Endoglin is a transmembrane accessory receptor for transforming growth factor-beta (TGF-beta) that is predominantly expressed on proliferating endothelial cells in culture and on angiogenic blood vessels in vivo. Endoglin, as well as other TGF-beta signalling components, is essential during angiogenesis. Mutations in endoglin and activin receptor-like kinase 1 (ALK1), an endothelial specific TGF-beta type I receptor, have been linked to the vascular disorder, hereditary haemorrhagic telangiectasia. However, the function of endoglin in TGF-beta/ALK signalling has remained unclear. Here we report that endoglin is required for efficient TGF-beta/ALK1 signalling, which indirectly inhibits TGF-beta/ALK5 signalling. Endothelial cells lacking endoglin do not grow because TGF-beta/ALK1 signalling is reduced and TGF-beta/ALK5 signalling is increased. Surviving cells adapt to this imbalance by downregulating ALK5 expression in order to proliferate. The ability of endoglin to promote ALK1 signalling also explains why ectopic endoglin expression in endothelial cells promotes proliferation and blocks TGF-beta-induced growth arrest by indirectly reducing TGF-beta/ALK5 signalling. Our results indicate a pivotal role for endoglin in the balance of ALK1 and ALK5 signalling to regulate endothelial cell proliferation.

Vascular development depends on transforming growth factor β (TGFβ), but whether signalling of this protein is required for the development of endothelial cells (ECs), vascular smooth muscle cells (VSMCs) or both is unclear. To address this, we selectively deleted the type I (ALK5, TGFBR1) and type II (TβRII, TGFBR2) receptors in mice. Absence of either receptor in ECs resulted in vascular defects in the yolk sac, as seen in mice lacking receptors in all cells, causing embryonic lethality at embryonic day (E)10.5. Deletion of TβRII specifically in VSMCs also resulted in vascular defects in the yolk sac; however, these were observed at later stages of development, allowing the embryo to survive to E12.5. Because TGFβ can also signal in ECs via ALK1 (ACVRL1), we replaced ALK5 by a mutant defective in SMAD2 and SMAD3 (SMAD2/3) activation that retained the ability to transactivate ALK1. This again caused defects in the yolk sac vasculature with embryonic lethality at E10.5, demonstrating that TGFβ/ALK1 signalling in ECs cannot compensate for the lack of TGFβ/ALK5-induced SMAD2/3 signalling in vivo. Unexpectedly, SMAD2 phosphorylation and α-smooth muscle actin (SMAα, ACTA2) expression occurred in the yolk sacs of ALK5–/– embryos and ALK5–/– embryonic stem cells undergoing vasculogenesis, and these processes could be blocked by an ALK4 (ACVR1B)/ALK5 inhibitor. Together, the data show that ALK5 is required in ECs and VSMCs for yolk sac vasculogenesis; in the absence of ALK5, ALK4 mediates SMAD2 phosphorylation and consequently SMAα expression.

Hereditary haemorrhagic telangiectasia (HHT) is an autosomal dominant disorder in humans that is characterised by multisystemic vascular dyplasia and recurrent haemorrhage. Germline mutations in one of two different genes,endoglin or ALK1 can cause HHT. Both are members of the transforming growth factor (TGF) β receptor family of proteins, and are expressed primarily on the surface of endothelial cells (ECs). Mice that lack endoglin or activin receptor like kinase (ALK) 1 die at mid-gestation as a result of defects in the yolk sac vasculature. Here, we have analyzed TGFβsignalling in yolk sacs from endoglin knockout mice and from mice with endothelial-specific deletion of the TGFβ type II receptor (TβRII)or ALK5. We show that TGFβ/ALK5 signalling from endothelial cells to adjacent mesothelial cells is defective in these mice, as evidenced by reduced phosphorylation of Smad2. This results in the failure of vascular smooth muscle cells to differentiate and associate with endothelial cells so that blood vessels remain fragile and become dilated. Phosphorylation of Smad2 and differentiation of smooth muscle can be rescued by culture of the yolk sac with exogenous TGFβ1. Our data show that disruption of TGFβsignalling in vascular endothelial cells results in reduced availability of TGFβ1 protein to promote recruitment and differentiation of smooth muscle cells, and provide a possible explanation for weak vessel walls associated with HHT.

Endoglin is an auxiliary receptor for TGFbeta signalling. Heterozygous germline Endoglin mutations have been identified in patients with the vascular abnormality, Hereditary Haemorrhagic Telangiectasia. Endoglin is upregulated in endothelial cells during angiogenesis and loss of Endoglin in the mouse results in embryonic lethality at mid-gestation. This phenotype points to an important role of Endoglin in new blood vessel formation but precludes analysis at later stages in development and in postnatal life. To bypass this limitation and allow further investigations of the function of Endoglin we have generated a floxed Endoglin allele in which loxP sites flank exons 5 and 6. Mice homozygous for this allele are normal and in the presence of appropriate Cre lines will allow time and cell specific Endoglin deletion for in vivo analysis of function in cardiovascular development and disease.

Transforming growth factor beta (TGFbeta) inhibits proliferation and promotes the migration of primordial germ cells (PGCs) towards explants of gonadal ridges in vitro. However, its effects in vivo are still unclear. Here, we analyzed the behavior of PGCs in embryos lacking TGFbeta signaling via the type I receptor ALK5. TGFbeta in vivo was neither a chemoattractant for PGCs, nor did it affect their proliferation during migration towards the gonadal ridges up to embryonic day (E)10. Unexpectedly, the absence of TGFbeta signaling in fact resulted in significant facilitation of PGC migration out of the hindgut, due to the reduced deposition of collagen type I surrounding the gut of Alk5-deficient mutant embryos. Migratory PGCs adhere strongly to collagen; therefore, reduced collagen type I along the gut may result in reduced adhesion, facilitating migration into the dorsal mesenterium and gonadal ridges. Our results provide new evidence for the role of TGFbeta signaling in migration of PGCs in vivo distinct from that described previously.