QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online September 5, 2008
doi: 10.1242/10.1242/dev.025627

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Screen, M. Articles by Hemberger, M. Search for Related Content PubMed PubMed Citation Articles by Screen, M. Articles by Hemberger, M. Social Bookmarking                         What's this?

Cathepsin proteases have distinct roles in trophoblast function and vascular remodelling

¹ Laboratory of Developmental Genetics and Imprinting, The Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, UK.
² Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, 3330 Hospital Drive N.W., Calgary, Alberta T2N 4N1, Canada.
³ Centre for Trophoblast Research, University of Cambridge, Cambridge CB2 3EG, UK.

View larger version (100K): [in this window] [in a new window]   Fig. 1. Expression analysis of Cts7 and Cts8 on early post-implantation conceptuses. (A-C) In situ hybridization on E6.5 implantation sites. Cts7 and Cts8 expression (blue staining) is present in a subset of parietal trophoblast giant cells labelled by placental lactogen-I (Prl3d1). (D-F) Serial sections of E7.0 conceptuses. Cts7 and Cts8 are expressed by some parietal giant cells that are positive for Prl3d1, and in invasive giant cells at the margins of the ectoplacental cone (arrow). (G,H) E7.5 conceptuses. Cts7 and Cts8 staining is largely restricted to secondary invasive giant cells of the ectoplacental cone. The red arrowheads indicate that Cts7 expression in parietal giant cells extends slightly more distally compared with that of Cts8. (I) Every Cts8-positive giant cell (arrows) is in contact with a maternal blood vessel (asterisks). (J-L) Ectoplacental cone area of E8.5 conceptuses. Cts7 (J) and Cts8 (K) expression is restricted to a subset of trophoblast giant cells compared to the pan-giant cell marker proliferin (Prl2c2) (inset in L). Ctsl (L) is not expressed in this cell population. Scale bars: 200 µm in A-H,J-L; 100 µm in I.

View larger version (61K): [in this window] [in a new window]   Fig. 2. Cytological characterization and effects of Cts7/Cts8 expression in TS cells. (A) Northern blot hybridization on trophoblast stem (TS) cells grown in media promoting stem cell maintenance (+FGF/CM) or differentiation (-FGF/CM). Expression of Cts7 and Cts8 correlates with the profile of giant cell markers (Cdx2, stem cells; Ascl2, ectoplacental cone and spongiotrophoblast; Prl3d1, primary, parietal giant cells; Prl3b1, secondary giant cells; Prl2c2, all giant cells). (B) In situ hybridization on TS cell grown for 4 days in differentiation medium. Some giant cells (blue, arrows) are positive. (C) Immunostaining of CTS7 showing localization to the perinuclear area, the Golgi, and to the cytoplasm in a granular pattern indicative of endo- and lysosomal localization. (D) Western blots of transfected TS cells and their supernatants. CTS7 and CTS8 are secreted into the medium; intracellular control proteins (PFPL) were not detected in the supernatant. (E,F) Relative cell size measurements of TS cells 2 days after transfection with empty GFP-expression vector and Cts7-GFP (E) or Cts8-GFP (F). Cathepsin expression causes a significant shift towards larger cell sizes. Scale bars: 40 µm in B; 20 µm in C.

View larger version (45K): [in this window] [in a new window]   Fig. 3. Transgenic mouse models for inducible expression of Cts7. (A) General structure of the construct used. (B) β-Galactosidase staining proves strong, ubiquitous transgene activity before activation with Cre recombinase. (C) Northern blot demonstrating strong Cts7 expression in the embryo after ubiquitous activation with Cre. (D) E12.5 wild-type and tgCts7 (Sox2-Cre.tgCts7) placentas hybridized with Cts7, Tpbpa and Prl3b1. (E) Northern blot analysis and relative expression levels of marker genes in wild-type and transgenic (Sox2-Cre.tgCts7) placentas. *P<0.05. Scale bars: 500 µm in B; 1 mm in D.

View larger version (77K): [in this window] [in a new window]   Fig. 4. Nuclear CTS7 causes trophoblast proliferation and differentiation defects. (A) Differentiation of sinusoidal giant cells marked by Prl3b1 (blue) is reduced in the labyrinth layer of E12.5 Sox2-Cre.tgCts7 placentas. The border to the spongiotrophoblast is indicated. (B) Isolectin BSI-B4 staining (brown) on E12.5 placentas labelling glycogen cells (Glc, encircled in red). Bracket indicates the spongiotrophoblast (SpTr) layer. Broken black lines indicate the borders to the decidua (Dec) and labyrinth (Lab). (C) Anti-phosphohistone staining (H3S10-P, brown) on E9.5 trophoblast of CMV-CrextgCts7 matings and (D) quantification of signals. The arrow indicates a cell in metaphase of mitosis. Arrowheads indicate some nuclei with a speckled punctate staining in the tgCts7 sample. (E,F) Ki-67 staining (brown) of E12.5 placentas shows denser clusters and less differentiated (negative) trophoblast cells in transgenic (Sox2-Cre.tgCts7) placentas. Quantification in F. (G) Proportion of H3S10-P positive TS cells 2 days post-transfection with empty vector control (vec), wild-type Cts7, nuclear localization signal-mutant (NLS) and active site-mutant Cts7 (AS). Accumulation of H3S10-P staining depends on nuclear localization and proteolytic activity. Transfected cells were identified by GFP expression. *P<0.05. (H) Confocal sectioning of TS cells expressing Cts7 or Cts7-NLS. CTS7 protein can be found inside the nucleus (arrows) of some cells, whereas the NLS-mutant variant is clearly excluded from this compartment. Scale bars: 200 µm in A,B; 20 µm in C; 100 µm in E; 20 µm in H.

View larger version (99K): [in this window] [in a new window]   Fig. 5. Cts8 overexpression promotes giant cell differentiation. (A) Northern blot hybridization with Cts8 on wild-type and tgCts8 placentas showing approximately equal amounts of endogenous (endo. Cts8) and transgenic (tg Cts8) Cts8 mRNA. The transgenic product is a bicistronic Cts8-GFP RNA. (B) Marker expression analysis of E9.5 placentas after ubiquitous transgene activation (Sox2-Cre.tgCts8). Note the particularly strong transgene expression in extra-embryonic mesoderm and parietal endoderm. No striking phenotype is observed at this stage. (C) Prl3b1 expression on E16.5 placentas after trophoblast-specific Cts8-activation by mating to Tpbp-Cre mice. The giant cell/spongiotrophoblast layer is enlarged. (D) Trophoblast tissue dissected from E9.5 control and ubiquitously induced Sox2-Cre.tgCts8 placentas after 2 days culture stained with DAPI. Fewer diploid but more and bigger giant cells are observed. (E) Quantification of nuclear sizes. No giant cell larger than 1200 µm2 was found in controls. Scale bars: 500 µm in B; 1 mm in C; 1 mm in D.

View larger version (95K): [in this window] [in a new window]   Fig. 6. Cts8 causes vascular disintegration associated with perivascular smooth muscle degradation. (A) Phenotype of E10.5 tgCts8 conceptuses upon ubiquitous activation of the transgene (Sox2-Cre.tgCts8). No mature vitelline vessels are observed. (B) Wild-type E10.5 embryo and ubiquitously activated tgCts8 embryos (Sox2-Cre.tgCts8 and CMV-Cre.tgCts8) at E10.5 (left-hand panels) and E9.5 (right-hand panels). Transgenic embryos are severely retarded and display an inflated amnion (arrowheads), pericardial oedema (arrows) and lack of normal heart chamber formation. Asterisks indicate the occurrence of blood pools. (C) Yolk sac phenotype of Cts8-overexpressing (Sox2-Cre.tgCts8) conceptuses. Pecam/CD31-positive endothelial cells are present in transgenic samples; however, spaces between the endodermal and mesodermal layers are dilated. Smooth muscle -actin (SMA) staining surrounds vessels in wild-type yolk sacs, but is absent from transgenic samples (arrowheads). (D,E) Laminin (Lam) and smooth muscle -actin (SMA) staining on consecutive sections (D) and by double immunofluorescence (E) of chorioallantoic vessels in the E9.5 placenta. Laminin staining reveals disorganized vessel structures that lack smooth muscle cells in transgenic samples (Sox2-Cre.tgCts8). Arrows indicate vessel structures that display a striking reduction of SMA staining in transgenic samples. The arrowhead indicates a site of vessel rupture where blood cells (round, DAPI positive) can be seen exiting into the surrounding tissue. (F) Quantitative RT-PCR analysis of SMA mRNA levels in embryos (E) and placentas (P). (G) Consecutive sections of E9.5 placentas stained for Cts8 by RNA in situ hybridization (blue) and SMA by immunohistochemistry (brown) showing lack of SMA in the vicinity of Cts8-expressing giant cells. Scale bars: 1 mm in A; 500 µm in B; 50 µm in C; 50 µm in D; 100 µm in E; 500 µm in G.

View larger version (36K): [in this window] [in a new window]   Fig. 7. Model of Cts7 and Cts8 function. Spiral artery remodelling is a concerted action between uterine natural killer (uNK) cells and trophoblast giant cells. Cts8 endows trophoblast giant cells with smooth muscle-degrading functions. This activity facilitates endovascular trophoblast invasion to complete the remodelling process. In addition, Cts7 and Cts8 may have complementary roles in promoting differentiation of the spiral artery-associated giant cell subtype from precursors within the ectoplacental cone.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter