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Wilson, V.

Publications and source records attributed to Wilson, V..

11 recordsLinked to original sources

Axolotl tail regeneration emerges during a defined embryonic window

How regenerative capacity originates during development remains poorly understood, even in vertebrates with exceptional adult regenerative ability. Using the axolotl, we identify a defined embryonic window between stages 30 and 34 during which the tail region transitions from a regeneration-incompetent to a regeneration-competent state. Amputations across staged embryos reveal that earlier embryos entirely fail to regenerate, whereas later embryos regenerate functional tails. Notably, tail stumps from nonregenerating embryos can recover the ability to regenerate when reamputated at later stages, demonstrating that early regenerative failure does not permanently impair regenerative capacity. This differs from the transient refractory period described in Xenopus, where regenerative competence is lost and reacquired around the end of tail outgrowth, and indicates that staged acquisition of regenerative competence is a broadly shared but mechanistically distinct feature of amphibian development. To determine whether this transition reflects changes in progenitor composition, we analysed the single-cell transcriptional landscapes of axolotl tail buds across this window. Tail bud progenitors, including neuromesodermal progenitors, persist through the transition, indicating that the onset of regenerative competence is unlikely to be explained by the loss of embryonic progenitors. Finally, using Tbxt (Brachyury) crispant axolotls with severe axial defects, we show that tail regeneration occurs effectively despite earlier abnormal embryonic tail development, with functional uncoupling of the mechanisms of tail development and regeneration. This framework provides new opportunities for identifying the drivers of regenerative competence and understand why this capacity is lost in other vertebrate species.

developmental biology↗

Punarnavayolepa Choornam in Iron Deficiency Anaemia management: Pharmaceutic insights and biological activity.

Background and AimIndigenous medical systems employ unique pharmaceutical techniques to meet the therapeutic needs. In the Indian System of Medicine, Ayurveda, a unique method called "Ayolepam" is described that facilitates efficient iron absorption from its source to the formulation. Punarnavayolepa Choornam (PC), prepared from Boerhavia diffusa L., is one such novel Ayolepam formulation developed by a traditional Ayurveda school of South India, widely used in the clinical management of iron deficiency anaemia. This study aims at scientifically evaluating the Ayolepam technique by assessing the iron binding and bioavailability properties of PC, prepared from the leaves, root and whole plant of B. diffusa, using in-vitro model systems.. Experimental ProcedureThe iron content in both raw and processed samples of PC was quantified using Inductively Coupled Plasma Mass Spectrometry (ICP-MS). A simulated in-vitro digestion model employed to assess the release of bioavailable iron from the formulation. Subsequently, iron bioavailability was evaluated using the Caco-2 cell model of human intestinal epithelium following the ferrozine method. Results and ConclusionPC preparation from leaves, whole plant and roots of B. diffusa, showed significant increase in iron content compared to the raw material. In-vitro digestion studies confirmed the efficient release of bioavailable iron from these formulations, and subsequent Caco-2 cell assays confirmed their iron bioavailability properties. In conclusion, these findings provide a preliminary scientific basis for this novel and unique pharmaceutical design from a traditional school of Ayurveda, supporting its clinical application.

pharmacology and toxicology↗

Brachyury expression levels predict lineage potential and axis-forming ability of in vitro derived neuromesodermal progenitors

Neuromesodermal progenitors (NMPs) produce the spinal cord and musculoskeleton in the elongating anterior-posterior axis. In vivo, NMPs possess dual potency, coinciding with regions coexpressing SOX2 and Brachyury (TBXT). In vitro, SOX2/TBXT co-expressing cells can be produced from pluripotent cells and, like their in vivo counterparts, can produce neural tube and somitic mesoderm. However, the functional characteristics of in vitro SOX2/TBXT co-expressing cells remain unclear, confounding comparisons with in vivo data. To address this, we developed a dual Sox2/Tbxt reporter mouse ESC line. SOX2/TBXT reporter-positive cells emerge in vitro from pluripotent populations with dynamics that mirror their appearance in the embryo. Purified SOX2/TBXT co-expressing populations can differentiate towards neurectoderm or mesoderm, including lateral mesoderm upon BMP stimulation. In gastruloids, quantitative live imaging shows that WNT or NOTCH inhibition rapidly leads to downregulation of TBXT expression and diminished axial extension. We show that clonally plated SOX2/TBXT co-expressing cells are bipotent NMPs that can also self-propagate. By combining clonal analysis with mathematical modelling, we identify two thresholds of SOX2/TBXT expression, switching clonal output from neural- to mesoderm-biased, and from mesoderm-biased to mesoderm-specified. Media and substrate composition alter the lineage outcomes of in vitro derived mouse NMPs. Thus, this Sox2/Tbxt double reporter cell line provides support for unsuspected heterogeneity in NMPs, together with evidence for a role of these transcription factors in directing cell fate to drive axis elongation.

developmental biology↗

A toolkit for mapping cell identities in relation to neighbours reveals Notch-dependent heterogeneity within neuromesodermal progenitor populations

Patterning of cell fates is central to embryonic development, tissue homeostasis, and disease. Quantitative analysis of patterning reveals the logic by which cell-cell interactions orchestrate changes in cell fate. However, it is challenging to quantify patterning when graded changes in identity occur over complex 4D trajectories, or where different cell states are intermingled. Furthermore, comparing patterns across multiple individual embryos, tissues, or organoids is difficult because these often vary in shape and size. Here we present a toolkit of computational approaches to tackle these problems. These strategies are based on measuring properties of each cell in relation to the properties of its neighbours to quantify patterning, and on using embryonic landmarks in order to compare these patterns between embryos. We use this toolkit to characterise patterning of cell identities within the caudal lateral epiblast of E8.5 embryos, revealing local patterning in emergence of early mesoderm cells that is sensitive to inhibition of Notch activity.

developmental biology↗

Capture of Human Neuromesodermal and Posterior Neural Tube Axial Stem Cells

The spinal cord, nerves, and skeletal muscles arise from neuromesodermal progenitors (NMPs). We have developed a growth-factor screening strategy, utilizing ES and iPS cells, facilitating the indefinite self-renewal of two types of human axial stem cells (AxSCs), closely resembling mouse NMPs (NM-AxSCs) and posterior neural tube progenitors (N-AxSCs). Under specific regimens-- Wnt/CHIR99021, FGF2, and TGF-{beta} inhibitor SB431542 (CFS) and excluding FGF2 (CS), respectively--these AxSCs self-renew and sustain telomeres. Single cell transcriptomics and proteomics have revealed expression of posterior growth-zone and dorsoventral neural tube markers in NM-AxSCs, and correspondingly, differentiation to a wide spectrum of neural tube neurons and myocytes. N-AxSCs rapidly matured into dorsal sensory subsets and neural crest. Crucially, neither AxSC type produces teratomas, and analogous mouse NM-AxSCs integrated successfully into the neural tube and somites. Capturing of AxSCs from patient and GMP ES / iPS cells without transgenesis unveils ontogeny and promises modeling and therapy in neuropathies.

developmental biology↗

A TgfβRI/Snai1-dependent developmental module at the core of vertebrate axial elongation

Formation of the vertebrate postcranial body axis follows two sequential but distinct phases. The first phase generates pre-sacral structures (the so-called primary body) through the activity of the primitive streak (PS) on axial progenitors within the epiblast. The embryo then switches to generate the secondary body (post-sacral structures), which depends on axial progenitors in the tail bud. Here we show that the mammalian tail bud is generated through an independent developmental module, concurrent but functionally different to that generating the primary body. This module is triggered by convergent Tgf{beta}RI and Snai1 activities that promote an incomplete epithelial to mesenchymal transition (EMT) on a subset of epiblast axial progenitors. This EMT is functionally different to that coordinated by the PS, as it does not lead to mesodermal differentiation but brings axial progenitors into a transitory state, keeping their progenitor activity to drive further axial body extension.

developmental biology↗

Development of a monkey avatar to study social perception in macaques

Following the expanding use and applications of virtual reality in every-day life, dynamic virtual stimuli are of increasing interest in cognitive studies. They allow for control of features such as gaze, expression and movement, which may help to overcome limitations of using either static or poorly controlled real stimuli. In using virtual stimuli however, one must be careful to avoid the uncanny valley effect - where realistic stimuli can be perceived as eerie, and induce an aversion response. At the same time, it is important to establish whether responses to virtual stimuli mirror responses to depictions of a real conspecific. In the current study, we describe the development of a new avatar with realistic features for nonhuman primates, the primatar. As a first step towards validation, we assessed how monkeys respond to images of this avatar compared to images of real monkeys, and an unrealistic avatar. We also compared responses between original images and scrambled as well as obfuscated versions of these images. We measured looking time to images in six free moving long-tailed macaques (Macaca fascicularis) and eye movement exploration behaviour in three rhesus macaques (Macaca mulatta). Both groups showed more of such signs of overt attention to original images than scrambled or obfuscated images. In addition, we assessed whether the realistic avatar created an uncanny valley effect through decreased looking time, finding that in both groups, monkeys did not differentiate between real, realistic or unrealistic images. These results provide support for further development of our avatar for use in social cognition studies, and more generally for cognitive research with virtual stimuli in nonhuman primates. Future research needs to shed light on the source of the inconsistent findings for the uncanny valley effect in macaques, to elucidate the roots of this mechanism in humans.

animal behavior and cognition↗

Cables2 Is a novel Smad2-regulatory factor essential for early embryonic development in mice

CDK5 and Abl enzyme substrate 2 (Cables2), a member of the Cables family that has a C-terminal cyclin box-like domain, is widely expressed in adult mouse tissues. However, the physiological role of Cables2 in vivo is unknown. We show here that Cables2-deficiency causes post-gastrulation embryonic lethality in mice. The mutant embryos progress to gastrulation, but then arrest, and fail to grow. Analysis of gene expression patterns reveals that formation of the anterior visceral endoderm and the primitive streak is impaired in Cables2-deficient embryos. Tetraploid complementation analyses support the critical requirement of Cables2 in both the epiblast and visceral endoderm for progression of embryogenesis. In addition, we show that Cables2 physically interacts with a key mediator of the canonical Nodal pathway, Smad2, and augments its transcriptional activity. These findings provide novel insights into the essential role of Cables2 in the early embryonic development in mice.

developmental biology↗

Human axial progenitors generate trunk neural crest cells

The neural crest (NC) is a multipotent embryonic cell population generating distinct cell types in an axial position-dependent manner. The production of NC cells from human pluripotent stem cells (hPSCs) is a valuable approach to study human NC biology. However, the origin of human trunk NC remains undefined and therefore current in vitro differentiation strategies induce only a modest yield of trunk NC cells. Here we show that hPSC-derived axial progenitors, the posteriorly-located drivers of embryonic axis elongation, give rise to trunk NC cells and their derivatives. Moreover, we define the molecular signatures associated with the emergence of human NC cells of distinct axial identities in vitro. Collectively, our findings indicate that there are two routes toward a human post-cranial NC state: the birth of cardiac and vagal NC is facilitated by retinoic acid-induced posteriorisation of an anterior precursor whereas trunk NC arises within a pool of posterior axial progenitors.

developmental biology↗

Distinct SoxB1 networks are required for naïve and primed pluripotency

Deletion of Sox2 from embryonic stem cells (ESCs) causes trophectodermal differentiation. While this can be prevented by enforced expression of the related SOXB1 proteins, SOX1 or SOX3, the roles of SOXB1 proteins in epiblast stem cell (EpiSC) pluripotency are unknown. Here we show that Sox2 can be deleted from EpiSCs with impunity. This is due to a shift in the balance of SoxB1 expression in EpiSCs, which have decreased Sox2 and increased Sox3 compared to ESCs. Consistent with functional redundancy, Sox3 can also be deleted from EpiSCs without eliminating self-renewal. However, deletion of both Sox2 and Sox3 prevents self-renewal. The overall SOXB1 levels in ESCs affect differentiation choices: neural differentiation of Sox2 heterozygous ESCs is compromised, while increased SOXB1 levels divert the ESC to EpiSC transition towards neural differentiation. Therefore, optimal SOXB1 levels are critical for each pluripotent state and for cell fate decisions during exit from naive pluripotency.

cell biology↗

Mapping transcription factor occupancy using minimal numbers of cells in vitro and in vivo

The identification of transcription factor (TF) binding sites in the genome is critical to understanding gene regulatory networks (GRNs). While ChIP-seq is commonly used to identify TF targets, it requires specific ChIP-grade antibodies and high cell numbers, often limiting its applicability. DNA adenine methyltransferase identification (DamID), developed and widely used in Drosophila, is a distinct technology to investigate protein-DNA interactions. Unlike ChIP-seq, it does not require antibodies, precipitation steps or chemical protein-DNA crosslinking, but to date it has been seldom used in mammalian cells due to technical impediments. Here we describe an optimised DamID method coupled with next generation sequencing (DamID-seq) in mouse cells, and demonstrate the identification of the binding sites of two TFs, OCT4 and SOX2, in as few as 1,000 embryonic stem cells (ESCs) and neural stem cells (NSCs), respectively. Furthermore, we have applied this technique in vivo for the first time in mammals. Oct4 DamID-seq in the gastrulating mouse embryo at 7.5 days post coitum (dpc) successfully identified multiple Oct4 binding sites proximal to genes involved in embryo development, neural tube formation, mesoderm-cardiac tissue development, consistent with the pivotal role of this TF in post-implantation embryo. This technology paves the way to unprecedented investigations of TF-DNA interactions and GRNs in specific cell types with limited availability in mammals including in vivo samples.

genomics↗