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First published online 3 August 2006
doi: 10.1242/dev.02526

Development 133, 3429-3440 (2006)
Published by The Company of Biologists 2006
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Requirement for Map2k1 (Mek1) in extra-embryonic ectoderm during placentogenesis

Vickram Bissonauth, Sophie Roy, Mathieu Gravel, Stéphanie Guillemette and Jean Charron*

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. The underdevelopment of the labyrinthine region in Map2k1-/- placenta results from reduced proliferation and increased apoptosis. (A) Hematoxylin-Eosin staining of E10.5 wild-type (Map2k1+/+) and Map2k1-/- placental sections. (B) Proliferation was detected by immunostaining with phospho-histone H3 antibody and (C) apoptosis was detected by TUNEL assays on paraffin sections from E8.5, E9.5 and E10.5 wild-type and Map2k1-/- placentas. Arrows indicate examples of mitotic cells (B) and apoptotic cells (C) in the labyrinthine region (or chorioallantoic region at E8.5). The percentage of labyrinthine trophoblasts in proliferation (D) and apoptosis (E) is represented. A statistically significant reduction of the proliferating cell ratio (D) was observed for E9.5 and E10.5 Map2k1-/- placentas. The apoptotic cell ratio was significantly increased in Map2k1-/- placenta at all stages analyzed (E). al, allantois; d, deciduum; g, trophoblast giant cells; l, labyrinth. Scale bars: 100 µm in A; 50 µm in B,C.

 

Figure 2
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  		Fig. 2. ERK/MAPK activation in Map2k1-/- placentas and embryos. Expression and phosphorylation levels of MAPK1/MAPK3 and MAP2K1/MAP2K2 were evaluated by western blot analysis of total protein extracts from E9.5 (A) and E10.5 (B) wild-type and Map2k1-/- embryos and placentas. Phosphorylation of MAPK1 and MAPK3 was significantly reduced in E10.5 Map2k1-/- embryos and placentas, and remained unchanged in E9.5 Map2k1-/- embryos. A reduction was also observed in the corresponding E9.5 placentas.

 

Figure 3
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  		Fig. 3. Localization of phospho-MAPK1/MAPK3 and phospho-MAP2K1/MAP2K2 in wild-type and Map2k1-/- placentas. Anti-phospho-MAPK1/MAPK3 and anti-phospho-MAP2K1/MAP2K2 staining of E9.5 (A) and E10.5 (B) wild-type, Map2k1-/- and Map2k2-/- placentas. (A) At E9.5, MAP2K1/MAP2K2 were phosphorylated in cells lining the maternal sinuses (black arrows), in cells of the allantois (red arrowheads), and in some labyrinthine trophoblasts of wild-type, Map2k1-/- and Map2k2-/- placentas. However, phospho-MAPK1/MAPK3 signal was primarily found around maternal sinuses (black arrows) and in some labyrinthine trophoblasts in wild-type and Map2k2-/- specimens. In Map2k1-/- placentas, MAPK1/MAPK3 were activated only in the allantois (red arrowhead). (B) At E10.5, phospho-MAP2K1/MAP2K2 signal was also observed in the vascular endothelial cells lining the embryonic blood vessels (black arrowheads). Despite the high activation of MAP2K2 at the chorioallantoic interface (red arrows) and in the allantois of Map2k1-/- placentas (red arrowhead), phospho-MAPK1/MAPK3 were detected only in the allantois (red arrowhead). al, allantois; ev, embryonic blood vessel; l, labyrinth; ma, maternal sinus. Scale bar: 50 µm.

 

Figure 4
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  		Fig. 4. Abnormal vascularization of Map2k1-/- placental labyrinth. The embryonic vascular network of E10.5 wild-type (A) and Map2k1-/- (B) placentas was revealed by anti-CD31 staining (black arrows). In Map2k1-/- placentas, the fetal blood vessels were restricted at the chorioallantoic plate (B). Detection of p38 MAPK activation in wild-type (C) and Map2k1-/- (D) placentas revealed a high activation of p38 MAPK in the embryonic blood vessels of the labyrinth of wild-type specimens and at the chorioallantoic interface of Map2k1-/- placentas (black arrowheads). Vegf expression in wild-type (E) and Map2k1-/- (F) placentas was analyzed by in situ hybridization (red signal and blue arrows). High Vegf expression levels were detected in the allantoic region of Map2k1-/- placentas, indicating a response to hypoxic stress. For placental structure identification, near adjacent Hematoxylin and Eosin-stained sections of specimens E and F are presented in Fig. 1A. X-Gal staining of Map2k1+/- (G) and Map2k1-/- (H) placentas was used to define the Map2k1 expression profile in normal and mutant placentas. The labyrinthine region is presented. In Mapk2k1-/- specimens, X-Gal staining is restricted to the chorioallantoic plate. al, allantois; ch, chorionic plate; d, deciduum; g, tropohoblast giant cells; l, labyrinth. Scale bars: 50 µm in A-D,G,H; 100 µm in E,F.

 

Figure 5
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  		Fig. 5. Determination and differentiation of syncytiotrophoblasts in Map2k1-/- placentas. (A-F) The functional differentiation of chorioallantoic trophoblasts in syncytiotrophoblasts was assessed by Gcm1 in situ hybridization (red signal) in E8.5 (A,B), E9.5 (C,D) and E10.5 (E,F), wild-type (A,C,E) and Map2k1-/- (B,D,F) placentas. Gcm1-positive cells were detected as early as E8.5 in wild-type and Map2k1-/- placentas, with no major difference between the genotypes. However, at E9.5 and E10.5, Gcm1 labeling remained restricted to the chorioallantoic interface in Map2k1-/- specimens (D,F; arrowheads), whereas in control placentas, Gcm1-positive cells invaded the labyrinth to line the maternal sinuses (C,E; arrows). (G-J) Alkaline phosphatase activity in E10.5 wild-type (G,I) and Map2k1-/- (H,J) placentas. In wild-type specimens (I), cells surrounding the maternal blood sinuses produced high levels of alkaline phosphatase activity (arrowheads). There is a correlation between the punctuated localization of alkaline phosphatase activity (J; arrowheads) and the Gcm1 expression in the chorioallantoic interface of Map2k1-/- placentas (F; arrowhead). Scale bars: 100 µm in A-H; 50 µm in I,J.

 

Figure 6
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  		Fig. 6. Tetraploid rescue of Map2k1-deficient placentas and fetuses. (A,B) Wild-type and Map2k1-/- embryos are presented for comparison. (C-E) Tetraploid-aggregation chimeras were prepared from tetraploid wild-type embryos and diploid Map2k1+/- embryos (D) or Map2k1-/- ES cells (C,E). After transfer into foster mothers, chimeras were recovered at E11.5 (C) or at E13.5 (D,E) for analysis. Corresponding placentas (F-J) were analyzed by Hematoxylin and Eosin staining. (K-O) Higher magnification images of F-J. (P-T) Higher magnification images of K-O. (C,H,M,R,W) E11.5 chimeras from wild-type tetraploid embryo and Map2k1-/- ES cells. For comparison, E10.5 wild-type (A,F,K,P,U) and Map2k1-/- (B,G,L,Q,V) specimens are shown. The normal appearance of E11.5 Map2k1-/- tetraploid-aggregated embryos (C) and the histology of the placenta (M,R,W) indicated the tetraploid rescue of the Map2k1-/- phenotype. The tetraploid chimera obtained at E13.5 (E) also showed normal vascularization of the labyrinth region of the placenta (T) when compared to tetraploid control (D,S). However, the gross morphology of the embryo (E) and the absence of embryonic blood cells in the labyrinth (arrows in S; T) suggested some developmental defects. (U-W) Anti-CD31 immunostaining. (X) PCR (upper panel) and western blot (lower panel) analyses were performed to confirm the genotype of the tetraploid embryos shown in C,E. Genotype of the tetraploid embryos and yolk sacs revealed that the embryos were Map2k1-/-, while the yolk sac, which received a contribution from the embryonic and the extra-embryonic tissues contained both Map2k1 alleles. Map2k1-/- ES cells (ES 26-4) and Map2k1+/- embryo DNAs were included as control. No MAP2K1 protein was detected in Map2k1-/- ES cells (ES 26-4) or in Map2k1-/- tetraploid chimera presented in C. d, deciduum; g, tropohoblast giant cells; l, labyrinth. y, yolk sac; e, embryo. Scale bars: 1 mm in A-J; 100 µm in K-O; 50 µm in P-W.

 

Figure 7
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  		Fig. 7. Generation of the Map2k1 conditional allele. (A) Map2k1 gene targeting strategy for the generation of the conditional allele. Map2k1 translated exons 2 to 4 are represented by the white boxes. A neo selection cassette flanked by loxP sites was inserted between the third and fourth Map2k1 exons. A third loxP site was inserted between the second and the third exons. The targeting vector contains 5.5 kb and 6.9 kb of Map2k1 homologous genomic sequences on the 5' and 3' sides of the neo insertion, respectively. The herpes simplex virus-thymidine kinase (HSV-TK) selection cassette (gray box) was added at the 5' end of the vector. (B) Southern blot analysis of tail DNA from a litter obtained after breeding a Map2k1+/floxed female with a Sox2Cre+/-;Map2k1+/{Delta} male. Tail DNA was digested with StuI, blotted and hybridized with a Map2k1 genomic probe (A; upper panel) and a Cre probe (lower panel). The position of the different alleles is indicated on the right side of the panels. (C) Western blot analyses of proteins extracted from different organs of 8-week-old wild-type (+/+) and Map2k1{Delta}/{Delta} ({Delta}/{Delta}) littermates. Triplicate blots were probed with antibodies directed against MAP2K1, MAPK1 and MAP2K2. Br, brain; He, heart; Ki, kidney; Li, liver; Lu, lung; Pa, pancreas; Th, thymus.

 

Figure 8
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  		Fig. 8. Normal activation of the ERK/MAPK cascade in rescued Map2k1{Delta}/{Delta} embryos. (A) Placental phenotype of Map2k1{Delta}/{Delta} embryos. Gross morphology of Map2k1{Delta}/{Delta} embryos revealed hemorrhages (white arrowhead) characteristic of the Map2k1-/- null phenotype. Comparative Hematoxylin-Eosin staining of E10.5 wild-type and Map2k1{Delta}/{Delta} placenta sections showed the absence of maternal sinuses and fetal blood vessels intermingling in mutant specimens. The black arrows and arrowheads indicate the fetal blood vessels and the maternal sinuses in the labyrinthine region, respectively. (B) ERK/MAPK cascade activation in Sox2Cre;Map2k1{Delta}/{Delta} embryos having a normal development of their extra-embryonic structures. E10.5 embryos produced from mating between Sox2Cre;Map2k1+/{Delta} males with Map2k1+/floxed females were used for protein extracts. Western blots were probed with antibodies against MAPK1, MAP2K1, MAP2K2, phospho-MAPK1/MAPK3 and phospho-MAP2K1/MAP2K2.

 

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© The Company of Biologists Ltd 2006