis genes in <i>Prdm6</i>-deficient yolk sacs.

<p>Real-time RT-PCR analysis of selected transcripts identified <i>via</i> global gene expression profiling analysis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081833#pone.0081833.s003" target="_blank">Fig. S3</a>). The expression values obtained from <i>Prdm6</i>-deficient (del/del) yolk sac samples were normalized to the expression values detected in wild type control samples. The housekeeping gene <i>Tbp</i> was expressed at equivalent levels in wild type and <i>Prdm6</i>-deficient yolk sacs.</p

<i>Prdm6</i> deficiency results in embryonic lethality.

<p>(<b>A</b>) <i>Prdm6</i>^wt/del mice were intercrossed. Pregnant mice were euthanized and embryos dissected and genotyped at defined developmental stages. The percentages of viable embryos of the respective genotypes at the different stages of embryonic development (dpc = days post coitum) are indicated; wild type <i>Prdm6</i>^wt/wt (wt/wt) and heterozygous <i>Prdm6</i>^wt/del (wt/del) mice are viable, whereas <i>Prdm6</i>-deficient <i>Prdm6</i>^{<i>del/del</i>} (del/del) embryos begin to die after E10.0, with no <i>Prdm6</i>^{<i>del/del</i>} embryos being found at developmental stages beyond E16.0. (<b>B</b>) Northern blot analysis of <i>Prdm6</i> expression using total embryonic RNA from different developmental stages from wild type embryos. <i>Gapdh</i> expression analysis served as a loading control. (<b>C</b>) Representative wild type control and <i>Prdm6</i>-deficient embryos (del/del) at the indicated developmental stages. White arrows indicate edematous swelling. (<b>D</b>) Transverse heart sections from wild type control and <i>Prdm6</i>-deficient embryos were stained with H&E and analyzed by microscopy. The thin myocardium of <i>Prdm6</i>-deficient embryos (del/del) is indicated by an arrow. Scale bars correspond to 200 µm. </p