Biology subjects

Cleaver, O.

Publications and source records attributed to Cleaver, O..

14 recordsLinked to original sources

bioRxiv2026

Functional divergence of WWC family proteins in human endothelial cells

The Hippo signaling pathway is increasingly recognized as a key regulator of endothelial cell (EC) proliferation, migration and vascular development. However, the roles of its upstream scaffold proteins remain poorly understood. Although WWC family proteins are widely regarded as functionally redundant activators of LATS1/2 kinases, the human genome contains a third family member, WWC3, that is absent from mice, raising the possibility of species-specific regulation of endothelial Hippo signaling. Here, we assessed the roles of WWC2 and WWC3 in human ECs using siRNA-mediated knockdown. Surprisingly, we found that WWC3 is the predominant regulator of canonical Hippo signaling, with a substantially greater effect than WWC2 on LATS1/2 phosphorylation, YAP/TAZ localization and expression of Hippo target genes. Loss of WWC3 also altered endothelial morphology and induced a partial endothelial-to-mesenchymal transition-like (EndoMT-like) phenotype. By contrast, WWC2 had a lesser effect on canonical Hippo signaling, but it was required for normal VEGF signaling dynamics. Despite these distinct molecular functions, depletion of either WWC2 or WWC3 impaired EC proliferation, migration, and cord formation in vitro. Together, our findings demonstrate that WWC family proteins perform overlapping but distinct functions in human ECs, with WWC3 acting as the predominant canonical Hippo regulator, whereas WWC2 more efficiently modulates VEGF signaling. These results reveal unexpected functional specialization among WWC proteins and suggest that regulation of Hippo signaling in human ECs differs from that inferred from mouse studies.

cell biology↗

bioRxiv2026

Transient architecture of the embryonic pancreas determines endocrine mass

During organogenesis, epithelial tissues undergo extensive three-dimensional (3D) remodeling while simultaneously generating specialized cell types. Whether these transient architectural states actively instruct lineage allocation remains unclear. Here we identify a morphogenetic stage in which resolution of epithelial stratification is required for lineage allocation and establishment of endocrine cell mass. We show that loss of the Hippo pathway regulator Merlin disrupts lumen morphogenesis and prevents formation of the transient 3D epithelial architecture that characterizes normal pancreas development. Failure to establish this architectural state alters lineage allocation, impairing acinar differentiation, markedly reducing adult endocrine cell mass, and disrupting glucose homeostasis. Mosaic analyses reveal that these lineage defects arise non-cell autonomously, demonstrating that epithelial architecture itself instructs cell fate decisions. Mechanistically, Merlin coordinates PI3K-regulated polarized membrane trafficking required for apical membrane biogenesis and lumen formation. Together, these findings identify Merlin-dependent membrane trafficking as a mechanism coupling epithelial morphogenesis to lineage allocation and demonstrate that transient developmental architectures can determine the cellular composition of mature organs.

developmental biology↗

bioRxiv2026

Endothelial adaptation to complex flow patterns in a novel in vitro model predicted by computational fluid dynamics

The endothelial cells (ECs) that line blood vessels continuously sense and respond to the physical forces exerted by blood flow. In vivo, pulsatile arterial flow interacts with vessel curvature, branching and other anatomical features to generate complex local hemodynamic environments that dictate the magnitude, direction, pulsatility, and oscillatory nature of wall shear stress experienced by ECs. Currently, accessible and reproducible in vitro models of complex pulsatile flow that recapitulate in vivo vascular anatomy remain limited. Here, we combine a novel rotational-flow endothelial culture platform with detailed computational fluid dynamics (CFD) modeling to characterize four well geometries designed to generate distinct hemodynamic environments. CFD analyses demonstrate that these geometries intrinsically generate pulsatile flow and produce reproducible spatially distinct regions of wall shear stress magnitude, pulsatility, and oscillatory shear within a single culture well. Endothelial alignment mapping and functional assays reveal region-specific cellular responses to the predicted local flow conditions that closely corresponded to the predicted local hemodynamic environment, linking complex flow patterns to endothelial adaptation. The technical advancements of our modeling efforts should support a faster, cheaper, simpler, and--importantly--validated framework for future investigation into EC mechanobiology under complex flow conditions. HIGHLIGHTSO_LISimple engineered well geometries generate distinct hemodynamic microenvironments, mimicking in vivo vascular structures, using a conventional orbital shaker. C_LIO_LIComputational fluid dynamics (CFD) reveals spatially distinct patterns of wall shear stress, pulsatility, and oscillatory shear applied to ECs within individual culture wells. C_LIO_LIHigh average wall shear stress and elevated oscillatory shear index induces a unique perpendicular alignment of ECs to the dominant flow vector. C_LI

cell biology↗

bioRxiv2025

Pulsatile flow dynamics determine pulmonary arterial architecture

Single ventricle congenital heart disease (SV-CHD) is a uniformly lethal condition requiring the Glenn surgery, which as a side effect eliminates arterial pulsatility and contributes to pulmonary vascular complications. In Glenn patients, we quantified pulsatility loss in each dimension of force (flow, pressure, and stretch) using cardiac catheterization and MRI. To model and investigate the individual impact of each dimension of pulsatility loss on the pulmonary vasculature, we applied isolated pulsatile and non-pulsatile mechanical stimuli to pulmonary arterial endothelial cells (ECs) in vitro. We found that each dimension of force triggered distinct transcriptional responses, revealing force-specific regulation of structural and signaling pathways. Pulsatile stretch uniquely stimulated EC secretion of PDGFB, a key driver of vascular smooth muscle cell (vSMC) recruitment. In a rat Glenn model, loss of pulsatility led to vascular wall thinning, confirming in vivo relevance. Our findings uncover a mechanistic link between endothelial stretch sensing and PDGFB-mediated EC-vSMC crosstalk, essential for maintaining pulmonary artery architecture. Clinically, these insights suggest that restoring or mimicking pulsatile forces may help preserve vascular integrity and prevent remodeling in SV-CHD patients.

cell biology↗

bioRxiv2025

Zonal endothelial cell heterogeneity underlies murine renal vascular development.

The renal vasculature consists of highly specialized blood vessels that carry out distinct physiological functions. Defining their transcriptional signatures and tracing their developmental ontogeny has thus far been challenging due to a lack of regionally specific endothelial biomarkers. Here, we performed single nuclear RNA sequencing (snucRNA-Seq) to interrogate embryonic renal endothelial cells. We identified nine different endothelial cell types, and validated expression of novel candidates using multiplex RNAScope. We interrogate biological characteristics, including cell cycle stage, of developmental blood vessels with a variety of in vivo tools. Using genetic tools based on expression of tip cell marker Esm1, we performed detailed lineage tracing, demonstrating multi-clonal and multi-directional endothelial contribution to glomerular vasculature. Together, this study provides the first validated, tool-focused developmental atlas of the murine renal vasculature and elucidates several novel cellular mechanisms of nephron vascularization.

developmental biology↗

bioRxiv2024

Integration of spatial and single nucleus transcriptomics to map gene expression in the developing mouse kidney

The kidney is a complex organ requiring tightly coordinated interactions between epithelial, endothelial, and mesenchymal cells during development. Congenital kidney defects can result in kidney disease and renal failure, highlighting the importance of understanding kidney formation mechanisms. Advances in RNA sequencing have revealed remarkable cellular heterogeneity, especially in the kidney stroma, though relationships between stromal, epithelial, and endothelial cells remain unclear. This study presents a comprehensive gene expression atlas of embryonic and postnatal kidneys, integrating single-nucleus and in situ RNA sequencing data. We developed the Kidney Spatial Transcriptome Analysis Tool (KSTAT), enabling researchers to identify cell locations, predict cell-cell communication, and map gene pathway activity. KSTAT revealed significant heterogeneity among embryonic kidney pericytes, providing a critical resource for hypothesis generation and advancing knowledge of kidney development and disease.

developmental biology↗

bioRxiv2024

Lats1/2 are essential for developmental vascular remodeling and biomechanical adaptation to shear stress

Blood vessels in mammalian embryos develop from initial aggregates of endothelial cell (EC) progenitors, which coordinate the opening and stabilization of central vascular lumens, all while under progressively increasing flow and pressure from blood circulation. Mechanical cues exerted by shear stress from the blood flow remodel an initial vascular plexus into a ramifying array of large and small vessels. As plasma starts to fill vascular lumens, these forces trigger changes in EC gene expression and dynamic alterations in cell shape and cell adhesion, as cuboidal angioblasts elongate and flatten into ECs. Little is known about how embryonic ECs sense and transduce hemodynamic signals as vessels form in vivo. Here, we report a critical requirement for the Lats1 and Lats2 Hippo pathway kinases during this process. We show that when Lats1/2 are genetically deleted in ECs, embryos develop severe defects in blood vessel formation, which lead to embryonic lethality by E11.5. We find that initial vessel patterning and circulation initiate properly, however remodeling of the initial vascular plexus fails due to lumen collapse and altered blood flow. When Lats1/2 are knocked down using siRNA approaches in cultured ECs, cells fail to elongate and polarize, similar to ECs in the mutant embryos. In addition, VE-cadherin (VEcad) based junctions fail to mature under shear stress. These data show that Lats1/2 deficient cells no longer respond to laminar shear stress, both in vivo and in vitro. This work identifies the Hippo pathway kinases Lats1 and Lats2 as critical transducers of biomechanical cues during the early steps of blood vessel remodeling. This study will provide new targets for treatment of vascular diseases and new directions for efforts to generate vascularized tissues for replacement therapies. HighlightsO_LILats1 and Lats2 mRNA and protein are expressed in murine embryonic endothelial cells (ECs). C_LIO_LIDeletion of Lats1/2 in embryonic endothelium results in severe vascular defects and embryonic lethality. C_LIO_LILoss of Lats1/2 leads to failure of both vascular remodeling and EC elongation upon exposure to flow, in vivo and in vitro. C_LIO_LILats1/2 are required for cell-cell VE-cadherin adhesion maturation under flow. C_LIO_LILoss of Lats1/2 results in cytoskeletal disorganization in response to shear stress. C_LI

developmental biology↗

bioRxiv2024

Spatiotemporal dynamics of primary and motile cilia throughout lung development

Cilia are specialized structures found on a variety of mammalian cells, with variable roles in the transduction of mechanical and biological signals (by primary cilia, PC), as well as the generation of fluid flow (by motile cilia). Their critical role in the establishment of a left-right axis in early development is well described, as is the innate immune function of multiciliated upper airway epithelium. By contrast, the dynamics of ciliary status during organogenesis and postnatal development is largely unknown. In this study, we define the progression of ciliary status within the endothelium, epithelium, and mesenchyme of the lung. Remarkably, we find that endothelial cells (ECs) lack PC at all stages of development, except in low numbers in the most proximal portions of the pulmonary arteries. In the lung epithelium, a proximodistal ciliary gradient is established over time, as the uniformly mono-ciliated epithelium transitions into proximal, multiciliated cells, and the distal alveolar epithelium loses its cilia. Mesenchymal cells, interestingly, are uniformly ciliated in early development, but with restriction to PDGFR+ fibroblasts in the adult alveoli. This dynamic process in multiple cellular populations both challenges prior assertions that PC are found on all cells, and highlights a need to understand their spatiotemporal functions. HighlightsO_LIPrimary cilia are found broadly throughout early embryonic tissues. C_LIO_LIPrimary cilia are observed in both epithelial and mesenchymal cells in the early lung. C_LIO_LIPulmonary endothelial cells largely do not possess primary cilia during embryonic development. C_LIO_LIDifferential multiciliation and loss of epithelial cilia in a proximal-distal axis. C_LIO_LIMaintenance of cilia in adult pulmonary PDGFR+ fibroblasts. C_LI

developmental biology↗

bioRxiv2024

Characterization of Hippo Signaling Components in the Early Dorsal Pancreatic Bud

All pancreatic lineages originate from a transitory structure known as the multipotent progenitor epithelium (MPE), which is a placode formed via epithelial stratification. Cells within the MPE undergo de novo lumenogenesis to give rise to an epithelial plexus, which serves as a progenitor niche for subsequent development of endocrine, ductal and acinar cell types. Recent evidence suggests that Hippo signaling is required for pancreatic cell differentiation, but little is known about the function of Hippo signaling in the development of the MPE. Here, we characterize the expression of YAP1, TAZ, and the Hippo regulators LATS1/2 kinases and MERLIN in early murine pancreatic epithelium, during epithelial stratification, plexus development and emergence of endocrine cells. We find that YAP1 expression is relatively low in the pancreas bud during stratification, but increases by E11.5. Intriguingly, we find that TAZ, but not YAP1, is expressed in early endocrine cells. We further find that MERLIN and LATS1/2 kinases are robustly expressed during the period of rapid stratification and become markedly apical at nascent lumens. To gain a better understanding of how Hippo signaling and lumen formation are connected, we analyzed the expression of Hippo signaling components in an in vitro model of lumen formation and found that they are dynamically regulated during lumenogenesis. Together, our results point to a relationship between Hippo signaling and lumen formation during pancreatic development. HIGHLIGHTSO_LIYAP1 expression in the early mouse pancreatic anlagen is low until approximately E11.5, when it becomes localized to cell nuclei in multipotent progenitor cells. At E14.5, we find nuclear YAP1 in ductal cells. C_LIO_LIYAP1 is not expressed in early and midgestation endocrine cells. By contrast, TAZ is expressed in first transition endocrine cells. C_LIO_LIHippo regulators MERLIN and LATS1/2 kinases are robustly expressed in the early pancreatic bud by E10.5. Both MERLIN and LATS1/2 exhibit strong apical localization in epithelial cells at nascent microlumens. C_LIO_LIUsing in vitro models of de novo pancreas lumen formation, we show that YAP1 nuclear localization is high in early phases of lumen formation and gradually decreases as lumens matures. C_LI

developmental biology↗

bioRxiv2023

Uncovering the transcriptomic heterogeneity of pancreatic endothelial cells using integrative and comparative single cell gene expression analysis

The pancreatic islet vasculature displays tissue-specific physiological and functional adaptations that support rapid glucose sensing and insulin response by {beta}-cells. To uncover the transcriptomic basis of this specialization, we performed a meta-analysis of multi-organ single cell RNA sequencing atlases employing a unique strategy to avoid transcriptomic contamination. We identified biologically relevant genes involved in sphingosine-1-phosphate-mediated insulin-secretion (PLPP1, RDX, CDC42EP1), islet basement membrane formation (SPARC, COL15A1), endothelial cell (EC) permeability (PLVAP, EHD4), membrane transporters (CD320, SLCO2A1) and developmental transcription factors (NKX2-3, AHR). These were validated in silico in independent datasets. We further established the first integrated transcriptomic atlas of human pancreatic ECs and described two unique capillary subpopulations: exocrine and endocrine pancreas ECs. We validated the spatial localization of key markers using RNAscope and immunofluorescence staining on mouse pancreatic tissue cross-sections. Our findings provide novel insights into pancreatic EC heterogeneity and islet EC function with potential implications in therapeutic strategies.

cell biology↗

bioRxiv2023

Stromal netrin-1 coordinates renal arteriogenesis and mural cell differentiation

The kidney vasculature has a uniquely complex architecture that is essential to proper renal function. Little is known about the molecular mechanisms that direct where and when blood vessels form during kidney development. We identified a regionally-restricted, stroma-derived signaling molecule, netrin-1 (Ntn1), as a putative regulator of vascular patterning. We generated a stromal progenitor-specific knockout of netrin-1 (Ntn1SPKO) that resulted in smaller postnatal kidneys with altered epithelial development and profound defects in arterial and capillary architecture. We also found significant loss of arterial vascular smooth muscle cell (vSMC) coverage and ectopic smooth muscle cell deposition at the kidney cortex. Transcriptomic analysis of Ntn1SPKO kidneys revealed downregulation of Klf4, which we find expressed in stromal progenitors. Deletion of Klf4 in the stroma largely phenocopies loss of Ntn1, and expression of Klf4 in Ntn1SPKO kidneys rescues ectopic vSMC deposition. Vascular defects observed in Ntn1SPKO are transient, as both arterial and smooth muscle coverage defects resolve late in development, however ectopic peripheral smooth muscle perdures perinatally. These data suggest a stromal-intrinsic Ntn1-Klf4 axis acting as an essential mediator of stromal crosstalk and vascular progenitor differentiation.

developmental biology↗

bioRxiv2022

Rab11 is essential to pancreas morphogenesis, lumen formation and endocrine mass.

The molecular links between tissue-level morphogenesis and the differentiation of cell lineages in the pancreas remain elusive despite a decade of studies. We previously showed that in pancreas both these processes depend on proper lumenogenesis. The Rab GTPase Rab11 has been shown to be essential to epithelial lumen formation in vitro, however few studies have addressed its functions in vivo and none have tested its requirement in pancreas. Here, we show that Rab11 is critical to proper pancreas development. Co-deletion of the Rab11 isoforms Rab11A and Rab11B in the developing pancreatic epithelium (Rab11pancDKO) results in ~50% neonatal lethality, and surviving adult Rab11pancDKO mice exhibit defective endocrine function. Loss of Rab11 in the embryonic pancreas results in morphogenetic defects of the epithelium linked to defective lumen formation and interconnection. In contrast to wildtype cells, Rab11pancDKO cells attempt to form multiple lumens, resulting in a failure to coordinate a single apical membrane initiation site (AMIS) between groups of cells. We show that these defects are due to failures in vesicle trafficking, as apical components remain trapped within Rab11pancDKO cells. Together, these observations suggest Rab11 directly regulates epithelial lumen formation and morphogenesis. Our report links intracellular trafficking to organ morphogenesis in vivo, and presents a novel framework for decoding pancreatic development. HIGHLIGHTSO_LIRab11Af/f;Rab11B-/-;Pdx1-Cre pancreas displays disruption of epithelial organization and reduction of endocrine cell mass. C_LIO_LILoss of Rab11 results in disruption of pancreatic lumen continuity due to a failure of lumen formation. C_LIO_LIEpithelial cells lacking Rab11 display abnormal polarity. C_LI

developmental biology↗

bioRxiv2021

Cyp26b1 restrains murine heart valve growth during development

Endothelial cells (ECs) are critical to proper heart valve development, directly contributing to the mesenchyme of the cardiac cushions, which progressively transform into mature valves. To date, investigators have lacked useful markers of valve ECs to fully evaluate their contributions during valve morphogenesis. As a result, it has been unclear whether the well-characterized regional differentiation of valves correlates with any endothelial domains in the heart. Furthermore, it has been difficult to ascertain whether endothelial heterogeneity in the heart influences underlying mesenchymal zones in an angiocrine manner. To identify regionally expressed EC genes in the heart valves, we screened publicly available databases and assembled a toolkit of endothelial-enriched genes. We identified Cyp26b1 as one of many endothelial enriched genes found to be expressed in the endocardium of the developing cushions and valves. Here, we show that Cyp26b1 is required for normal heart valve development. Genetic ablation of Cyp26b1 in mouse embryos leads to abnormally thickened aortic valve leaflets, which is due in part to increased endothelial and mesenchymal cell proliferation in the remodeling valves. In addition, Cyp26b1 mutant hearts display ventricular septal defects (VSDs) in a portion of null embryos. We show that loss of Cyp26b1 results in upregulation of retinoic acid (RA) target genes, supporting the observation that Cyp26b1 has RA-dependent roles. Together, this work identifies a novel role for Cyp26b1 in heart valve morphogenesis. Understanding the spatiotemporal expression dynamics of cardiac EC genes will likely prove useful to the investigation of both normal as well as dysfunctional heart valve development. HIGHLIGHTS{middle dot} A mouse heart valve gene expression atlas can be generated with publicly available online tools, such as Genepaint and other gene expression databases. {middle dot} Endothelium of developing mouse heart valves is regionally heterogeneous. {middle dot} Cyp26b1 is expressed in the endocardial/endothelial lining of developing heart valves. {middle dot} Loss of Cyp26b1 leads to significant enlargement of aortic valves and to ventricular septal defects. {middle dot} Cyp26b1 represses cell proliferation in valve mesenchyme. {middle dot} Retinoic acid targets are upregulated in Cyp26b1-/- heart valves, indicating dysregulation of RA metabolism.

developmental biology↗

bioRxiv2019

Cyp26b1 is required for proper airway epithelial differentiation during lung development

Proper organ development depends on coordinated communication between multiple cell types. Retinoic acid (RA) is an autocrine and paracrine signaling molecule critical for the development of most organs including the lung. Both RA excess and deficiency lead to drastic alterations in embryogenesis, often culminating in embryonic or neonatal lethality. Therefore, RA levels must be spatially and temporally titrated to ensure proper organogenesis. Despite extensive work detailing the effects of RA deficiency in early lung morphogenesis, little is known about how RA levels are modulated during late lung development. Here, we investigate the role of the RA catabolizing protein Cyp26b1 in lung development. Cyp26b1 is highly enriched in lung endothelial cells (ECs) throughout the course of development. We find that loss of Cyp26b1 impacts differentiation of the distal epithelium without appreciably affecting proximal airways, EC lineages, or stromal populations. Cyp26b1-/- lungs exhibit an increase in cellular density, with an expansion of distal progenitors at the expense of alveolar type 1 (AT1) cells, which culminates in neonatal death. Exogenous administration of RA in late gestation was able to partially reproduce this defect in epithelial differentiation; however, transcriptional analyses of Cyp26b1-/- lungs and RA-treated lungs reveal separate, but overlapping, transcriptional responses. These data suggest that the defects observed in Cyp26b1-/- lungs are caused by both RA-dependent and RA-independent mechanisms. This work highlights critical cellular crosstalk during lung development involving a crucial role for Cyp26b1-expressing endothelium, and identifies a novel RA rheostat in lung development.\n\nHIGHLIGHTSO_LICyp26b1 is highly expressed in lung ECs throughout development\nC_LIO_LICyp26b1-null lungs fail to undergo proper differentiation of distal epithelium leading to an increase in progenitors and AT2 cells at the expense of AT1 cells\nC_LIO_LIFunctional and transcriptional analyses suggest both RA-dependent and RA-independent mechanisms\nC_LI

developmental biology↗