Endothelial SMAD1/5 signaling couples angiogenesis to osteogenesis in juvenile bone.

Skeletal development depends on coordinated angiogenesis and osteogenesis. Bone morphogenetic proteins direct bone formation in part by activating SMAD1/5 signaling in osteoblasts. However, the role of SMAD1/5 in skeletal endothelium is unknown. Here, we found that endothelial cell-conditional SMAD1/5 depletion in juvenile mice caused metaphyseal and diaphyseal hypervascularity, resulting in altered trabecular and cortical bone formation. SMAD1/5 depletion induced excessive sprouting and disrupting the morphology of the metaphyseal vessels, with impaired anastomotic loop formation at the chondro-osseous junction. Endothelial SMAD1/5 depletion impaired growth plate resorption and, upon long-term depletion, abrogated osteoprogenitor recruitment to the primary spongiosa. Finally, in the diaphysis, endothelial SMAD1/5 activity was necessary to maintain the sinusoidal phenotype, with SMAD1/5 depletion inducing formation of large vascular loops and elevated vascular permeability. Together, endothelial SMAD1/5 activity sustains skeletal vascular morphogenesis and function and coordinates growth plate remodeling and osteoprogenitor recruitment dynamics in juvenile mouse bone.

Functional and topological analysis of PSENEN, the fourth subunit of the γ-secretase complex.

The γ-secretase complexes are intramembrane cleaving proteases involved in the generation of the Aß peptides in Alzheimer's disease. The complex consists of four subunits, with Presenilin harboring the catalytic site. Here, we study the role of the smallest subunit, PSENEN or Presenilin enhancer 2, encoded by the gene Psenen, in vivo and in vitro. We find a profound Notch deficiency phenotype in Psenen-/- embryos confirming the essential role of PSENEN in the γ-secretase complex. We used Psenen-/- fibroblasts to explore the structure-function of PSENEN by the scanning cysteine accessibility method. Glycine 22 and proline 27, which border the membrane domains 1 and 2 of PSENEN, are involved in complex formation and stabilization of γ-secretase. The hairpin structured hydrophobic membrane domains 1 and 2 are exposed to a water-containing cavity in the complex, while transmembrane domain 3 is not water exposed. We finally demonstrate the essential role of PSENEN for the cleavage activity of the complex. PSENEN is more than a structural component of the γ-secretase complex and might contribute to the catalytic mechanism of the enzyme.

The Wnt/TCF7L1 transcriptional repressor axis drives primitive endoderm formation by antagonizing naive and formative pluripotency.

Early during preimplantation development and in heterogeneous mouse embryonic stem cells (mESC) culture, pluripotent cells are specified towards either the primed epiblast or the primitive endoderm (PE) lineage. Canonical Wnt signaling is crucial for safeguarding naive pluripotency and embryo implantation, yet the role and relevance of canonical Wnt inhibition during early mammalian development remains unknown. Here, we demonstrate that transcriptional repression exerted by Wnt/TCF7L1 promotes PE differentiation of mESCs and in preimplantation inner cell mass. Time-series RNA sequencing and promoter occupancy data reveal that TCF7L1 binds and represses genes encoding essential naive pluripotency factors and indispensable regulators of the formative pluripotency program, including Otx2 and Lef1. Consequently, TCF7L1 promotes pluripotency exit and suppresses epiblast lineage formation, thereby driving cells into PE specification. Conversely, TCF7L1 is required for PE specification as deletion of Tcf7l1 abrogates PE differentiation without restraining epiblast priming. Taken together, our study underscores the importance of transcriptional Wnt inhibition in regulating lineage specification in ESCs and preimplantation embryo development as well as identifies TCF7L1 as key regulator of this process.

Efficient generation of ETX embryoids that recapitulate the entire window of murine egg cylinder development.

The murine embryonic-trophoblast-extra-embryonic endoderm (ETX) model is an integrated stem cell-based model to study early postimplantation development. It is based on the self-assembly potential of embryonic, trophoblast, and hypoblast/primitive/visceral endoderm-type stem cell lines (ESC, TSC, and XEN, respectively) to arrange into postimplantation egg cylinder-like embryoids. Here, we provide an optimized method for reliable and efficient generation of ETX embryoids that develop into late gastrulation in static culture conditions. It is based on transgenic Gata6-overproducing ESCs and modified assembly and culture conditions. Using this method, up to 43% of assembled ETX embryoids exhibited a correct spatial distribution of the three stem cell derivatives at day 4 of culture. Of those, 40% progressed into ETX embryoids that both transcriptionally and morphologically faithfully mimicked in vivo postimplantation mouse development between E5.5 and E7.5. The ETX model system offers the opportunity to study the murine postimplantation egg cylinder stages and could serve as a source of various cell lineage precursors.

Endothelial SMAD1/5 signaling couples angiogenesis to osteogenesis during long bone growth.

Skeletal development depends on coordinated angiogenesis and osteogenesis. Bone morphogenetic proteins direct bone development by activating SMAD1/5 signaling in osteoblasts. However, the role of SMAD1/5 in skeletal endothelium is unknown. Here, we found that endothelial cell-conditional SMAD1/5 depletion in juvenile mice caused metaphyseal and diaphyseal hypervascularity, resulting in altered cancellous and cortical bone formation. SMAD1/5 depletion induced excessive sprouting, disrupting the columnar structure of the metaphyseal vessels and impaired anastomotic loop morphogenesis at the chondro-osseous junction. Endothelial SMAD1/5 depletion impaired growth plate resorption and, upon long term depletion, abrogated osteoprogenitor recruitment to the primary spongiosa. Finally, in the diaphysis, endothelial SMAD1/5 activity was necessary to maintain the sinusoidal phenotype, with SMAD1/5 depletion inducing formation of large vascular loops, featuring elevated endomucin expression, ectopic tip cell formation, and hyperpermeability. Together, endothelial SMAD1/5 activity sustains skeletal vascular morphogenesis and function and coordinates growth plate remodeling and osteoprogenitor recruitment dynamics during bone growth.

The development of the amnion in mice and other amniotes.

The amnion is an extraembryonic tissue that evolutionarily allowed embryos of all amniotes to develop in a transient and local aquatic environment. Despite the importance of this tissue, very little is known about its formation and its molecular characteristics. In this review, we have compared the basic organization of the extraembryonic membranes in amniotes and describe the two types of amniogenesis, folding and cavitation. We then zoom in on the atypical development of the amnion in mice that occurs via the formation of a single posterior amniochorionic fold. Moreover, we consolidate lineage tracing data to better understand the spatial and temporal origin of the progenitors of amniotic ectoderm, and visualize the behaviour of their descendants in the extraembryonic-embryonic junctional region. This analysis provides new insight on amnion development and expansion. Finally, using an online-available dataset of single-cell transcriptomics during the gastrulation period in mice, we provide bioinformatic analysis of the molecular signature of amniotic ectoderm and amniotic mesoderm. The amnion is a tissue with unique biomechanical properties that deserves to be better understood. This article is part of the theme issue 'Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom'.

Keratin filaments mediate the expansion of extra-embryonic membranes in the post-gastrulation mouse embryo.

Mesoderm arises at gastrulation and contributes to both the mouse embryo proper and its extra-embryonic membranes. Two-photon live imaging of embryos bearing a keratin reporter allowed recording filament nucleation and elongation in the extra-embryonic region. Upon separation of amniotic and exocoelomic cavities, keratin 8 formed apical cables co-aligned across multiple cells in the amnion, allantois, and blood islands. An influence of substrate rigidity and composition on cell behavior and keratin content was observed in mesoderm explants. Embryos lacking all keratin filaments displayed a deflated extra-embryonic cavity, a narrow thick amnion, and a short allantois. Single-cell RNA sequencing of sorted mesoderm cells and micro-dissected amnion, chorion, and allantois, provided an atlas of transcriptomes with germ layer and regional information. It defined the cytoskeleton and adhesion expression profile of mesoderm-derived keratin 8-enriched cells lining the exocoelomic cavity. Those findings indicate a novel role for keratin filaments in the expansion of extra-embryonic structures and suggest mechanisms of mesoderm adaptation to the environment.

Endothelial Zeb2 preserves the hepatic angioarchitecture and protects against liver fibrosis.

AIMS: Hepatic capillaries are lined with specialized liver sinusoidal endothelial cells (LSECs) which support macromolecule passage to hepatocytes and prevent fibrosis by keeping hepatic stellate cells (HSCs) quiescent. LSEC specialization is co-determined by transcription factors. The zinc-finger E-box-binding homeobox (Zeb)2 transcription factor is enriched in LSECs. Here, we aimed to elucidate the endothelium-specific role of Zeb2 during maintenance of the liver and in liver fibrosis. METHODS AND RESULTS: To study the role of Zeb2 in liver endothelium we generated EC-specific Zeb2 knock-out (ECKO) mice. Sequencing of liver EC RNA revealed that deficiency of Zeb2 results in prominent expression changes in angiogenesis-related genes. Accordingly, the vascular area was expanded and the presence of pillars inside ECKO liver vessels indicated that this was likely due to increased intussusceptive angiogenesis. LSEC marker expression was not profoundly affected and fenestrations were preserved upon Zeb2 deficiency. However, an increase in continuous EC markers suggested that Zeb2-deficient LSECs are more prone to dedifferentiation, a process called 'capillarization'. Changes in the endothelial expression of ligands that may be involved in HSC quiescence together with significant changes in the expression profile of HSCs showed that Zeb2 regulates LSEC-HSC communication and HSC activation. Accordingly, upon exposure to the hepatotoxin carbon tetrachloride (CCl4), livers of ECKO mice showed increased capillarization, HSC activation, and fibrosis compared to livers from wild-type littermates. The vascular maintenance and anti-fibrotic role of endothelial Zeb2 was confirmed in mice with EC-specific overexpression of Zeb2, as the latter resulted in reduced vascularity and attenuated CCl4-induced liver fibrosis. CONCLUSION: Endothelial Zeb2 preserves liver angioarchitecture and protects against liver fibrosis. Zeb2 and Zeb2-dependent genes in liver ECs may be exploited to design novel therapeutic strategies to attenuate hepatic fibrosis.

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.

Prdm16 Supports Arterial Flow Recovery by Maintaining Endothelial Function.

[Figure: see text].

A SMAD1/5-YAP signalling module drives radial glia self-amplification and growth of the developing cerebral cortex.

The growth and evolutionary expansion of the cerebral cortex are defined by the spatial-temporal production of neurons, which itself depends on the decision of radial glial cells (RGCs) to self-amplify or to switch to neurogenic divisions. The mechanisms regulating these RGC fate decisions are still incompletely understood. Here, we describe a novel and evolutionarily conserved role of the canonical BMP transcription factors SMAD1/5 in controlling neurogenesis and growth during corticogenesis. Reducing the expression of both SMAD1 and SMAD5 in neural progenitors at early mouse cortical development caused microcephaly and an increased production of early-born cortical neurons at the expense of late-born ones, which correlated with the premature differentiation and depletion of the pool of cortical progenitors. Gain- and loss-of-function experiments performed during early cortical neurogenesis in the chick revealed that SMAD1/5 activity supports self-amplifying RGC divisions and restrains the neurogenic ones. Furthermore, we demonstrate that SMAD1/5 stimulate RGC self-amplification through the positive post-transcriptional regulation of the Hippo signalling effector YAP. We anticipate this SMAD1/5-YAP signalling module to be fundamental in controlling growth and evolution of the amniote cerebral cortex.

Author Correction: The epicardium obscures interpretations on endothelial-to-mesenchymal transition in the mouse atrioventricular canal explant assay.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Slc25a17 Gene Trapped Mice: PMP34 Plays a Role in the Peroxisomal Degradation of Phytanic and Pristanic Acid.

Mice lacking PMP34, a peroxisomal membrane transporter encoded by Slc25a17, did not manifest any obvious phenotype on a Swiss Webster genetic background, even with various treatments designed to unmask impaired peroxisomal functioning. Peroxisomal α- and ß-oxidation rates in PMP34 deficient fibroblasts or liver slices were not or only modestly affected and in bile, no abnormal bile acid intermediates were detected. Peroxisomal content of cofactors like CoA, ATP, NAD+, thiamine-pyrophosphate and pyridoxal-phosphate, based on direct or indirect data, appeared normal as were tissue plasmalogen and very long chain fatty acid levels. However, upon dietary phytol administration, the knockout mice displayed hepatomegaly, liver inflammation, and an induction of peroxisomal enzymes. This phenotype was partially mediated by PPARα. Hepatic triacylglycerols and cholesterylesters were elevated and both phytanic acid and pristanic acid accumulated in the liver lipids, in females to higher extent than in males. In addition, pristanic acid degradation products were detected, as wells as the CoA-esters of all these branched fatty acids. Hence, PMP34 is important for the degradation of phytanic/pristanic acid and/or export of their metabolites. Whether this is caused by a shortage of peroxisomal CoA affecting the intraperoxisomal formation of pristanoyl-CoA (and perhaps of phytanoyl-CoA), or the SCPx-catalyzed thiolytic cleavage during pristanic acid ß-oxidation, could not be proven in this model, but the phytol-derived acyl-CoA profile is compatible with the latter possibility. On the other hand, the normal functioning of other peroxisomal pathways, and especially bile acid formation, seems to exclude severe transport problems or a shortage of CoA, and other cofactors like FAD, NAD(P)+, TPP. Based on our findings, PMP34 deficiency in humans is unlikely to be a life threatening condition but could cause elevated phytanic/pristanic acid levels in older adults.

BMP-SMAD1/5 Signaling Regulates Retinal Vascular Development.

Vascular development is an orchestrated process of vessel formation from pre-existing vessels via sprouting and intussusceptive angiogenesis as well as vascular remodeling to generate the mature vasculature. Bone morphogenetic protein (BMP) signaling via intracellular SMAD1 and SMAD5 effectors regulates sprouting angiogenesis in the early mouse embryo, but its role in other processes of vascular development and in other vascular beds remains incompletely understood. Here, we investigate the function of SMAD1/5 during early postnatal retinal vascular development using inducible, endothelium-specific deletion of Smad1 and Smad5. We observe the formation of arterial-venous malformations in areas with high blood flow, and fewer and less functional tip cells at the angiogenic front. The vascular plexus region is remarkably hyperdense and this is associated with reduced vessel regression and aberrant vascular loop formation. Taken together, our results highlight important functions of SMAD1/5 during vessel formation and remodeling in the early postnatal retina.

Impaired SMAD1/5 Mechanotransduction and Cx37 (Connexin37) Expression Enable Pathological Vessel Enlargement and Shunting.

OBJECTIVE: Impaired ALK1 (activin receptor-like kinase-1)/Endoglin/BMP9 (bone morphogenetic protein 9) signaling predisposes to arteriovenous malformations (AVMs). Activation of SMAD1/5 signaling can be enhanced by shear stress. In the genetic disease hereditary hemorrhagic telangiectasia, which is characterized by arteriovenous malformations, the affected receptors are those involved in the activation of mechanosensitive SMAD1/5 signaling. To elucidate how genetic and mechanical signals interact in AVM development, we sought to identify targets differentially regulated by BMP9 and shear stress. Approach and Results: We identify Cx37 (Connexin37) as a differentially regulated target of ligand-induced and mechanotransduced SMAD1/5 signaling. We show that stimulation of endothelial cells with BMP9 upregulated Cx37, whereas shear stress inhibited this expression. This signaling was SMAD1/5-dependent, and in the absence of SMAD1/5, there was an inversion of the expression pattern. Ablated SMAD1/5 signaling alone caused AVM-like vascular malformations directly connecting the dorsal aorta to the inlet of the heart. In yolk sacs of mouse embryos with an endothelial-specific compound heterozygosity for SMAD1/5, addition of TNFα (tumor necrosis factor-α), which downregulates Cx37, induced development of these direct connections bypassing the yolk sac capillary bed. In wild-type embryos undergoing vascular remodeling, Cx37 was globally expressed by endothelial cells but was absent in regions of enlarging vessels. TNFα and endothelial-specific compound heterozygosity for SMAD1/5 caused ectopic regions lacking Cx37 expression, which correlated to areas of vascular malformations. Mechanistically, loss of Cx37 impairs correct directional migration under flow conditions. CONCLUSIONS: Our data demonstrate that Cx37 expression is differentially regulated by shear stress and SMAD1/5 signaling, and that reduced Cx37 expression is permissive for capillary enlargement into shunts.

EGFL7 Mediates BMP9-Induced Sprouting Angiogenesis of Endothelial Cells Derived from Human Embryonic Stem Cells.

Human embryonic stem cells (hESCs) are instrumental in characterizing the molecular mechanisms of human vascular development and disease. Bone morphogenetic proteins (BMPs) play a pivotal role in cardiovascular development in mice, but their importance for vascular cells derived from hESCs has not yet been fully explored. Here, we demonstrate that BMP9 promotes, via its receptor ALK1 and SMAD1/5 activation, sprouting angiogenesis of hESC-derived endothelial cells. We show that the secreted angiogenic factor epidermal growth factor-like domain 7 (EGFL7) is a downstream target of BMP9-SMAD1/5-mediated signaling, and that EGFL7 promotes expansion of endothelium via interference with NOTCH signaling, activation of ERK, and remodeling of the extracellular matrix. CRISPR/Cas9-mediated deletion of EGFL7 highlights the critical role of EGFL7 in BMP9-induced endothelial sprouting and the promotion of angiogenesis. Our study illustrates the complex role of the BMP family in orchestrating hESC vascular development and endothelial sprouting.

Astrocyte-derived Jagged-1 mitigates deleterious Notch signaling in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a late-onset devastating degenerative disease mainly affecting motor neurons. Motor neuron degeneration is accompanied and aggravated by oligodendroglial pathology and the presence of reactive astrocytes and microglia. We studied the role of the Notch signaling pathway in ALS, as it is implicated in several processes that may contribute to this disease, including axonal retraction, microgliosis, astrocytosis, oligodendrocyte precursor cell proliferation and differentiation, and cell death. We observed abnormal activation of the Notch signaling pathway in the spinal cord of SOD1G93A mice, a well-established model for ALS, as well as in the spinal cord of patients with sporadic ALS (sALS). This increased activation was particularly evident in reactive GFAP-positive astrocytes. In addition, one of the main Notch ligands, Jagged-1, was ectopically expressed in reactive astrocytes in spinal cord from ALS mice and patients, but absent in resting astrocytes. Astrocyte-specific inactivation of Jagged-1 in presymptomatic SOD1G93A mice further exacerbated the activation of the Notch signaling pathway and aggravated the course of the disease in these animals without affecting disease onset. These data suggest that aberrant Notch signaling activation contributes to the pathogenesis of ALS, both in sALS patients and SOD1G93A mice, and that it is mitigated in part by the upregulation of astrocytic Jagged-1.

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.