Biology subjects

Zwijsen, A.

Publications and source records attributed to Zwijsen, A..

7 recordsLinked to original sources

bioRxiv2024

Atheroprone Flow Activates SMAD-FOXO1 to drive Endothelial-to-Mesenchymal Transition and Atherosclerosis

BackgroundCardiovascular 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 (TGF{beta}) 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. MethodsWe 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. ResultsWe 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. ConclusionWe 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.

genomics↗

bioRxiv2023

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

The {gamma}-secretase complexes are intramembrane cleaving proteases involved in the generation of the A{beta} peptides in Alzheimers 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 {gamma}-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 {gamma}-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 {gamma}-secretase complex and might contribute to the catalytic mechanism of the enzyme.

molecular biology↗

bioRxiv2023

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.

developmental biology↗

bioRxiv2022

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, deletion of Tcf7l1 abrogates PE differentiation without restraining epiblast priming. Taken together, our study underscores the importance of transcriptional Wnt inhibition in regulating lineage segregation in ESCs and preimplantation embryo development as well as identifies TCF7L1 as key regulator of this process.

developmental biology↗

bioRxiv2021

Keratin dynamics govern the establishment of the maternal-fetal interface.

After implantation, the mouse embryo undergoes gastrulation and forms mesoderm and endoderm. Mesoderm participates in embryonic and extra-embryonic tissues including the amnion, yolk sac, chorion and allantois, the umbilical cord precursor. Extra-embryonic mesoderm is rich in intermediate filaments. Two-photon live imaging of keratin 8-eYFP knock-in embryos allowed recording nucleation and elongation of keratin filaments, which formed apical cables coordinated across multiple cells in amnion, allantois, and blood islands. Embryos lacking all keratins displayed a deflated exocoelomic cavity, a narrow thick amnion, and a short allantois, indicating a hitherto unknown role for keratin filaments in post-implantation extra-embryonic membranes morphogenesis. Single-cell RNA sequencing of mesoderm cells, microdissected amnion, chorion, and allantois provided an interactive atlas of transcriptomes with germ layer and regional information. Keratin 8high mesenchymal cells in contact with the exocoelom shared a cytoskeleton and adhesion expression profile that might explain the adaptation of extra-embryonic structures to the increasing mechanical pressure. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/438772v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@30dfe4org.highwire.dtl.DTLVardef@bb19d6org.highwire.dtl.DTLVardef@19d6758org.highwire.dtl.DTLVardef@ebd3a5_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2019

Evolutionary conserved canonical BMP activity controls cortical neurogenesis

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 restrain the neurogenic ones. Furthermore, we demonstrate that SMAD1/5 stimulate RGC self-amplification through the positive post-transcriptional regulation of the Hippo signaling effector YAP. We anticipate this SMAD1/5-YAP signaling module to be fundamental in controlling growth and evolution of the amniote cerebral cortex.

developmental biology↗

bioRxiv2018

RLIM enhances BMP signalling mediated fetal lung development in mice

RLIM, also called RNF12, is an X-linked E3 ubiquitin ligase. It is a pleotropic protein acting as a negative regulator of LIM-homeodomain transcription factors and of SMAD7, an antagonist of the BMP and TGF{beta} pathways. Mutations in RLIM are associated with variable congenital malformations in human. The molecular mechanism causing these malformations is still poorly understood.\n\nIn this study, we used a conventional knockout (KO) mouse model to phenotypically characterize male mutants and to investigate the effect of Rlim mutation on BMP pathway signalling. Only 50% of KO male neonates survive and are significantly smaller. We show that Rlim deletion alters lung branching and maturation resulting in severe respiratory distress and neonatal death. Except for a single case of cardiac interventricular septal defect, no other malformations that mimic the human condition were observed. Western blot analysis shows that RLIM deficiency is associated with an increase of its target SMAD7 levels and a loss of SMAD1/5/9 activation. Interestingly, SMAD5 levels were diminished, suggesting another regulatory loop between RLIM and the BMP pathway.\n\nIn conclusion, RLIM is important for lung development in mice and exerts this function in part through intracellular propagation of BMP signalling.\n\nAuthor summaryRLIM is an X-linked gene encoding an enzyme that regulates a number of proteins including SMAD7, an antagonist of BMP pathway. RLIM mutations are associated with intellectual disability and several congenital malformations in boys. In mice, RLIM is thought to be nonessential for embryos development. Using a mouse model in which Rlim is deleted, we show that 50% of males carrying the deletion display respiratory distress and die short after birth. No congenital malformations mimicking the human phenotype have been observed. Histological analysis of lung samples show that Rlim is critical for late lung development and maturation. The analysis of lung samples by western blot, a technique that quantifies proteins, is consistent with a dysregulation of BMP pathway. These finding confirm the importance of RLIM for murine embryos development and provide a link between human RLIM pathology and BMP pathway.

genetics↗