Biology subjects

Greene, N.

Publications and source records attributed to Greene, N..

13 recordsLinked to original sources

bioRxiv2026

Extracellular matrix defects destabilise apical cytoarchitecture and mechanical properties during early Down syndrome neurodevelopment

Down syndrome (DS) is associated with altered brain development, especially in the cerebellum. However, how trisomy 21 (TS21) is linked to cerebellar defects remains poorly understood. Cerebellar organoids reveal that TS21 leads to extracellular matrix (ECM) alterations, perturbing downstream pathways linked to reduction of apical RAB11+ endosomes. These changes lead to impaired apical maintenance and altered progenitor composition. Significantly, ECM-enriched culture rescues apical defects in TS21 organoids, supporting a functional role for ECM in preserving apical organisation and progenitor niche integrity. In the developing human DS cerebellum and 2D neural cultures, where exogenous ECM is present, severe epithelial disorganisation is attenuated. Nevertheless, ECM organisation and mechanical properties remain altered in 2D cultures. Our findings identify TS21-driven ECM abnormalities as a mechanism impairing apical integrity, progenitor composition and mechanical proprieties in neural stem cell models of DS. Altogether, we established apical maintenance instability as an early developmental defect contributing for DS neuropathology.

Developmental Biology↗

bioRxiv2025

Metabolic immunity to infection is driven by mitochondrial one-carbon metabolism

As large consumers of cellular metabolites, mitochondria are positioned to compete with invading microbes for the nutrients they require to grow. Yet, little is known of whether cells weaponize mitochondrial metabolism during infection. We found that the transcription factor ATF4 activated a mitochondrial metabolic defense based on the essential B vitamin folate. During infection with the human parasite Toxoplasma gondii, ATF4 increased mitochondrial DNA (mtDNA) levels by driving the one-carbon (1C) metabolism processes that occur in mitochondria and use folate. The activation of ATF4 depended on host detection of parasite effector proteins, and restricted parasite growth by limiting its access to folate(s) required for dTMP synthesis. Impairing host mitochondrial 1C metabolism downstream of ATF4 promoted parasite growth, while forcing host dependence on mitochondrial 1C metabolism had the opposite effect. ATF4 activation also promoted a host-protective response in a mouse model of Toxoplasma infection. Thus, ATF4 rewires mitochondrial metabolism to activate a folate-based metabolic immunity against Toxoplasma. Our work paves the way for future studies exploring noncanonical defense strategies mediated by mitochondria and the role of folate metabolism during infectious disease. One-Sentence SummaryATF4 rewires mitochondrial metabolism during infection to drive a host-protective response based on folate competition.

cell biology↗

bioRxiv2025

Chiari II brain malformation is secondary to open spina bifida

Chiari II brain malformation affects 90% of children with open spina bifida. Hindbrain herniation leads to hydrocephalus, together with higher brain anomalies including cerebral cortical defects implicated in learning disability, which affects 20-25% of children with spina bifida. The causal link between Chiari II and spina bifida has long been debated, and we aimed to determine whether Chiari II arises secondary to spina bifida, rather than as a separate effect of shared genetic or non-genetic factor(s). Pax3 gene function was conditionally deleted by Cdx2cre specifically in the lower body of mice, leaving the head genetically intact. Open spina bifida is seen in all Cdx2cre/+; Pax3fl/flfetuses, together with many features of Chiari II in the wild-type brain and skull. These include: hindbrain herniation, callosal and hippocampal hypogenesis, cortical thinning with neuronal heterotopia, a thickened ventricular zone, and posterior skull defects. Hence, the brain and skull defects of Chiari II arise secondary to open spina bifida, with likely disturbance of neurogenesis and neuronal migration early in gestation. The Cdx2cre/+; Pax3fl/fl mouse provides a model for improved understanding of Chiari II pathogenesis.

developmental biology↗

bioRxiv2024

Humidity as a zeitgeber for circadian entrainment of insect systems

Humidity levels, like light and temperature, fluctuate daily yet are less predictable; however, whether humidity can entrain circadian clocks and synchronize animal behaviors with environmental variations remains unknown. Here, we investigate the circadian humidity entrainment in various insects across multiple orders. Insect species respond to humidity cycles with distinct patterns, some active during wet periods or at the arid-humid transition. When the humidity cue is removed, most species continue to show rhythmic activity associated with the previous arid-humid (AH) cycles. Fruit flies shift their activity accordingly when humidity cycles are altered and remain in the new rhythms under the following free-running conditions (FRC; constant humidity, HH). Moreover, Drosophila clock and hygrosensation mutants lack rhythmic activity during (AH) and after humidity entrainment (FRC with HH), indicating that core clock components and hygrosensors are essential for circadian entrainment. Our findings provide strong evidence that humidity is likely to serve as a potential zeitgeber for circadian entrainment in most, but not all, insect systems and will likely have broad applicability and importance across animal systems. While light and temperature act as the primary zeitgebers, understanding the mechanisms of humidity entrainment will help us better interpret the behavioral patterns of terrestrial animals, particularly those susceptible to dehydration. One Sentence Summary: Humidity entrainment of the circadian clock synchronizes insect activity to environmental changes.

physiology↗

bioRxiv2024

A Decade in a Systematic Review: The Evolution and Impact of Cell Painting

High-content image-based assays have fueled significant discoveries in the life sciences in the past decade (2013-2023), including novel insights into disease etiology, mechanism of action, new therapeutics, and toxicology predictions. Here, we systematically review the substantial methodological advancements and applications of Cell Painting. Advancements include improvements in the Cell Painting protocol, assay adaptations for different types of perturbations and applications, and improved methodologies for feature extraction, quality control, and batch effect correction. Moreover, machine learning methods recently surpassed classical approaches in their ability to extract biologically useful information from Cell Painting images. Cell Painting data have been used alone or in combination with other - omics data to decipher the mechanism of action of a compound, its toxicity profile, and many other biological effects. Overall, key methodological advances have expanded Cell Paintings ability to capture cellular responses to various perturbations. Future advances will likely lie in advancing computational and experimental techniques, developing new publicly available datasets, and integrating them with other high-content data types.

cell biology↗

bioRxiv2024

PKSmart: An Open-Source Computational Model to Predict in vivo Pharmacokinetics of Small Molecules

Drug exposure is a key contributor to the safety and efficacy of drugs. It can be defined using human pharmacokinetics (PK) parameters that affect the blood concentration profile of a drug, such as steady-state volume of distribution (VDss), total body clearance (CL), half-life (t[1/2]), fraction unbound in plasma (fu) and mean residence time (MRT). In this work, we used molecular structural fingerprints, physicochemical properties, and predicted animal PK data as features to model the human PK parameters VDss, CL, t[1/2], fu and MRT for 1,283 unique compounds. First, we predicted animal PK parameters [VDss, CL, fu] for rats, dogs, and monkeys for 372 unique compounds using molecular structural fingerprints and physicochemical properties. Second, we used Morgan fingerprints, Mordred descriptors and predicted animal PK parameters in a hyperparameter-optimised Random Forest algorithm to predict human PK parameters. When validated using repeated nested cross-validation, human VDss was best predicted with an R2 of 0.55 and a Geometric Mean Fold Error (GMFE) of 2.09; CL with accuracies of R2=0.31 and GMFE=2.43, fu with R2=0.61 and GMFE=2.81, MRT with R2=0.28 and GMFE=2.49, and t[1/2] with R2=0.31 and GMFE=2.46 for models combining Morgan fingerprints, Mordred descriptors and predicted animal PK parameters. We evaluated models with an external test set comprising 315 compounds for VDss (R2=0.33 and GMFE=2.58) and CL (R2=0.45 and GMFE=1.98). We compared our models with proprietary pharmacokinetic (PK) models from AstraZeneca and found that model predictions were similar with Pearson correlations ranging from 0.77-0.78 for human PK parameters of VDss and fu and 0.46-0.71 for animal (dog and rat) PK parameters of VDss, CL and fu. To the best of our knowledge, this is the first work that publicly releases PK models on par with industry-standard models. Early assessment and integration of predicted PK properties are crucial, such as in DMTA cycles, which is possible with models in this study based on the input of only chemical structures. We developed a webhosted application PKSmart (https://broad.io/PKSmart) which users can access using a web browser with all code also downloadable for local use. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=73 SRC="FIGDIR/small/578658v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1a4b673org.highwire.dtl.DTLVardef@c64c5aorg.highwire.dtl.DTLVardef@bec0b3org.highwire.dtl.DTLVardef@15d5756_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure:C_FLOATNO For TOC Only C_FIG

bioinformatics↗

bioRxiv2023

AAV-mediated gene therapy in a GLDC-deficient mouse model of Non-Ketotic Hyperglycinemia

Non-Ketotic Hyperglycinemia (NKH) is a rare inborn error of metabolism caused by impaired function of the glycine cleavage system (GCS) and characterised by accumulation of glycine in body fluids and tissues. NKH is an autosomal recessive condition and the majority of affected individuals carry mutations in GLDC (glycine decarboxylase). Current treatments for NKH are not effective or curative. As a monogenic condition with known genetic causation, NKH is potentially amenable to gene therapy. An AAV9-based expression vector was designed to target sites of GCS activity. Using an ubiquitous promoter to drive expression of a GFP reporter, transduction of liver and brain was confirmed following intra-venous and/or intra-cerebroventricular administration to neonatal mice. Using the same capsid and promoter with transgenes to express mouse or human GLDC, vectors were then tested in a GLDC-deficient mice that provide a model of NKH. GLDC-deficient mice exhibited elevated plasma glycine concentration and accumulation of glycine in liver and brain tissues as previously observed. Moreover, the folate profile indicated suppression of folate one carbon metabolism (FOCM) in brain tissue, as found at embryonic stages, and reduced abundance of FOCM metabolites including betaine and choline. Neonatal administration of vector achieved reinstatement of GLDC mRNA and protein expression in GLDC-deficient mice. Treated GLDC-deficient mice showed significant lowering of plasma glycine, confirming functionality of vector expressed protein. AAV9-GLDC treatment also led to lowering of brain tissue glycine, and normalisation of the folate profile indicating restoration of glycine-derived one-carbon supply. These findings support the hypothesis that AAV-mediated gene therapy may offer potential in treatment of NKH.

molecular biology↗

bioRxiv2023

CFL1-dependent dynamicity of surface ectoderm filopodia-like protrusions increases neurulation zippering speed in mice

Epithelial fusion is critical for formation of many embryonic tissues. The developing mammalian spinal cord fuses by zippering, through cell behaviours dependent on F-actin turnover. We propose a caudal-to-rostral sequence of surface ectoderm cell behaviours which drive zippering progression in mice, and test the requirement for the F-actin severing protein CFL1 in this process. Key zipper-advancing behaviours are i. constriction of supracellular actomyosin cables; ii. extension of short-lived (<2 min) filopodial or more persistent lamellipodial protrusions, which we live-image establishing pioneering contacts across the midline; remodelling of iii. cell-cell and iv. cell-ECM adhesions; v. zipper advancement through constriction of leading-edge cell borders. In wildtype embryos CFL1 is enriched at the leading edge of surface ectoderm cells adjacent to the zippering point and localised within some protrusions. Conditional Cfl1 deletion in the surface ectoderm produces more prominent F-actin stress fibres, makes filopodial protrusions excessively stable, diminishes their exploratory movements which may establish nascent contacts, and impairs junctional constriction of cells at the point of fusion. Zippering speed is reduced [~]30% in conditional Cfl1-deleted embryos and [~]30% develop spina bifida. We therefore propose that impaired filopodial dynamicity limits the sequence of cell behaviours driving spinal zippering in mice, predisposing to spina bifida.

developmental biology↗

bioRxiv2022

Synchronisation of apical constriction and cell cycle progression is a conserved behaviour of pseudostratified neuroepithelia informed by their tissue geometry

Neuroepithelial cells balance tissue growth requirement with the morphogenetic imperative of closing the neural tube. They apically constrict to generate mechanical forces which elevate the neural folds, but are thought to apically dilate during mitosis. However, we previously reported that mitotic neuroepithelial cells in the mouse posterior neuropore have smaller apical surfaces than non-mitotic cells. Here, we document progressive apical enrichment of non-muscle myosin-II in mitotic, but not non-mitotic, neuroepithelial cells with smaller apical areas. Live-imaging of the chick posterior neuropore confirms apical constriction synchronised with mitosis, reaching maximal constriction by anaphase, before division and re-dilation. Mitotic apical constriction amplitude is significantly greater than interphase constrictions. To investigate conservation in humans, we characterised early stages of iPSC differentiation through dual SMAD-inhibition to robustly produce pseudostratified neuroepithelia with apically enriched actomyosin. These cultured neuroepithelial cells achieve an equivalent apical area to those in mouse embryos. iPSC-derived neuroepithelial cells have large apical areas in G2 which constrict in M phase and retain this constriction in G1/S. Given that this differentiation method produces anterior neural identities, we studied the anterior neuroepithelium of the elevating mouse mid-brain neural tube. Instead of constricting, mid-brain mitotic neuroepithelial cells have larger apical areas than interphase cells. Tissue geometry differs between the apically convex early midbrain and flat posterior neuropore. Culturing human neuroepithelia on equivalently convex surfaces prevents mitotic apical constriction. Thus, neuroepithelial cells undergo high-amplitude apical constriction synchronised with cell cycle progression but the timing of their constriction if influenced by tissue geometry.

cell biology↗

bioRxiv2021

Severe neural tube defects due to failure of closure initiation can arise without abnormality of neuroepithelial convergent extension

Planar cell polarity (PCP) signalling is vital for initiation of neural tube closure in mice, with diminished convergent extension (CE) cell movements leading to a severe form of neural tube defect (NTD), termed craniorachischisis (CRN). Some human NTDs are also associated with PCP gene mutations, but affected individuals are generally heterozygous, whereas PCP homozygosity or compound heterozygosity is needed to produce CRN in mice. This suggests human NTDs may involve other genetic or environmental factors, that interact with partial loss of PCP function. We found that reduced sulfation OF glycosaminoglycans (GAGs) interacts with heterozygosity for the Lp allele of Vangl2 (a core PCP gene), to cause CRN in mice. Here, we hypothesised that this GAG-PCP interaction may regulate convergent extension movements, and hence lead to severe NTDs in the context of only partial loss of PCP function. Both Lp and null alleles of Vangl2 gave similar findings. Culture of E8.5 embryos in the presence of chlorate (a GAG sulfation inhibitor), or enzymatic cleavage of GAG chains, led to failure of NT closure initiation in the majority of Lp/+ embryos, whereas few +/+ littermates exhibited CRN. The chlorate effect was rescued by exogenous sulphate. Surprisingly, DiO labeling of the embryonic node demonstrated no abnormality of midline axial extension in chlorate-treated Lp/+ embryos that developed CRN. In contrast, positive control Lp/Lp embryos displayed severe convergent extension defects in this assay. Morphometric analysis of the closure initiation site revealed abnormalities in the size and shape of somites that flank the closing neural tube in chlorate-treated Lp/+ embryos. We conclude that severe NTDs involving failure of closure initiation can arise by a mechanism other than faulty neuroepithelial convergent extension. Matrix-mediated expansion of somites, flanking the closing neural tube, may be required for closure initiation.

developmental biology↗

bioRxiv2020

Hindbrain neuropore tissue geometry determines asymmetric cell-mediated closure dynamics

Gap closure is a common morphogenetic process. In mammals, failure to close the embryonic hindbrain neuropore (HNP) gap causes fatal anencephaly. We observed that surface ectoderm cells surrounding the mouse HNP assemble high-tension actomyosin purse-strings at their leading edge and establish the initial contacts across the embryonic midline. The HNP gap closes asymmetrically, faster from its rostral than caudal extreme, while maintaining an elongated aspect ratio. Cell-based physical modelling identifies two closure mechanisms sufficient to describe tissue-level HNP closure dynamics; purse-string contraction and directional cell crawling. Combining both closure mechanisms hastens gap closure and produces a constant rate of gap shortening. Purse-string contraction reduces, whereas crawling increases gap aspect ratio, and their combination maintains it. Closure rate asymmetry can be explained by embryo tissue geometry, namely a narrower rostral gap apex. At the cellular level, our model predicts highly directional cell migration with a constant rate of cells leaving the HNP rim. These behaviours are reproducibly live-imaged in mouse embryos. Thus, mammalian embryos coordinate cellular and tissue-level mechanics to achieve this critical gap closure event.

biophysics↗

bioRxiv2019

Novel mouse model of encephalocele: post-neurulation origin and relationship to open neural tube defects

Encephalocele is a clinically important birth defect that can lead to severe disability in childhood and beyond. The embryonic pathogenesis of encephalocele is poorly understood and, while usually classified as a neural tube defect, there is conflicting evidence on whether encephalocele results from defective neural tube closure, or is a post-neurulation defect. It is also unclear whether encephalocele can result from the same causative factors as anencephaly and open spina bifida, or whether it is aetiologically distinct. This lack of information results largely from the scarce availability of animal models of encephalocele, particularly ones that resemble the commonest, non-syndromic human defects. Here we report a novel mouse model of occipito-parietal encephalocele, in which the small GTPase Rac1 is conditionally ablated in the (non-neural) surface ectoderm. Most mutant fetuses have open spina bifida, and some also exhibit exencephaly/anencephaly. However, a large proportion of mutant fetuses exhibit encephalocele affecting the occipito-parietal region. The encephalocele phenotype does not result from a defect in neural tube closure, but rather from a later disruption of the surface ectoderm covering the already closed neural tube, allowing the brain to herniate. The neuroepithelium itself shows no down-regulation of Rac1 and appears morphologically normal until late gestation. A large skull defect develops overlying the region of brain herniation. Our work provides a new genetic model of occipito-parietal encephalocele, particularly resembling non-syndromic human cases. While encephalocele has a different, later-arising pathogenesis than open neural tube defects, both can share the same genetic causation.\n\nSUMMARY STATEMENTEncephalocele - a severe brain defect - arises after neural tube closure, but can share a common genetic cause with anencephaly, a defect of neural tube closure.

developmental biology↗

bioRxiv2018

Genetic approaches in mice demonstrate that neuro-mesodermal progenitors express T/Brachyury but not Sox2

Neural tube and somites have long been thought to derive from separate germ layers: the ectoderm and mesoderm. This concept was challenged by the discovery of neuro-mesodermal progenitors, a bi-potent cell population that gives rise to both spinal neural tube and somites. In line with their proposed potency, these cells are considered to co-express the neural marker Sox2 and the mesodermal marker T/Brachyury. We performed genetic lineage tracing in mouse embryos and confirmed that T-expressing cells give rise to both neural tube and mesoderm. Surprisingly, however, Sox2-expressing cell derivatives colonise only the neural tube after embryonic day 8.5. Deletion of Sox2 in T-expressing cells was compatible with an otherwise normal neural tube and paraxial mesoderm. Moreover, Sox2 expression is absent from the chordoneural hinge, where neuro-mesodermal progenitors are located. Our findings demonstrate that neuro-mesodermal progenitors express T but not Sox2, suggesting the need for re-evaluation of the neuro-mesodermal progenitor hypothesis.

developmental biology↗