Amniotic ectoderm expansion in mouse occurs via distinct modes and requires SMAD5-mediated signalling.

Upon gastrulation, the mammalian conceptus transforms rapidly from a simple bilayer into a multilayered embryo enveloped by its extra-embryonic membranes. Impaired development of the amnion, the innermost membrane, causes major malformations. To clarify the origin of the mouse amnion, we used single-cell labelling and clonal analysis. We identified four clone types with distinct clonal growth patterns in amniotic ectoderm. Two main types have progenitors in extreme proximal-anterior epiblast. Early descendants initiate and expand amniotic ectoderm posteriorly, while descendants of cells remaining anteriorly later expand amniotic ectoderm from its anterior side. Amniogenesis is abnormal in embryos deficient in the bone morphogenetic protein (BMP) signalling effector SMAD5, with delayed closure of the proamniotic canal, and aberrant amnion and folding morphogenesis. Transcriptomics of individual Smad5 mutant amnions isolated before visible malformations and tetraploid chimera analysis revealed two amnion defect sets. We attribute them to impairment of progenitors of the two main cell populations in amniotic ectoderm and to compromised cuboidal-to-squamous transition of anterior amniotic ectoderm. In both cases, SMAD5 is crucial for expanding amniotic ectoderm rapidly into a stretchable squamous sheet to accommodate exocoelom expansion, axial growth and folding morphogenesis.

The BMP Pathway in Blood Vessel and Lymphatic Vessel Biology.

Bone morphogenetic proteins (BMPs) were originally identified as the active components in bone extracts that can induce ectopic bone formation. In recent decades, their key role has broadly expanded beyond bone physiology and pathology. Nowadays, the BMP pathway is considered an important player in vascular signaling. Indeed, mutations in genes encoding different components of the BMP pathway cause various severe vascular diseases. Their signaling contributes to the morphological, functional and molecular heterogeneity among endothelial cells in different vessel types such as arteries, veins, lymphatic vessels and capillaries within different organs. The BMP pathway is a remarkably fine-tuned pathway. As a result, its signaling output in the vessel wall critically depends on the cellular context, which includes flow hemodynamics, interplay with other vascular signaling cascades and the interaction of endothelial cells with peri-endothelial cells and the surrounding matrix. In this review, the emerging role of BMP signaling in lymphatic vessel biology will be highlighted within the framework of BMP signaling in the circulatory vasculature.

Smad1/5 is acetylated in the dorsal aortae of the mouse embryo before the onset of blood flow, driving early arterial gene expression.

AIMS: During embryonic development, arteriovenous (AV) differentiation ensures proper blood vessel formation and maturation. Defects in arterial or venous identity cause inappropriate fusion of vessels, resulting in atypical shunts, so-called arteriovenous malformations (AVM). Currently, the mechanism behind AVM formation remains unclear and treatment options are fairly limited. Mammalian AV differentiation is initiated before the onset of blood flow in the embryo; however, this pre-flow mechanism is poorly understood. Here, we aimed to unravel the role of Smad1/5 signalling in pre-flow arterial identity, and in the process uncovered an unexpected control mechanism of Smad1/5 signalling. METHODS AND RESULTS: We establish that despite Notch1 being expressed in the pre-flow mouse embryo, it is not activated, nor is it necessary for the expression of the earliest arterial genes in the dorsal aortae (i.e., Hey1 and Gja4). Furthermore, interrupting blood flow by using the Ncx1 KO model completely prevents the activation of Notch1 signalling, suggesting a strong role of shear stress in maintaining arterial identity. We demonstrate that early expression of Hey1 and Gja4 requires SMAD1/5 signalling. Using embryo cultures, we show that Smad1/5 signalling is activated through the Alk1/Alk5/TGFßR2 receptor complex, with TGFß1 as a necessary ligand. Furthermore, our findings demonstrate that early arterial gene expression requires the acetylation of Smad1/5 proteins, rendering them more sensitive to TGFß1 stimulation. Blocking acetyl-CoA production prevents pre-flow arterial expression of Hey1 and Gja4, while stabilizing acetylation rescues their expression. CONCLUSIONS: Our findings highlight the importance of the acetyl-CoA production in the cell and provide a novel control mechanism of Smad1/5 signalling involving protein acetylation. As disturbed canonical Smad1/5 signalling is involved in several vascular conditions, our results offer new insights in treatment options for circumventing canonical Smad1/5 signalling.