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First published online 17 November 2004
doi: 10.1242/dev.01529

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Defective paracrine signalling by TGFß in yolk sac vasculature of endoglin mutant mice: a paradigm for hereditary haemorrhagic telangiectasia

Rita L. C. Carvalho¹, Leon Jonker², Marie-José Goumans^3,*, Jonas Larsson^4,, Peter Bouwman¹, Stefan Karlsson⁴, Peter ten Dijke³, Helen M. Arthur² and Christine L. Mummery^1,

¹ Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Uppsalalaan 8, 3584CT Utrecht, The Netherlands
² Institute of Human Genetics, International Centre for Life, University of Newcastle, Newcastle upon Tyne, NE1 3BZ, UK
³ Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066CX Amsterdam, The Netherlands
⁴ Molecular Medicine and Gene Therapy, Institute of Laboratory Medicine and Department of Medicine, Lund University Hospital, 221 00 Lund, Sweden

View larger version (160K): [in a new window]   Fig. 1. Expression of endothelial cell markers in the yolk sacs. (A) lacZ staining for endoglin, in Eng+/– mice with ß-gal knocked into the endoglin locus. (B-D) Immunohistochemistry for PECAM1 (B), Flk1 (C) and endoglin (D) in wild-type yolk sacs. Yolk sac was sectioned in plastic following staining as whole mounts in all cases. Scale bar: 0.5 mm. Abbreviations: EC, endothelial cell layer; end, endoderm; mes, mesothelial cell layer.

View larger version (114K): [in a new window]   Fig. 2. TGFß induces Smad2 phosphorylation in the yolk sacs of wild-type and Eng mutant embryos. PSmad1/5/8 (PS1) was detected by immunohistochemistry in both endothelial and mesothelial cells of wild-type yolk sacs (A). In Eng mutant yolk sacs, Smad1 was phosphorylated in mesothelial cells (green arrow) but not in endothelial cells (B); after a 1 hour treatment with BMP2 (C) or TGFß1 (D) PSmad1 was detected in endothelial cells. PSmad2 (PS2) was detected by immunohistochemistry in the mesothelial layer of both untreated (E) and TGFß1-treated (F) wild-type yolk sacs. In the Eng null yolk sac, PSmad2 was not detectable in the mesothelial layer (G) but phosphorylation of Smad2 was restored after TGFß1 treatment (green arrow, H) for 1 hour. To confirm the specificity of PSmad2 antibody, Tgfbr2 null yolk sac were analysed. PSmad2 was not present in either endothelial or mesothelial cell layers (I) and its phosphorylation was not restored after TGFß1 treatment (J). (K) Percentage of endothelial cells expressing PSmad1/5/8 and PSmad2 in sections from wild-type and Eng mutant treated with BMP2 or treated with TGFß1. Bars represent the mean±s.e.m. of five independent experiments. (L) Percentage of mesothelial cells expressing PSmad2 in sections from wild-type, Eng mutant and tie-1-Cre/TßRIIfl/fl yolk sacs with or without 1 hour TGFß1 treatment. Bars represent the mean±s.e.m. of cells from four independent experiments (*P<0.05; **P<0.01). Scale bar: 0.5 mm. Abbreviations: EC, endothelial cell layer; end, endoderm; mes, mesothelial cell layer.

View larger version (105K): [in a new window]   Fig. 3. Smad2 phosphorylation is absent in endothelial cells of Eng mutant embryos. Transverse sections of E9.5 embryos stained with PECAM1 antibody showing endothelial cells in the brain (A), dorsal aorta and cardinal vein (B), heart (C), and vitelline artery and umbilical vein (D). Transverse sections of E9.5 wild-type embryos stained with PSmad2 (PS2) antibody showing positive endothelial cells in the brain (E,E'), dorsal aorta and branchial arch artery (F,F'), endothelium of the heart (G,G'), dorsal aorta and vitelline artery (H,H'). In the endoglin null embryos, PSmad2 was not detectable in endothelial cells of arteries and veins (I,J,I',J') but was observed in the endothelium of the heart (K,K'). (L,L') Scale bars: 0.5 mm. Red arrowheads in I',J',L' indicate negative ECs. Black arrowheads in K' indicate PS2-positive ECs. Abbreviations: bba, branchial arch artery; cv, cardinal vein; da, dorsal aorta; da/baa, communication between da and baa; fg, foregut, ht, heart; nt, neural tube; pvp, perioptic vascular plexus; s, somite; uv, umbilical vein; va, vitelline artery.

View larger version (137K): [in a new window]   Fig. 4. Extracellular matrix components in wild-type and Eng mutant yolk sac. Immunostaining for fibronectin and collagen in sectioned whole mounts of yolk sacs. In wild-type (A) and Eng mutant (B) yolk sacs, fibronectin was observed between the layers of the yolk sac. No striking difference was observed in collagen expression in the wild-type (C) and the Eng mutant yolk sacs (D). Scale bar: 0.5 mm. Abbreviations: EC, endothelial cell layer; end, endoderm; mes, mesothelial cell layer.

View larger version (45K): [in a new window]   Fig. 5. Effect of TGFß1 in smooth muscle actin expression in Eng mutant yolk sacs. Caldesmon and -smooth muscle actin was detected by immunohistochemistry in sectioned yolk sacs, stained as whole mounts. (A) Caldesmon was expressed in mesothelial cells of wild-type yolk sacs but was not detected in Eng mutant yolk sacs (B). (C) In wild-type embryos, -sma was observed in cells surrounding the vessels while in (D) Eng knockout yolk sac its expression was absent. (E) -sma expression was partially restored after 3 hours of TGFß treatment (red arrowheads). In Eng mutant yolk sacs -sma was absent (F) while after treatment with high concentrations of TGFß1 (5 ng/ml) for 8 hours its expression was clearly restored (G, red arrowheads). (H) Real-time RT-PCR analysis of -sma RNA expression in wild-type and Eng mutant yolk sacs. Expression of -sma mRNA was compared with ß-actin (as loading control) in wild-type and Eng mutant yolk sac. E9.5 yolk sac endoderm was used as a negative control. The number of yolk sacs analysed is shown (n). Contamination with genomic DNA was not evident in samples without RT (not shown). The Wilcoxon Test was used for statistical analysis of significance; medians are shown as black horizontal bar (*P<0.1; **P<0.05). Scale bar: 0.5 mm. Abbreviations: EC, endothelial cell layer; end, endoderm; mes, mesothelial cell layer.

View larger version (86K): [in a new window]   Fig. 6. TGFß1 mRNA and protein expression in wild-type and Eng mutant yolk sacs. Expression of Tgfb1 mRNA was examined by whole-mount in situ hybridization in wild type (not shown) and (A) Eng mutant yolk sacs, and detected specifically in endothelial cells. (B) Higher magnification of A, showing Tgfb1-positive ECs (red arrow). TGFß1 protein was detected by immunohistochemistry following whole-mount staining and sectioning of yolk sacs from (C) wild-type and (D) Eng mutant embryos. In the wild-type yolk sac, TGFß1 was present in the endoderm as well as in both endothelial and mesothelial cell layers (C,D). (E,F) Higher magnification of the yolk sac layers. Positive endothelial cells are indicated by black arrows and positive mesothelial cells are indicated by green arrows. (E) In the Eng null yolk sac, TGFß1 was present in the endoderm but was almost absent in the endothelial and mesothelial cell layers. A TGFß1-positive endothelial cell is indicated by black arrow and a TGFß1-positive mesothelial cell is indicated by a green arrow. (F) Higher magnification of the yolk sac. TGFß1-negative endothelial cell is indicated by black arrow, TGFß1-negative mesothelial cell is indicated by green arrow and TGFß1-positive mesothelial cell is indicated by red arrow. (G) Real-time RT-PCR analysis of Tgfb1 mRNA expression in wild-type and Eng mutant yolk sacs. Expression of Tgfb1 mRNA compared with ß-actin (loading control) in wild-type and Eng mutant yolk sacs. The number of yolk sacs analysed is shown (n). Samples were corrected for loading difference using ß-actin primers. Contamination with genomic DNA was not evident in samples without RT (not shown). The Wilcoxon Test was used for statistical analysis of significance; medians are shown as a black stripe (*P>0.5). (H) The decreased expression of TGFß1 in Eng–/– yolk sacs was confirmed by Western blot analysis. *Unspecific band. Scale bars: 0.5 mm. Abbreviations: EC, endothelial cell layer; end, endoderm; mes, mesothelial cell layer.

View larger version (70K): [in a new window]   Fig. 7. Smad2 phosphorylation and TGFß1 protein expression in tie-1-Cre/TßRIIfl/fl and tie-1-Cre/ALK5fl/fl double transgenic mutant yolk sacs. PSmad2 was analysed by immunohistochemistry in sectioned whole mounts of both untreated (A-C) and TGFß1-treated (D-F) yolk sacs. Phosphorylation of Smad2 was observed in wild-type yolk sacs (A,D). In the yolk sacs of tie-1-Cre/TßRIIfl/fl (B) and tie-1-Cre/ALK5fl/fl (C) mice, PSmad2 was not present in the mesothelial cell layer (green arrow) but phosphorylation of Smad2 was restored after TGFß1 treatment (E,F, green arrows) for 1 hour. To determine the expression of functional Cre in vivo, lacZ expression was analysed in double transgenic yolk sacs. ß-Gal staining was observed in the endothelial cells of tie-1-Cre/TßRIIfl/fl (B and E, red arrows) and tie-1-Cre/ALK5fl/fl (not shown). TGFß1 protein expression was analysed by immunohistochemistry on whole-mount staining of yolk sacs from (G) wild-type, (H) tie-1-Cre/TßRIIfl/fl mutant embryos and (I) tie-1-Cre/ALK5fl/fl mutant embryos. In wild-type yolk sacs, normal expression of TGFß1 was observed in the endothelial (black arrow) and mesothelial (green arrow) cell layers, while in both tie-1-Cre/TßRIIfl/fl and tie-1-Cre/ALK5fl/fl mutant yolk sacs, TGFß1 was no longer detected in the mesothelial cell layer (green arrows). Scale bars: 0.5 mm. Abbreviations: EC, endothelial cell layer; end, endoderm; mes, mesothelial cell layer.

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