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First published online 4 April 2007
doi: 10.1242/dev.02843

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The Mrj co-chaperone mediates keratin turnover and prevents the formation of toxic inclusion bodies in trophoblast cells of the placenta

Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, T2N 4N1, Canada.

View larger version (104K): [in this window] [in a new window]   Fig. 1. Mrj-/- embryos fail to undergo chorioallantoic attachment because of defects within the chorionic trophoblast layer. (A,B) Expression of the ß-galactosidase (ß-gal; A; blue) and Mrj (B; red) proteins in E8.25 chorions. (C) Aggregation of a diploid Mrj-/- embryo (blue) with a wild-type tetraploid embryo expressing the Egfp transgene (green) results in a genetically mosaic chorionic trophoblast, ectoplacental cone and trophoblast giant cell layer (Rossant and Cross, 2001). The chorionic mesothelium, allantois and embryo proper are almost exclusively composed of Mrj-deficient diploid cells. (D,E) Rescued chorioallantoic attachment in an Egfp:Mrj-/- chimeric conceptus at E9.5. (E) Higher-magnification of boxed region in D. (F-H) Initiation of branching morphogenesis in the placental labyrinth layer (arrowheads) at E9.5 in Egfp:Mrj+/+ (F) and Egfp:Mrj-/- (H) chimeric placentas, but not in a Mrj-/- chorion (G), which remains flat. al, allantois; ch, chorion; em, embryo; me, chorionic mesothelial layer; tr, chorionic trophoblast layer. Scale bars: 10 µm in A,B,F-H; 1 mm in D; 0.5 mm in E.

View larger version (99K): [in this window] [in a new window]   Fig. 2. The keratin cytoskeleton is collapsed in primary cultures of Mrj-/- trophoblast cells. (A) Keratinfilament (red) organization in trophoblast giant cells derived from wild-type and Mrj-/- chorionectoplacental cone explants. (B) Quantification of the types of keratin-filament organization observed in trophoblast giant cells derived from wild-type and Mrj-/- chorionectoplacental cone explants. (C) Ultrastructure of a keratin aggregate (surrounded by broken line) in a Mrj-/- trophoblast giant cell at E8.25. (D) Mrj expression (red) in trophoblast giant cells of a wild-type conceptus at E8.25. Right, higher-magnification of boxed region in the left panel. (E) Nuclear (left), cytoplasmic (center), and both nuclear and cytoplasmic (right) Mrj expression in wild-type trophoblast giant cells in vitro. Arrowheads indicate trophoblast giant cells with undetectable Mrj expression. Broken line indicates the periphery of a trophoblast giant cell. (A,D,E) DNA is blue. ch, chorion; em, embryo; pan-CK, pan-cytokeratin; TGC, trophoblast giant cells. Scale bars: 10 µm in A,E; 0.5 µm in C; 20 µm (left) and 10 µm (right) in D.

View larger version (109K): [in this window] [in a new window]   Fig. 3. Keratin inclusion bodies disrupt cell-cell adhesion and disrupt the organization of Mrj-deficient chorionic trophoblast cells. (A) Mrj expression (red) in a wild-type chorion at E8.25. (B,C) K18 expression (red) in wild-type (B; +/+) and Mrj-deficient (C; -/-) trophoblast stem (TS) cells. DNA is blue. (D) Ultrastructure of a keratin inclusion body (arrowheads) within a Mrj-/- chorionic trophoblast cell at E8.25. (E-H) K18 expression (red) in wild-type (E,F) and Mrj-/- (G,H) chorionic trophoblast cells at E8.25. (F,H) Higher-magnifications of boxed regions in E and G, respectively. (I-L) Morphology of Mrj mutant chorionic trophoblast cells in vivo (J) and in vitro (L) in contrast to wild-type cells (I,K). (M-P) Desmoplakin (Dp) expression (red) in wild-type (M,N) and Mrj-/- (O,P) chorionic trophoblast cells at E8.25. (N,P) Higher-magnifications of boxed regions in M and O, respectively. (Q,R) Desmoplakin expression (green) in wild-type (Q) and Mrj-/- (R) TS cells. (S,T) Electron micrographs of desmosomes (arrowheads) in wild-type (S) and Mrj-/- (T) chorionic trophoblast cells at E8.25. ch, chorion; DP, desmoplakin; EPC, ectoplacental cone; me, chorionic mesothelium; TGCs, trophoblast giant cells; tr, chorionic trophoblast. Scale bars: 10 µm in A-C,E-R; 0.5 µm in D,S,T.

View larger version (136K): [in this window] [in a new window]   Fig. 4. Mrj protein expression does not co-localize to K18-containing filaments. (A,B) Deconvolved images of a trophoblast giant cell immunostained for Mrj (red), K18 (green) and DNA (blue). (B) Higher-magnification of the boxed region in A. A cross-section (broken line) appears on the far right side of B. Scale bar: 10 µm in A.

View larger version (114K): [in this window] [in a new window]   Fig. 5. Mrj is required for proteasome degradation of keratin filaments. (A) Expression of Mrj (red) and 20S proteasome core subunits (green) in wild-type trophoblast giant cells in vitro. (B) K18 expression (green) in trophoblast giant cells cultured with (right) or without (left) the proteasome inhibitor clasto-lactacystin ß-lactone. (C) Mrj expression (red) in trophoblast giant cells treated with clasto-lactacystin ß-lactone. K18 is green. (B,C) DNA is blue. Scale bars: 10 µm.

View larger version (128K): [in this window] [in a new window]   Fig. 6. Chorioallantoic attachment is rescued in Mrj-/-;K18+/- and Mrj-/-;K18-/- conceptuses. (A) An unattached allantois (arrow) of an E9.5 Mrj-/- embryo appears as a bud at the posterior end of the embryo. Broken line outlines the chorion (Ch). (B-D) Chorioallantoic attachment of K18-/- (B,C) and K18-/-;K19-/- (D) conceptuses at E9.5. (C) Higher-magnification of the boxed region in B. (E,F) Rescued chorioallantoic attachment in Mrj-/-;K18+/- (E) and Mrj-/-;K18-/- (F) conceptuses at E9.5. (G-I) Branching morphogenesis in Mrj-/-;K18+/- (H) and Mrj-/-;K18-/- (I) placentas with rescued chorioallantoic attachment at E9.5 compared to a Mrj+/-;K18+/+ littermate placenta (G). White arrowheads, fetal blood space/branchpoint; asterisks, maternal blood space; Ch, chorion; Al, allantois; black arrowhead, allantois. Scale bars: 1 mm in A,B; 0.5 mm in C,D; 0.5 mm in E,F; 20 µm in G-I.

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