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First published online March 20, 2009
doi: 10.1242/10.1242/dev.031872

Development 136, 1363-1374 (2009)
Published by The Company of Biologists 2009
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Map2k1 and Map2k2 genes contribute to the normal development of syncytiotrophoblasts during placentation

Valérie Nadeau*, Stéphanie Guillemette*, Louis-François Bélanger, Olivier Jacob, Sophie Roy and Jean Charron{dagger}

Centre de recherche en cancérologie de l'Université Laval, Centre Hospitalier Universitaire de Québec, L'Hôtel-Dieu de Québec, Québec, QC, G1R 2J6, Canada.


Figure 1
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  		Fig. 1. Underdevelopment of the labyrinth region in the placenta from Map2k1Map2k2 DH mutants. Hematoxylin and Eosin staining of E10.5, E12.5 and E14.5 control (Map2k1+/+Map2k2+/-), type I and type II DH (Map2k1+/-Map2k2+/-) placenta sections. The type I DH specimens show a reduction in the thickness of the labyrinth region (double-headed arrowheads). Large maternal blood sinuses (arrows) and MTG cells (*) are also observed in type I DH mutants. Scale bar: 100 µm. g, trophoblast giant cells.

 

Figure 2
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  		Fig. 2. The underdevelopment of the labyrinth region in Map2k1+/-Map2k2+/- placenta results from reduced proliferation and increased apoptosis. (A) Proliferation was assessed by immunostaining with phospho-histone H3 antibody on paraffin sections of E10.5, E12.5 and E14.5 control (Map2k1+/+Map2k2+/-), type I and type II DH (Map2k1+/-Map2k2+/-) placentas. The percentage of labyrinth trophoblasts in proliferation is presented. A statistically significant reduction in proliferation was observed for type I (filled circles) and type II (open circles) DH placentas when compared with control specimens (triangles) at all ages analyzed. (B) Apoptosis was detected by TUNEL assay and the percentage of labyrinth trophoblasts in apoptosis is presented. The apoptotic cell ratio was significantly increased at E12.5 for DH placentas. Moreover, the type I DH mutant (filled circles) showed an important increase in apoptosis at E14.5 when compared with type II DH (open circles) and control specimens (triangles).

 

Figure 3
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  		Fig. 3. Abnormal vascularization of Map2k1+/-Map2k2+/- placenta labyrinth. (A) The embryonic vascular network of E10.5, E12.5 and E14.5 control (Map2k1+/+Map2k2+/-) and type I DH placentas was revealed by anti-CD31 staining. (B) The maternal sinuses were detected using alkaline phosphatase activity, which labels the cells lining the maternal blood network. In type I DH placentas (Map2k1+/-Map2k2+/-), the fetal blood vessels as well as the maternal sinuses were less abundant when compared with the control specimens at all stages analyzed. Scale bar: 50 µm.

 

Figure 4
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  		Fig. 4. Placenta phenotype of Map2k1Map2k2 compound mutants. Hematoxylin and Eosin staining of E10.5 placenta sections from embryos carrying all possible allelic combinations except the Map2k1-/-Map2k2-/- mutants that were never obtained. (A-D) Map2k1 Map2k2 genotypes that conferred a normal labyrinth development. (E-H) Compound mutants exhibiting placenta defects, the severity of which increases as seen from left to right. The most severe phenotype was observed in Map2k1{Delta}/{Delta}Map2k2+/- specimens (H). Scale bar: 100 µm.

 

Figure 5
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  		Fig. 5. ERK/MAPK activation in Map2k1Map2k2 compound mutants and differential expression of MAP2K1 and MAP2K2 proteins in the placenta. (A) Expression of MAPK1, MAP2K1 and MAP2K2 proteins, and phosphorylation levels of MAPK1/MAPK3 were evaluated by western blot analysis of total protein extracts from E.10.5 wild-type and Map2k1Map2k2 compound mutant placentas and embryos. The analyses of embryonic and placenta extracts from compound mutants without Map2k1 or Map2k2 gene function revealed that the placenta could be contaminated by maternal tissues during dissection, as traces of MAP2K1 or MAP2K2 proteins were observed in few samples. Phosphorylation of MAPK1 and MAPK3 was significantly reduced in Map2k1{Delta}/{Delta}Map2k2+/+ and Map2k1+/{Delta}Map2k2-/- embryos and placentas. MAPK1 detection was used as loading control. (B) MAP2K1 and MAP2K2 immunostaining was performed on E10.5 wild-type, Map2k1-/- and Map2k2-/- placentas. Controls were done without primary antibody (not shown), and the specificity of each antibody was confirmed on Map2k1-/- or Map2k2-/- specimens. In wild-type specimens, MAP2K1 was mainly detected in the cells lining the maternal sinuses most likely the SynT (arrow) and no expression was detected in the mononuclear giant cells (arrowheads). MAP2K2 expression was ubiquitous in the placenta. Scale bar: 50 µm.

 

Figure 6
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  		Fig. 6. Characterization of the MTG cells in Map2k1+/-Map2k2+/- placentas. (A) In E12.5 type I DH mutant placenta sections, the MTG cells, indicated by an asterisk, were devoid of fibrin deposits stained in red with MSB (arrows). (B) Hematoxylin and Eosin staining showed the presence of MTG cells with multiple nuclei located at the periphery (arrow). (C) The multinucleated nature of the MTG cells was also revealed by the DAPI/Phalloidin staining (arrowhead). The absence of Gcm1 expression (D) and alkaline phosphatase activity (E) in MTG cells suggested that they were not SynT. The arrows indicate positive signals. (F) No PAS staining was detected in MTG cells, indicating that they were not glycogen trophoblast cells (arrow). (G) The MTG cells were also negative for CD31, indicating that they were not vascular endothelial cells (arrowhead). (H) No BrdU incorporation in the nuclei of the MTG cells (arrow) was observed, indicating that these cells are post-mitotic as the SynT. Positive nuclei adjacent to the nuclei of the MTG cells were rarely observed (arrowhead). Scale bars: 200 µm in A; 100 µm in B,C,D,F,H; 50 µm in E,G.

 

Figure 7
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  		Fig. 7. The MTG cells derive from Gcm1-positive SynT and result from a cell-autonomous defect. (A-C) lacZ expression following Gcm1Cre expression in R26R Cre reporter mouse line. Sagittal cryosections of Gt(ROSA)26Sor+/tm1SorTg+/Gcm1Cre placentas at E8.5 (A) and E10.5 (B) were tested for β-galactosidase activity. (A) At E8.5, lacZ expression was restricted to cells at the chorioallantoic interface (arrow). (B) At E10.5, the staining was limited to the cells lining the maternal sinuses (arrowheads). (C) β-galactosidase activity in Map2k1+/{Delta}Map2k2+/-Gt(ROSA)26Sor+/tm1SorTg+/Gcm1Cre placentas was detected in MTG cells (arrows). (D-G) Hematoxylin and Eosin staining of placenta sections from E12.5 Map2k1+/-Map2k2+/- (D), Map2k1+/floxMap2k2+/-Tg+/+ (E), Map2k1+/floxMap2k2+/-Tg+/Gcm1Cre (F) and Map2k1+/floxMap2k2+/-Tg+/Sox2Cre (G) specimens. (D) MTG cells indicated by the asterisk were detected in Map2k1+/-Map2k2+/- positive control. The specific inactivation of one Map2k1 allele in Map2k2+/- SynT-II (Map2k1+/floxMap2k2+/-Tg+/Gcm1Cre) led to the formation of MTG cells (F). By contrast, when the Map2k1 deletion was restricted to the embryo (Map2k1+/floxMap2k2+/-Tg+/Sox2Cre), no MTG cells were observed in placenta (G). Scale bars: 50 µm in A; 100 µm in B-G.

 

Figure 8
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  		Fig. 8. Lack of MAPK1 and MAPK3 activation in MTG cells. MAP2K1 (A,B), MAP2K2 (C,D), phospho-MAP2K1/2 (E,F) and phospho-MAPK1/3 (G,H) immunostainings were performed on E12.5 wild-type (A,C,E,G) and Map2k1+/{Delta}Map2k2+/- (B,D,F,H) placentas. MAP2K1, MAP2K2 and phospho-MAP2K1/2 were detected in MTG (*) cells. Phospho-MAPK1/3 was detected in SynT lining the maternal sinuses (arrows) from wild-type (G) and Map2k1+/{Delta}Map2k2+/- (H) specimens but not in the MTG. Scale bar: 50 µm.

 

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© The Company of Biologists Ltd 2009