James Sharpe

CRG Centre for Genomic Regulation, Barcino, Catalonia, Spain

Are you James Sharpe?

Claim your profile

Publications (64)404.05 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: During limb development, digits emerge from the undifferentiated mesenchymal tissue that constitutes the limb bud. It has been proposed that this process is controlled by a self-organizing Turing mechanism, whereby diffusible molecules interact to produce a periodic pattern of digital and interdigital fates. However, the identities of the molecules remain unknown. By combining experiments and modeling, we reveal evidence that a Turing network implemented by Bmp, Sox9, and Wnt drives digit specification. We develop a realistic two-dimensional simulation of digit patterning and show that this network, when modulated by morphogen gradients, recapitulates the expression patterns of Sox9 in the wild type and in perturbation experiments. Our systems biology approach reveals how a combination of growth, morphogen gradients, and a self-organizing Turing network can achieve robust and reproducible pattern formation.
    Science (New York, N.Y.). 08/2014; 345(6196):566-70.
  • [Show abstract] [Hide abstract]
    ABSTRACT: We have almost no understanding of how our joints take on their range of distinctive shapes, despite the clinical relevance of joint morphogenesis to postnatal skeletal malformations such as developmental dysplasia of the hip (DDH). In this study, we investigate the role of spontaneous prenatal movements in joint morphogenesis using pharmacological immobilization of developing chicks, and assess the system as a suitable model for early-onset hip dysplasia. We show that, prior to joint cavitation, the lack of dynamic muscle contractions has little impact on the shape of the hip joint. However, after the timepoint at which cavitation occurs, a dramatic effect on hip joint morphogenesis was observed. Effects in the immobilized chicks included flattening of the proximal femur, abnormal orientation of the pelvis relative to the femur and abnormal placement and coverage of the acetabulum. Although many clinical case studies have identified reduced or restricted movement as a risk factor for DDH, this study provides the first experimental evidence of the role of prenatal movements in early hip joint development. We propose that the immobilized chick embryo serves as a suitable model system for the type of early-onset DDH which arises due to neuromuscular conditions such as spinal muscular atrophy. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.
    Journal of Orthopaedic Research 03/2014; · 2.88 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Mesoscopic 3D imaging has become a widely used optical imaging technique to visualize intact biological specimens. Selective plane illumination microscopy (SPIM) visualizes samples up to a centimeter in size with micrometer resolution by 3D data stitching but is limited to fluorescent contrast. Optical projection tomography (OPT) works with fluorescent and nonfluorescent contrasts, but its resolution is limited in large samples. We present a hybrid setup (OPTiSPIM) combining the advantages of each technique. The combination of fluorescent and nonfluorescent high-resolution 3D data into integrated datasets enables a more extensive representation of mesoscopic biological samples. The modular concept of the OPTiSPIM facilitates incorporation of the transmission OPT modality into already established light sheet based imaging setups.
    Optics Letters 02/2014; 39(4):1053-6. · 3.39 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Interpreting a morphogen gradient into a single stripe of gene-expression is a fundamental unit of patterning in early embryogenesis. From both experimental data and computational studies the feed-forward motifs stand out as minimal networks capable of this patterning function. Positive feedback within gene networks has been hypothesised to enhance the sharpness and precision of gene-expression borders, however a systematic analysis has not yet been reported. Here we set out to assess this hypothesis, and find an unexpected result. The addition of positive-feedback can have different effects on two different designs of feed-forward motif- it increases the parametric robustness of one design, while being neutral or detrimental to the other. These results shed light on the abundance of the former motif and especially of mutual-inhibition positive feedback in developmental networks.
    Scientific Reports 01/2014; 4:5003. · 5.08 Impact Factor
  • Source
    Niamh C Nowlan, James Sharpe
    [Show abstract] [Hide abstract]
    ABSTRACT: The biology and mechanobiology of joint cavitation have undergone extensive investigation, but we have almost no understanding of the development of joint shape. Joint morphogenesis, the development of shape, has been identified as the 'least understood aspect of joint formation' (2005, Birth Defects Res C Embryo Today 75, 237), despite the clinical relevance of shape morphogenesis to postnatal skeletal malformations such as developmental dysplasia of the hip. In this study, we characterise development of early hip joint shape in the embryonic chick using direct capture 3D imaging. Contrary to formerly held assumptions that cavitation precedes morphogenesis in joint development, we have found that the major anatomical features of the adult hip are present at Hamburger Hamilton (HH)32, a full day prior to cavitation of the joint at HH34. We also reveal that the pelvis undergoes significant changes in orientation with respect to the femur, despite the lack of a joint cavity between the rudiments. Furthermore, we have identified the appearance of the ischium and pubis several developmental stages earlier than was previously reported, illustrating the value and importance of direct capture 3D imaging.
    Journal of Anatomy 11/2013; · 2.36 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Senescence is a form of cell-cycle arrest linked to tumor suppression and aging. However, it remains controversial and has not been documented in nonpathologic states. Here we describe senescence as a normal developmental mechanism found throughout the embryo, including the apical ectodermal ridge (AER) and the neural roof plate, two signaling centers in embryonic patterning. Embryonic senescent cells are nonproliferative and share features with oncogene-induced senescence (OIS), including expression of p21, p15, and mediators of the senescence-associated secretory phenotype (SASP). Interestingly, mice deficient in p21 have defects in embryonic senescence, AER maintenance, and patterning. Surprisingly, the underlying mesenchyme was identified as a source for senescence instruction in the AER, whereas the ultimate fate of these senescent cells is apoptosis and macrophage-mediated clearance. We propose that senescence is a normal programmed mechanism that plays instructive roles in development, and that OIS is an evolutionarily adapted reactivation of a developmental process.
    Cell 11/2013; · 31.96 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Growth and Differentiation Factor 5 (GDF5) is a secreted growth factor that belongs to the Bone Morphogenetic Protein (BMP) family and plays a pivotal role during limb development. GDF5 is a susceptibility gene for osteoarthritis (OA) and mutations in GDF5 are associated with a wide variety of skeletal malformations ranging from complex syndromes such as acromesomelic chondrodysplasias to isolated forms of brachydactylies or multiple synostoses syndrome 2 (SYNS2). Here, we report on a family with an autosomal dominant inherited combination of SYNS2 and additional brachydactyly type A1 (BDA1) caused by a single point mutation in GDF5 (p.W414R). Functional studies, including chondrogenesis assays with primary mesenchymal cells, luciferase reporter gene assays and Surface Plasmon Resonance analysis, of the GDF5(W414R) variant in comparison to other GDF5 mutations associated with isolated BDA1 (p.R399C) or SYNS2 (p.E491K) revealed a dual pathomechanism characterized by a gain- and loss-of-function at the same time. On the one hand insensitivity to the main GDF5 antagonist NOGGIN (NOG) leads to a GDF5 gain of function and subsequent SYNS2 phenotype. Whereas on the other hand, a reduced signaling activity, specifically via the BMP receptor type IA (BMPR1A), is likely responsible for the BDA1 phenotype. These results demonstrate that one mutation in the overlapping interface of antagonist and receptor binding site in GDF5 can lead to a GDF5 variant with pathophysiological relevance for both, BDA1 and SYNS2 development. Consequently, our study assembles another part of the molecular puzzle of how loss and gain of function mutations in GDF5 affect bone development in hands and feet resulting in specific types of brachydactyly and SYNS2. These novel insights into the biology of GDF5 might also provide further clues on the pathophysiology of OA.
    PLoS Genetics 10/2013; 9(10):e1003846. · 8.52 Impact Factor
  • Source
  • Source
    Dataset: TMI2[1]
  • [Show abstract] [Hide abstract]
    ABSTRACT: Key points CXCR5, but not CXCR4 or CCR7, acts with LFA-1 to mediate random B cell migration in T cell area and B cell folliclesIn contrast, stromal guidance during B cell migration is LFA-1- and CXCR5-independent.
    Blood 04/2013; · 9.78 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Direct videomicroscopic visualization of organ formation and regeneration in toto is a powerful strategy to study cellular processes that often cannot be replicated in vitro. Intravital imaging aims at quantifying changes in tissue architecture or subcellular organization over time during organ development, regeneration or degeneration. A general feature of this approach is its reliance on the optical isolation of defined cell types in the whole animals by transgenic expression of fluorescent markers. Here we describe a simple and robust method to analyze sensory hair-cell development and regeneration in the zebrafish lateral line by high-resolution intravital imaging using laser-scanning confocal microscopy (LSCM) and selective plane illumination microscopy (SPIM). The main advantage of studying hair-cell regeneration in the lateral line is that it occurs throughout the life of the animal, which allows its study in the most natural context. We detail protocols to achieve continuous videomicroscopy for up to 68 hours, enabling direct observation of cellular behavior, which can provide a sensitive assay for the quantitative classification of cellular phenotypes and cell-lineage reconstruction. Modifications to this protocol should facilitate pharmacogenetic assays to identify or validate otoprotective or reparative drugs for future clinical strategies aimed at preserving aural function in humans.
    Frontiers in Neuroanatomy 01/2013; 7:33. · 4.06 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: By adapting OPT to include the capability of imaging in the near infrared (NIR) spectrum, we here illustrate the possibility to image larger bodies of pancreatic tissue, such as the rat pancreas, and to increase the number of channels (cell types) that may be studied in a single specimen. We further describe the implementation of a number of computational tools that provide: 1/ accurate positioning of a specimen's (in our case the pancreas) centre of mass (COM) at the axis of rotation (AR)(2); 2/ improved algorithms for post-alignment tuning which prevents geometric distortions during the tomographic reconstruction(2) and 3/ a protocol for intensity equalization to increase signal to noise ratios in OPT-based BCM determinations(3). In addition, we describe a sample holder that minimizes the risk for unintentional movements of the specimen during image acquisition. Together, these protocols enable assessments of BCM distribution and other features, to be performed throughout the volume of intact pancreata or other organs (e.g. in studies of islet transplantation), with a resolution down to the level of individual islets of Langerhans.
    Journal of Visualized Experiments 01/2013;
  • Source
    James Cotterell, James Sharpe
    [Show abstract] [Hide abstract]
    ABSTRACT: The extent and the nature of the constraints to evolutionary trajectories are central issues in biology. Constraints can be the result of systems dynamics causing a non-linear mapping between genotype and phenotype. How prevalent are these developmental constraints and what is their mechanistic basis? Although this has been extensively explored at the level of epistatic interactions between nucleotides within a gene, or amino acids within a protein, selection acts at the level of the whole organism, and therefore epistasis between disparate genes in the genome is expected due to their functional interactions within gene regulatory networks (GRNs) which are responsible for many aspects of organismal phenotype. Here we explore epistasis within GRNs capable of performing a common developmental function - converting a continuous morphogen input into discrete spatial domains. By exploring the full complement of GRN wiring designs that are able to perform this function, we analyzed all possible mutational routes between functional GRNs. Through this study we demonstrate that mechanistic constraints are common for GRNs that perform even a simple function. We demonstrate a common mechanistic cause for such a constraint involving complementation between counter-balanced gene-gene interactions. Furthermore we show how such constraints can be bypassed by means of "permissive" mutations that buffer changes in a direct route between two GRN topologies that would normally be unviable. We show that such bypasses are common and thus we suggest that unlike what was observed in protein sequence-function relationships, the "tape of life" is less reproducible when one considers higher levels of biological organization.
    PLoS ONE 01/2013; 8(4):e61178. · 3.53 Impact Factor
  • Luciano Marcon, James Sharpe
    [Show abstract] [Hide abstract]
    ABSTRACT: For many years Turing patterns-the repetitive patterns which Alan Turing proved could arise from simple diffusing and interacting factors-have remained an interesting theoretical possibility, rather than a central concern of the developmental biology community. Recently however, this has started to change, with an increasing number of studies combining both experimental and theoretical work to reveal how Turing models may underlie a variety of patterning or morphogenetic processes. We review here the recent developments in this field across a wide range of model systems.
    Current opinion in genetics & development 12/2012; · 8.99 Impact Factor
  • James Briscoe, James Sharpe
    Current opinion in genetics & development 12/2012; · 8.99 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The formation of repetitive structures (such as stripes) in nature is often consistent with a reaction-diffusion mechanism, or Turing model, of self-organizing systems. We used mouse genetics to analyze how digit patterning (an iterative digit/nondigit pattern) is generated. We showed that the progressive reduction in Hoxa13 and Hoxd11-Hoxd13 genes (hereafter referred to as distal Hox genes) from the Gli3-null background results in progressively more severe polydactyly, displaying thinner and densely packed digits. Combined with computer modeling, our results argue for a Turing-type mechanism underlying digit patterning, in which the dose of distal Hox genes modulates the digit period or wavelength. The phenotypic similarity with fish-fin endoskeleton patterns suggests that the pentadactyl state has been achieved through modification of an ancestral Turing-type mechanism.
    Science 12/2012; 338(6113):1476-80. · 31.20 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Guanine-rich nucleic acid sequences can form four-stranded structures called G-quadruplexes. Previous studies showed that transfecting G-quadruplex DNA oligonucleotides inhibits proliferation in many cancer cell lines and can induce apoptosis. However, little is known about the effects of transfecting RNA quadruplexes. In this study, we transfected a G-quadruplex RNA oligonucleotide (GqRNA) into HEK293T cells and observed that it did not alter cell viability. Subsequent transcriptome expression profiling revealed that only two genes, EGR1 and FOS, were significantly altered in the presence of GqRNA (upregulated 2- to 4-fold). Sequence analysis showed that both genes contained putative quadruplex sequences (PQS) in their 3'-UTRs, immediately adjacent to the stop codons. Transfection of the EGR1 PQS as an RNA oligonucleotide also caused an increase in EGR1 expression. Similar motifs are found in a variety of genomes, but are relatively rare and have been missed by previous annotations. A bioinformatic analysis revealed stop codon-proximal enrichment of such motifs compared with the rest of the 3'-UTR, although these genes were not affected by RNA quadruplex transfection, and their function remains unknown. Overall, transfecting RNA quadruplexes results in relatively few alterations in gene expression.
    RNA biology 12/2012; 10(2). · 5.56 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The immune system exhibits an enormous complexity. High throughput methods such as the "-omic'' technologies generate vast amounts of data that facilitate dissection of immunological processes at ever finer resolution. Using high-resolution data-driven systems analysis, causal relationships between complex molecular processes and particular immunological phenotypes can be constructed. However, processes in tissues, organs, and the organism itself (so-called higher level processes) also control and regulate the molecular (lower level) processes. Reverse systems engineering approaches, which focus on the examination of the structure, dynamics and control of the immune system, can help to understand the construction principles of the immune system. Such integrative mechanistic models can properly describe, explain, and predict the behavior of the immune system in health and disease by combining both higher and lower level processes. Moving from molecular and cellular levels to a multiscale systems understanding requires the development of methodologies that integrate data from different biological levels into multiscale mechanistic models. In particular, 3D imaging techniques and 4D modeling of the spatiotemporal dynamics of immune processes within lymphoid tissues are central for such integrative approaches. Both dynamic and global organ imaging technologies will be instrumental in facilitating comprehensive multiscale systems immunology analyses as discussed in this review.
    European Journal of Immunology 12/2012; 42(12):3116-25. · 4.97 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: We present the implementation of a combined digital scanned light-sheet microscope (DSLM) able to work in the linear and nonlinear regimes under either Gaussian or Bessel beam excitation schemes. A complete characterization of the setup is performed and a comparison of the performance of each DSLM imaging modality is presented using in vivoCaenorhabditis elegans samples. We found that the use of Bessel beam nonlinear excitation results in better image contrast over a wider field of view.
    Biomedical Optics Express 07/2012; 3(7):1492-505. · 3.18 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Deep tissue imaging has become state of the art in biology, but now the problem is to quantify spatial information in a global, organ-wide context. Although access to the raw data is no longer a limitation, the computational tools to extract biologically useful information out of these large data sets is still catching up. In many cases, to understand the mechanism behind a biological process, where molecules or cells interact with each other, it is mandatory to know their mutual positions. We illustrate this principle here with the immune system. Although the general functions of lymph nodes as immune sentinels are well described, many cellular and molecular details governing the interactions of lymphocytes and dendritic cells remain unclear to date and prevent an in-depth mechanistic understanding of the immune system. We imaged ex vivo lymph nodes isolated from both wild-type and transgenic mice lacking key factors for dendritic cell positioning and used software written in MATLAB to determine the spatial distances between the dendritic cells and the internal high endothelial vascular network. This allowed us to quantify the spatial localization of the dendritic cells in the lymph node, which is a critical parameter determining the effectiveness of an adaptive immune response.
    Computational and Mathematical Methods in Medicine 01/2012; 2012:128431. · 0.79 Impact Factor

Publication Stats

2k Citations
404.05 Total Impact Points

Institutions

  • 2007–2014
    • CRG Centre for Genomic Regulation
      Barcino, Catalonia, Spain
  • 2013
    • Imperial College London
      Londinium, England, United Kingdom
  • 2006–2013
    • Umeå University
      • Umeå Centre for Molecular Medicine (UCMM)
      Umeå, Vaesterbotten, Sweden
  • 2012
    • MRC National Institute for Medical Research
      Londinium, England, United Kingdom
  • 2011–2012
    • University Pompeu Fabra
      • Center for Genomic Regulation (CRG)
      Barcelona, Catalonia, Spain
  • 2009–2012
    • Catalan Institution for Research and Advanced Studies
      Barcino, Catalonia, Spain
  • 2008–2009
    • Trinity College Dublin
      • Department of Zoology
      Dublin, L, Ireland
  • 2003–2009
    • Western General Hospital
      Edinburgh, Scotland, United Kingdom
  • 2002–2004
    • Medical Research Council (UK)
      • • MRC Human Genetics Unit
      • • Genetics and Development
      London, ENG, United Kingdom