Biology subjects

Storey, K. G.

Publications and source records attributed to Storey, K. G..

10 recordsLinked to original sources

bioRxiv2026

Evidence that the protein phosphatase activity of PTEN contributes to embryonic development and tumour suppression in mice

PTEN (phosphatase and tensin homologue deleted on chromosome ten) is a tumour suppressor, the function of which is impaired in many diverse cancers. It has phosphoinositide lipid phosphatase activity by which it suppresses activation of the oncogenic PI3K signalling network but in vitro also displays activity against protein substrates and is able to auto-dephosphorylate its Thr366 residue. Here we generate germline knock-in mice expressing PTEN-Y138L, a mutant enzyme which selectively lacks protein phosphatase activity and retains lipid phosphatase activity. Homozygous PtenY138L/Y138L mice die in utero before E10.5. Primary MEFs and thymocytes with only a single PtenY138L allele display normal low levels of AKT phosphorylation indicating effective regulation of PI3K signalling by endogenous PTEN-Y138L in vivo. Heterozygous Pten+/Y138L mice have reduced overall survival compared to wild type littermates and develop tumours in multiple organs. Our data imply that in addition to its lipid phosphatase activity, the protein phosphatase activity of PTEN is also required for normal embryonic development and tumour suppression.

cancer biology↗

bioRxiv2024

Engineering fluorescent reporters in human pluripotent cells and strategies for live imaging human neurogenesis

Investigation of cell behaviour and cell biological processes underlying human development is facilitated by creation of fluorescent reporters in human pluripotent stem cells, which can be differentiated into cell types of choice. Here we report use of a piggyBac transposon-mediated stable integration strategy to engineer human pluripotent stem cell reporter lines. These express a plasma membrane localised protein tagged with the fluorescent proteins eGFP or mKate2, the photoconvertible nuclear marker H2B-mEos3.2, or the cytoskeletal protein F-tractin tagged with mKate2. Focussing on neural development these lines were used to live image and quantify cell behaviours, including cell cycle progression and cell division orientation in spinal cord rosettes. Further, lipofection-mediated introduction of piggyBac constructs into human neural progenitors labelled single cells and small cell groups within rosettes, allowing individual cell behaviours including neuronal delamination to be monitored. Finally, using the F-tractin-mKate2 hiPSC line, novel actin dynamics were captured during proliferation in cortical neural rosettes. This study presents and validates new tools and techniques with which to interrogate human cell behaviour and cell biology using live imaging approaches.

developmental biology↗

bioRxiv2022

Ependymal cell maturation is heterogeneous and ongoing in the mouse spinal cord and dynamically regulated in response to injury

The spinal cord neural stem cell potential resides within the ependymal cells lining the central canal. These cells are, however, heterogeneous, and we know little about the biological diversity this represents. Here we use single-cell RNA-sequencing to profile adult mouse spinal cord ependymal cells. We uncover transcriptomes of known subtypes and a new mature ependymal cell state, that becomes more prominent with age. Comparison of ependymal cell transcriptomes from the brain and spinal cord revealed that ongoing cell maturation distinguishes spinal cord ependymal cells from their postmitotic brain counterparts. Using an ex vivo model of spinal cord injury, we show that ependymal cell maturation is reversible but also highly regulated. We revisit ependymal cell identities in adult human spinal cord and uncover evidence for their maturation and surprising ventralisation with age. This first in-depth characterisation of spinal cord ependymal cells paves the way to manipulation of distinct ependymal subtypes, provides insights into ependymal cell maturation dynamics and informs strategies for coaxing ependymal cell-driven spinal cord repair.

developmental biology↗

bioRxiv2021

A lateral protrusion latticework connects neuroepithelial cells and is regulated during neurogenesis

Dynamic contacts between cells within the developing neuroepithelium are poorly understood but play important roles in cell and tissue morphology and cell signalling. Here, using live-cell imaging and electron microscopy we reveal multiple protrusive structures in neuroepithelial apical endfeet of the chick embryonic spinal cord, including sub-apical protrusions that extend laterally within the tissue, and observe similar structures in human neuroepithelium. We characterise the dynamics, shape, and cytoskeleton of these lateral protrusions and distinguish these structures from cytonemes/filopodia and tunnelling nanotubes. We demonstrate that lateral protrusions form a latticework of membrane contacts between non-adjacent cells, depend on actin but not microtubule dynamics and provide a lamellipodial-like platform for further extending fine actin-dependent filipodia. We find that lateral protrusions depend on the actin-binding protein WAVE1: mutant-WAVE1 misexpression attenuated protrusion and generated a round-ended apical endfoot morphology. However, this did not alter apico-basal cell polarity nor reduce tissue integrity. During normal neuronal delamination sub-apical protrusions were withdrawn, but mutant-WAVE1-induced precocious protrusion loss was insufficient to trigger neurogenesis. This study uncovers a new form of cell-cell contact within the developing neuroepithelium regulation of which prefigures neuronal delamination.

developmental biology↗

bioRxiv2021

Human spinal cord differentiation proceeds rapidly in vitro and only initially maintains differentiation pace in a heterologous environment

Species-specific differentiation pace in in vitro assays indicates that some aspects of neural differentiation are driven by cell-autonomous processes. Here we describe a novel in vitro human neural rosette assay that recapitulates the temporal sequence of dorsal spinal cord differentiation but proceeds more rapidly than in the human embryonic spinal cord, suggesting that in vitro conditions lack endogenous signalling dynamics. To test the extent to which this in vitro assay represents a cell intrinsic differentiation programme, human iPSC-derived neural rosettes were homo-chronically grafted into the faster differentiating chicken embryonic neural tube. Strikingly, in vitro human differentiation pace was not accelerated, even in single host-integrated cells. Moreover, rosette differentiation eventually stalled in a neural progenitor cell state. These findings demonstrate the requirement for timely extrinsic signalling to accurately recapitulate human neural differentiation tempo, and suggest that while intrinsic properties limit differentiation pace, such signals are also required to maintain differentiation progression.

developmental biology↗

bioRxiv2019

Multiple steps mediate ventricular layer attrition to form the adult mouse spinal cord central canal

The ventricular layer of the spinal cord is remodelled during embryonic development and ultimately forms the adult central canal, which retains neural stem cell potential. This anatomical transformation involves the process of dorsal collapse, however, accompanying changes in tissue organization and cell behaviour as well as the origin of cells contributing to the adult central canal are not well understood. Here we describe sequential localised cell rearrangements which contribute to the gradual attrition of the spinal cord ventricular layer during development. This includes local breakdown of the pseudostratified organisation of the dorsal ventricular layer prefiguring dorsal collapse and evidence for a new phenomenon, ventral dissociation, during which the ventral-most floor plate cells separate from a subset that are retained in the central canal. Using cell proliferation markers and cell-cycle reporter mice, we further show that following dorsal collapse, ventricular layer attrition involves an overall reduction in cell proliferation, characterised by an intriguing increase in the percentage of cells in G1/S. In contrast, programmed cell death does not contribute to ventricular layer remodelling. By analysing transcript and protein expression patterns associated with key signalling pathways, we provide evidence for a gradual decline in ventral sonic hedgehog activity and an accompanying ventral expansion of initial dorsal bone morphogenetic protein signalling, which comes to dominate the forming central canal. This study identifies multiple steps that contribute to spinal cord ventricular layer attrition and adds to increasing evidence for the heterogenous origin of the adult spinal cord central canal, which includes cells from the floor plate and the roof plate as well as ventral progenitor domain.

developmental biology↗

bioRxiv2019

ERK1/2 signalling dynamics promote neural differentiation by regulating the polycomb repressive complex

Fibroblast Growth Factor (FGF) is a neural inducer in many vertebrate embryos, but how it regulates chromatin organization to coordinate the activation of neural genes is unclear. Moreover, for differentiation to progress FGF signalling has to decline. Why this signalling dynamic is required has not been determined. Here we show that dephosphorylation of the FGF effector kinase ERK1/2 rapidly increases chromatin accessibility at neural genes in mouse embryos and, using ATAC-seq in human embryonic stem cell derived spinal cord precursors, we demonstrate that this occurs across hundreds of neural genes. Importantly, while Erk1/2 inhibition induces precocious neural gene transcription, this step involves dissociation of the polycomb repressive complex from gene loci and takes places independently of subsequent loss of the repressive histone mark H3K27me3 and transcriptional onset. We find that loss of ERK1/2 activity but not its occupancy at neural genes is critical for this mechanism. Moreover, transient ERK1/2 inhibition is sufficient for polycomb protein dissociation and this is not reversed on resumption of ERK1/2 signalling. These data indicate that ERK1/2 signalling maintains polycomb repressive complexes at neural genes, that its decline coordinates their increased accessibility and that this is a directional molecular mechanism, which initiates the process of neural commitment. Furthermore, as the polycomb repressive complexes repress but also ready genes for transcription, these findings suggest that ERK1/2 promotion of these complexes is a rite of passage for subsequent differentiation.

developmental biology↗

bioRxiv2018

Lineage tracing axial progenitors using Nkx1.2CreERT2 mice defines their trunk and tail contributions

The vertebrate body forms by continuous generation of new tissue from progenitors at the posterior end of the embryo. In mice, these axial progenitors initially reside in the epiblast, from where they form the trunk; and later relocate to the chordo-neural hinge of the tail bud to form the tail. Among them, a small group of bipotent neuromesodermal progenitors (NMPs) are thought to generate the spinal cord and paraxial mesoderm to the end of axis elongation. The study of these progenitors, however, has proven challenging in vivo due to their small numbers and dynamic nature, and the lack of a unique molecular marker to identify them. Here, we report the generation of the Nkx1.2CreERT2 transgenic mouse line in which the endogenous Nkx1.2 promoter drives tamoxifen-inducible CreERT2 recombinase. We show that Nkx1.2CreERT2 targets axial progenitors, including NMPs and early neural and mesodermal progenitors. Using a YFP reporter, we demonstrate that Nkx1.2-expressing epiblast cells contribute to all three germ layers, mostly neuroectoderm and mesoderm excluding notochord; and continue contributing neural and paraxial mesoderm tissues from the tail bud. This study identifies the Nkx1.2-expressing cell population as the source of most trunk and tail tissues in the mouse; and provides a key tool to genetically label and manipulate this progenitor population in vivo.

developmental biology↗

bioRxiv2017

MYC activity is required for maintenance of the Neuromesodermal Progenitor signalling network and for correct timing of segmentation clock gene oscillations

The Myc transcriptional regulators are implicated in a range of cellular functions, including proliferation, cell cycle progression, metabolism and pluripotency maintenance. Here, we investigated the expression, regulation and function of Myc during mouse embryonic axis elongation and segmentation. Expression of both cMyc and MycN in the domains where neuromesodermal progenitors (NMPs) and underlying caudal pre-somitic mesoderm (cPSM) cells reside is coincident WNT and FGF signals; factors known to maintain progenitors in an undifferentiated state. Pharmacological inhibition of MYC activity, downregulates expression of WNT/FGF components. In turn, we find that cMyc expression is WNT, FGF and NOTCH regulated, placing it centrally in the signalling circuit that operates in the tail end that both sustains progenitors and drives maturation of the PSM into somites. Interfering with MYC function in the PSM, where it displays oscillatory expression, delays the timing of segmentation clock oscillations and thus of somite formation. In summary, we identify Myc as a component that links NMP maintenance and PSM maturation during the body axis elongation stages of mouse embryogenesis.\n\nSummary StatementMYC operates in a positive feedback loop with WNT/FGF signals to maintain the progenitors which facilitate body axis elongation while its activity is crucial for timing of the segmentation clock.

developmental biology↗

bioRxiv2017

Generation, selection and transcriptomic profiling of human neuromesodermal and spinal cord progenitors in vitro

Robust protocols for directed differentiation of human pluripotent cells are needed to establish the extent to which mechanisms operating in model organisms are relevant to our own development. Recent work in vertebrate embryos has identified neuromesodermal progenitors as a bipotent cell population that contributes to paraxial mesoderm and spinal cord. However, precise protocols for in vitro differentiation of human neuromesodermal progenitors are lacking. Informed by signalling activities during spinal cord generation in amniote embryos, we show here that transient dual-SMAD inhibition, together with retinoic acid (dSMADi-RA), provides rapid and reproducible induction of human spinal cord progenitors from neuromesodermal progenitors. We use CRISPR-Cas9 to engineer a GFP-reporter for a neuromesodermal progenitor-associated transcription factor Nkx1.2 in human embryonic stem cells, to facilitate selection of this cell population. RNA-sequencing (RNA-Seq) was then used to identify human and conserved neuromesodermal progenitor transcriptional signatures, validate this differentiation protocol and implicate new pathways and processes in human neural differentiation. This optimised protocol, novel reporter line and transcriptomic data are useful resources with which to dissect cellular and molecular mechanisms regulating the generation of human spinal cord, allow scale-up of distinct cell populations for global analyses, including proteomic, biochemical and chromatin interrogation and open up translational opportunities.

developmental biology↗