Number, size and aspect of lipid droplets are affected by overexpression of miR-30b during lactation.

<p>A/ Lipid droplets staining with Nile Red (green) and nuclei staining with DAPI (blue) on frozen histological sections of mammary gland from transgenic (Tg) and wild-type (WT) mice at day-12 of lactation. B/ The relative number of lipid droplets to number of nuclei, counted using ImageJ, is significantly lower in transgenic (Tg) than in wild-type (WT) mice. C/ The total area (measured in pixels with ImageJ) of the lipid droplets relative to their number is significantly higher in transgenic (Tg) than in wild-type (WT) mice. Data represent means ± S.E. obtained from day-12 lactating mammary glands frozen sections of 8 transgenic and 6 wild-type mice (10 pictures analyzed per individual). a, b: indicate a significant difference among lines (p<0.05, ANOVA).</p

Prion Protein and Shadoo Are Involved in Overlapping Embryonic Pathways and Trophoblastic Development

<div><p>The potential requirement of either the Prion or Shadoo protein for early mouse embryogenesis was recently suggested. However, the current data did not allow to precise the developmental process that was affected in the absence of both proteins and that led to the observed early lethal phenotype. In the present study, using various <em>Prnp</em> transgenic mouse lines and lentiviral vectors expressing shRNAs that target the Shadoo-encoding mRNA, we further demonstrate the specific requirement of at least one of these two PrP-related proteins at early developmental stages. Histological analysis reveals developmental defect of the ectoplacental cone and important hemorrhage surrounding the <em>Prnp</em>-knockout-<em>Sprn</em>-knockdown E7.5 embryos. By restricting the RNA interference to the trophoblastic cell lineages, the observed lethal phenotype could be attributed to the sole role of these proteins in this trophectoderm-derived compartment. RNAseq analysis performed on early embryos of various <em>Prnp</em> and <em>Sprn</em> genotypes indicated that the simultaneous down-regulation of these two proteins affects cell-adhesion and inflammatory pathways as well as the expression of ectoplacental-specific genes. Overall, our data provide biological clues in favor of a crucial and complementary embryonic role of the prion protein family in <em>Eutherians</em> and emphasizes the need to further evaluate its implication in normal and pathological human placenta biology.</p> </div

miR-30 family targets validated in the literature.

<p>miR-30 family targets validated in the literature.</p

Effect of trophoblastic-restricted ShRNA- mediated <i>Sprn</i> knockdown on embryo resorption at E13.5.

*<p>p<0.05 (x² test) when compared to any of the other results.</p><p>These data are a compilation of at least 2 independent experiments. No statistically significant variability was observed between the analyzed litters (more than 4 for each lentiviral infections).</p

Apoptosis and proliferation processes are modified by overexpression of miR-30b during lactation.

<p>A/ Apoptosis is evaluated by TUNEL analysis: the number of TUNEL-positive cells (red) relative to the nuclei (blue) is significantly higher in transgenic (Tg) than in wild-type (WT) mice. B/ Cell proliferation activity is detected by Ki67 immunostaining: Ki67 labeled cells (green) are detected in transgenic mammary gland (Tg) samples whereas it is absent in wild-type (WT) ones. Nuclei (blue) were stained with DAPI and myoepithelial cells (red) were stained with α-SMA. Data represent means ± S.E. obtained from day-12 lactating mammary glands paraffin sections (5 pictures analyzed per individual) of 3 transgenic and 3 wild-type mice for TUNEL analysis and 6 transgenic and 7 wild-type mice for Ki67 immunostaining. a, b: indicate a significant difference among lines (p<0.05, ANOVA).</p

miR-30b overexpression affects the mammary gland structure during lactation and involution.

<p>Histological analyses (HES) of mammary gland from transgenic (Tg) and wild-type (WT) mice at day-12 of lactation (A) and day-6 of involution (B).</p

Ingenuity Pathway Analysis-Top Networks during lactation day-12.

<p>Ingenuity Pathway Analysis-Top Networks during lactation day-12.</p

Ingenuity Pathway Analysis-Top Networks-Involution day-6.

<p>Ingenuity Pathway Analysis-Top Networks-Involution day-6.</p

Genes showing the greatest degree of change at involution day-6.

<p>Genes showing the greatest degree of change at involution day-6.</p

Schematic representation of the embryonic phenotype induced by the lack of PrP and Shadoo.

<p>The lethal consequence of the absence of PrP and Shadoo during early mouse embryogenesis is indicated.</p