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First published online 30 April 2008
doi: 10.1242/dev.020099

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Early patterning of the chorion leads to the trilaminar trophoblast cell structure in the placental labyrinth

David G. Simmons^1,*, David R. C. Natale^1,*, Valerie Begay², Martha Hughes¹, Achim Leutz² and James C. Cross^1,

¹ Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, The University of Calgary, 3330 Hospital Drive NW, Calgary, AB, T2N 4N1, Canada.
² Max Delbrueck Center for Molecular Medicine, Robert-Roessle-Str. 10, 13092 Berlin, Germany.

View larger version (92K): [in this window] [in a new window]   Fig. 1. Expression of mouse labyrinth trophoblast markers from E7.5-14.5. In situ hybridizations for the labyrinth trophoblast markers Gcm1, Synb, Cebpa, Syna, Cebpb, Dlx3, Nr6a1 and Esx1 at E7.5, E8.5, E9.0 and E14.5. Low-magnification images (top row) are of Gcm1 expression. Black boxes indicate the areas shown at high-magnification in the panels beneath. al, allantois; ch, chorion; dec, decidua; epc, ectoplacental cone; f, fetal blood space; lab, labyrinth; m, maternal blood space; spt, spongiotrophoblast; tgc, trophoblast giant cell. Scale bars: black, 100 µm; red, 1.4 mm.

View larger version (75K): [in this window] [in a new window]   Fig. 2. Gcm1, Synb and Cebpa are expressed in SynT-II cells and Syna is expressed in SynT-I cells. (A) Electron micrograph of the trilaminar trophoblast structure that separates the maternal and fetal blood compartments. The black box indicates the area shown at high-magnification on the right. S-TGC, sinusoidal trophoblast giant cell; SynT-I, syncytiotrophoblast layer I; SynT-II, syncytiotrophoblast layer II; Endo, fetal endothelial cell; m, maternal blood space; f, fetal blood space; frbc, fetal red blood cell. (B) Double in situ hybridization for Syna (brown) and Synb (purple) in the labyrinth at E14.5 indicating that Syna expression is restricted to SynT-I cells and Synb expression is restricted to SynT-II cells. Note that Syna/Gcm1 and Syna/Cebpa double in situ hybridizations produced similar results (data not shown). Black arrowhead indicates unstained S-TGC which lines maternal blood spaces. Brown arrowhead indicates Syna-positive SynT-I cells located closer to maternal blood spaces. Blue arrowhead indicates Synb-positive SynT-II cells located closest to fetal blood spaces. (C) Double in situ hybridization for Ctsq (brown) and Synb (purple) in the labyrinth at E14.5. Black arrows indicate unstained SynT-I cell between Ctsq-positive S-TGC and Synb-positive SynT-II cell. (D) Double in situ hybridization for Syna (purple) and Synb (brown) in the labyrinth at E9.5. Syna and Synb expression do not co-localize. Note that Syna/Gcm1 and Syna/Cebpa double in situ hybridizations showed similar results (data not shown). (E) Double in situ hybridization for Syna (purple) and Synb (brown) in chorion at E8.5. Syna and Synb expression do not co-localize at E8.5 and demarcate distinct trophoblast populations within the chorion. Note that Syna/Gcm1 and Syna/Cebpa double in situ hybridizations showed similar results (data not shown). Scale bars: 100 µm (left); 50 µm (right).

View larger version (86K): [in this window] [in a new window]   Fig. 3. Early patterning of the mouse chorion. (A) In situ hybridizations for Hand1 and Pl1 at E9.0 indicate that Hand1+/Pl1- cells line the maternal blood sinuses located on the apical side of the chorion. At E8.5, Syna expression is restricted to cells near the top of the chorion, whereas Gcm1, Synb and Cebpa staining (on serial sections) indicates co-localization within a subset of trophoblast located on the basal side of the chorion (confirmed by double in situ hybridization, data not shown). The black arrows indicate Hand1+/Pl1- cells lining maternal sinusoids. (B) Higher magnification images of Hand1 and Syna in situ hybridizations on E8.5 chorion. Note that Syna-positive cells are separated from the maternal blood sinuses by a single layer of cells, typically Hand1-positive. Black arrows indicate the cell layer lining the maternal sinusoids. (C) Ultrathin epoxy section of E8.5 chorion (2 µm) stained with Tissue Epoxy Stain (EMS). Note the unique morphology of the different layers of trophoblast cells in the chorion. Cells that line the maternal sinusoids (Hand1+/Pl1- at E8.5) are positive for Ctsq expression by E12.5 (data not shown). Scale bars: black, 100 µm; red, 50 µm.

View larger version (147K): [in this window] [in a new window]   Fig. 4. Synb and Cebpa are downstream of Gcm1, but Synb is not downstream of Cebpa. In situ hybridizations for Gcm1, Synb, Cebpa, Cebpb and Syna on 10 µm sections of E9.5 Gcm1+/- (Het) and Gcm1-/- (Gcm1 KO) mouse placentas and Cebpa+/-;Cebpb+/- (Double Het), Cebpa-/- (Cebpa KO), Cebpb-/- (Cebpb KO) and Cebpa-/-;Cebpb-/- (DoubleKO) placentas. al, allantois; ch, chorion; f, fetal blood space; m, maternal blood space. Scale bars: 100 µm.

View larger version (50K): [in this window] [in a new window]   Fig. 5. Gcm1/Synb/Cebpa-expressing cells are not required to induce or maintain Syna or Hand1 expression. (Left) In situ hybridizations for Hand1, Syna, Gcm1, Cebpa and Synb were performed on E8.5 and E9.5 wild-type and Gcm1-/- mouse placentas. (Right) Northern blot analysis of Hand1, Ctsq, Syna, Gcm1, Cebpa and Synb expression in differentiating TS cultures following the removal of human recombinant FGF4, heparin and embryonic fibroblast-conditioned media. 18s rRNA was used as a loading control.

View larger version (24K): [in this window] [in a new window]   Fig. 6. Model for mouse labyrinth formation from a pre-patterned chorion. Note that Ctsq expression within S-TGCs is not fully evident until E12.5. No exclusive marker has been identified for S-TGC precursors, although cells lining the early maternal sinusoids are Hand1+/Pl1-. However, Hand1 expression is not restricted to cells lining the maternal sinusoids in the way that Ctsq is restricted to S-TGCs in mid-gestation; Hand1 is expressed in all TGC subtypes and within the ectoplacental cone and chorion (not shown). It is the combined Hand1+/Pl1- nature of the cells lining the maternal sinusoids, together with their location, that implies they are S-TGC progenitors. For simplicity, cell bodies of Hand1+/Pl1- cells are shown only along the top of the chorion. Light gray, allantois; gray, chorion; dark gray, ectoplacental cone (EPC); red, extraembryonic mesoderm-derivatives; yellow, Gcm1+ chorionic trophoblast cells; orange, Gcm1/Synb/Cebpa+ chorionic trophoblast cells; green, Syna+ trophoblast cells; blue, developing sinusoidal giant cells (S-TGCs) which will subsequently express Ctsq; mbs, maternal blood sinus.

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