An Zwijsen

Endothelial-to-mesenchymal transition (EndMT) contributes to vascular remodeling and endothelial dysfunction in diseases such as atherosclerosis and pulmonary arterial hypertension. This process describes a gradual shift in which endothelial cells (ECs) lose expression of classical EC markers and acquire mesenchymal-like gene expression patterns and phenotypic traits. Although SMAD transcription factors are key regulators of EndMT, driven by mechanical cues from disturbed flow that activate BMP or TGFβ signaling, their weak DNA-binding affinity invoked the need for additional transcriptional co-factors to specify gene targets under pathophysiological flow conditions. Here, we identified FOXO1 as a transcriptional co-factor that cooperates with SMADs to regulate mesenchymal gene expression in ECs exposed to irregular flow. FOXO1 and SMADs co-occupy genomic loci and form transcriptional complexes. Although SMAD DNA binding is FOXO1-independent, FOXO1 activity is required for full induction of SMAD target genes. Notably, FOXO1 DNA-binding activity is essential for robust activation of EndMT-associated genes, as both pharmacological inhibition and siRNA-mediated depletion of FOXO1 attenuate SMAD target gene expression. Functionally, combined targeting of FOXO1 and SMAD signaling rescues EndMT-associated endothelial dysfunction in carotid arteries of mice. Our results describe FOXO1 as key integrator of SMAD-mediated EndMT signaling, linking it to pathogenic SMAD activation in disease contexts.

Metabolic dysfunction-associated steatotic liver disease (MASLD), formerly known as non-alcoholic fatty liver disease, features liver sinusoidal endothelial cell (LSEC) alterations with ill-defined driving factors. Zinc-Finger E-Box-binding Homeobox (ZEB)2 in LSECs preserves their specialized features, prevents capillarization and protects against liver fibrosis. To investigate a potential protective role against steatosis, the initial MASLD stage, we fed EC-specific Zeb2 knockout (EC ^Zeb2KO ) mice a western-type diet (WD). In healthy and steatotic wild-type livers, Zeb2 was ubiquitously and similarly expressed across blood-vascular EC types. LSEC RNA sequencing revealed ZEB2 deficiency-triggered expression changes greatly overlapping with those evoked by WD-feeding. Endothelial ZEB2-loss and WD-feeding interacted to boost capillarization and fat metabolism, shown by increased expression of continuous EC markers and peroxisome proliferator-activated receptor (PPAR)α signaling components, respectively. Altered communication among LSECs after combined endothelial ZEB2-loss and WD-exposure revealed similar functional repercussions. Endothelial ZEB2-loss eventually corrected WD-induced liver hypo-vascularization while ameliorating hepatic damage and steatosis. Thus, endothelial ZEB2-loss amplifies WD-induced LSEC fat metabolism and capillarization, while decreasing steatosis, in part through altered LSEC-LSEC communication. The disease-promoting role of endothelial ZEB2 in early MASLD as opposed to its protective role in fibrosis underscores a context-dependent effect in liver disease.

Background: Cardiovascular diseases are the leading cause of death worldwide with atherosclerosis as the main underlying pathology. A hallmark of atherosclerotic lesion formation is endothelial-to-mesenchymal transition (EndoMT) triggered by perturbed blood flow patterns at arterial bifurcations and curvatures. SMAD transcription factors (TFs), activated by bone morphogenetic protein (BMP) 9/10 or transforming growth factor beta (TGFβ) signaling, are indispensable for endothelial homeostasis. Yet, they also play a significant role in stimulating EndoMT. How different interacting co-factors mediate the shift towards a pathological SMAD response remains elusive. Methods: We generated endothelial cell (EC)-specific SMAD1/5 or SMAD2/3 knock-out mice and performed assay for transposase accessible chromatin sequencing (ATAC-Seq) of EC nuclei from regions of atheroprone (aortic arch) and atheroprotective (descending thoracic aorta) flow to identify transcriptional co-regulators of SMADs. We validated this using single-cell (sc)ATAC-Seq and immunofluorescence staining data from wild-type mice. To assess conservation of our findings for the human situation, we performed co-immunoprecipitation and proximity ligation assays in human aortic ECs (HAoECs). We exposed HAoECs to pathological or physiological ( i . e . oscillatory or pulsatile) flow and performed ATAC- and RNA-Seq. Next, transcriptomic and chromatin accessibility data were integrated and motif enrichment and TF footprinting analysis were performed. Finally, we used siRNA-mediated approaches, TF inhibition, and luciferase-based reporter gene assays to analyze the transcriptional response of target TFs and explored their presence in plaques of atheroprone low-density lipoprotein receptor-deficient mice. Results: We observed enrichment of FOXO TF family motifs in DNA loci with increased accessibility in response to atheroprone flow in vitro and in vivo . These motifs were associated with genes displaying enhanced mRNA expression. We observed that FOXO motifs are enriched in peaks lost upon EC-specific SMAD KO in mice. We identified SMADs and FOXO1 as interacting partners that form complexes upon atheroprone flow stimulation. Inhibitor experiments revealed that FOXO1 and SMADs mediate EndoMT upon atheroprone flow exposure by upregulating SNAI2 . Conclusion: We identified SMAD/FOXO1 complexes that mediate EndoMT in response to atheroprone flow. Targeting this interaction can potentially reduce atherosclerotic burden by interfering with pathological flow-induced EndoMT and thus disease progression.

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. 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/TGFβR2 receptor complex, with TGFβ1 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 TGFβ1 stimulation. Blocking acetyl-CoA production prevents pre-flow arterial expression of Hey1 and Gja4, while stabilizing acetylation rescues their expression. 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.

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.

The γ-secretase complexes are intramembrane cleaving proteases involved in the generation of the Aβ 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 (PEN-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. Glycine22 and Proline27 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.

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.

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.

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 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’.