Figure 1.
Expression of Mst1 and Mst2 in E9.5 and E10.5 mouse placentas.
(A and B) Images of E9.5 and E10.5 placenta sections of wild type (129) mouse embryos immunohistochemically stained with anti-Mst1 (A) and anti-Mst2 (B) antibodies. High magnification images of the cropped regions in (A) and (B) are shown under each panel, respectively. White dashed lines indicate the boundary between the giant trophoblast cell layer and maternal decidua and red dashed lines designate the boundaries between the labyrinth and allantois. Black dashed lines mark the spongiotrophoblast layer. (C) The negative control for immunohistochemistry. Images of placenta sections from E10.5 Mst1−/− Mst2+/− (left) or Mst1+/− Mst2−/− (right) embryos stained with anti-Mst1 or anti-Mst2 antibody, respectively. Images are representative sections from at least three placentas from three pregnant mice per group. Al, allantois; De, decidua; Gi, trophoblast giant cells; La, labyrinth; Sp, spongiotrophoblast layer.
Figure 2.
Impaired differentiation of trophoblast giant cells and spongiotrophoblast cells and labyrinthine development in the Mst1/2 DKO placenta.
(A, C and D) Images of hematoxylin/eosin-stained placental sections of the control (Ctr) and Mst1/2 DKO embryos at E8.5 (A), E9.5 (C) and E10.5 (D). (B) Statistical analysis of TGC in the ectoplacental cone of E8.5 and E9.5 placental sections. Data are expressed as the means ± SEM (three sections/placenta, three placentas from three pregnant mice per group). ** P<0.01. (E) CD31-immunofluoresence staining of cryosections of E9.5 Ctr and Mst1/2 DKO placentas. High magnification images of the cropped region in (A), (C), (D) and (E) are shown under each panel, respectively. Dashed lines indicate spongiotrophoblast layers. Arrows and arrowheads indicate fetal nucleated and maternal enucleated red blood cells, respectively. (F) RNA in situ hybridization of E9.5 placental cryosections with a probe for the Prl3d1 gene which specifically marks TGC (upper panel) and a probe for the Tpbpa gene which marks spongiotrophoblast cells (lower panel). Inset in lower panel is an enlarged photo to show the existence of a weaker Tpbpa signal in Mst1/2 DKO placentas. Images are representative sections from at least three placentas from three pregnant mice per group. Al, allantois; Ctr, control; De, decidua; Gi, trophoblast giant cells; La, labyrinth; Sp, spongiotrophoblast layer.
Figure 3.
Downregulation of Mash2 expression in Mst1/2 DKO placentas.
(A and D) Images of Ki67 immunofluoresence staining (A) and TUNEL assay (D) of the placental cryosections of the indicated genotypes and ages. High magnification images of the cropped region of the ectoplacental cone in (A) were shown above each panel respectively. (B) Statistical analysis of cell proliferation in the ectoplacental cone (left panel) and the labyrinth (right) of E8.5 or E9.5 placentas of the indicated genotypes. (C) Positive control of TUNEL assay with the Dlic1−/− retina section. (E) RNA in situ hybridization analysis of E9.5 placental cryosections with a probe of Mash2 gene. (F) Quantitative RT-PCR analysis of Hand1 (left panel) and Stra13 (right panel) mRNA levels in E9.5 placentas of the indicated genotypes. (n = 3 for each group). Images in (A), (D) and (E) are representative sections from at least three placentas from three pregnant mice per group. Data in (B) and (F) are expressed as the means ± SEM, ns: not significant.
Figure 4.
Defective labyrinthine vascularization/morphogenesis in Mst1/2 CKO placentas.
(A) RT-PCR analysis of Mst1/2 expression in the purified endothelial cells from E10.5 wild type embryos and yolk sacs (left panel) and Flow cytometry analysis of CD31 expression of the purified endothelial cells (right panel). (B) Images of X-gal-stained E9.5 placental cryosections with indicated genotypes. Note: β-gal gene was expressed on blood vessels containing nucleated fetal red blood cells. Black dashed lines indicate the spongiotrophoblast layer. (C) Images of whole-mount X-gal-stained E9.5 yolk sacs with indicated genotypes. (D and E) Images of HE-stained cryosections of Ctr and Mst1/2 CKO placentas at E9.5 (D) and E10.5 (E). High magnification images of the cropped region in (D) and (E) are shown under each panel, respectively. The white dashed lines are the boundaries between maternal decidua and trophoblast giant cell layer. Black dashed lines mark the spongiotrophoblast layer. (F) Statistical analysis of the percentage of fetal blood vessels and maternal sinusoids in the labyrinth layer of control and Mst1/2 CKO placentas at E10.5. Data are expressed as the means ± SEM (three sections/placenta, three placentas from three pregnant mice per group, ** P<0.01). (G) CD31 immunofluoresence staining of cryosections of E10.5 control and Mst1/2 CKO placentas. The labyrinth layer is defined by the red dashed lines. Arrows and arrowheads indicate fetal nucleated and maternal enucleated red blood cells, respectively. Images are representative sections from at least three placentas per group. Al, allantois; Ctr, control; De, decidua; Gi, trophoblast giant cells; Iso, Isotype control; La, labyrinth; Sp, spongiotrophoblast layer.
Figure 5.
The effects of endothelial-specific deletion of Mst1/2 CKO on cardiac and vascular development during midgestation.
(A and B) Photographs of freshly dissected embryos proper (A) and yolk sacs containing embryos (B) with indicated genotypes and ages. Large vitelline blood vessels (white arrows) are observed on both the control and mutant yolk sacs. (C-E) Vasculature in E10.5 yolk sacs (C), head and intersomitic regions of E9.5 (D) and E10.5 (E) embryos of the indicated genotypes visualized by whole-mount anti-CD31 immunohistochemistry. (F and G) HE-stained transverse paraffin sections of E9.5 (F) and E10.5 (G) hearts of the indicated genotypes. Black arrows indicate atrioventricular cusions. Images are representatives from at least three embryos per group.
Table 1.
Numbers of Mst1/2 CKO and Ctr offsprings.