Quantitative cell polarity imaging defines leader-to-follower transitions during collective migration and the key role of microtubule-dependent adherens junction formation.
Revenu, C., Streichan, S., Dona, E., Lecaudey, V., Hufnagel, L. & Gilmour, D.
Development. 2014 Mar;141(6):1282-91. doi: 10.1242/dev.101675.
The directed migration of cell collectives drives the formation of complex organ systems. A characteristic feature of many migrating collectives is a 'tissue-scale' polarity, whereby 'leader' cells at the edge of the tissue guide trailing 'followers' that become assembled into polarised epithelial tissues en route. Here, we combine quantitative imaging and perturbation approaches to investigate epithelial cell state transitions during collective migration and organogenesis, using the zebrafish lateral line primordium as an in vivo model. A readout of three-dimensional cell polarity, based on centrosomal-nucleus axes, allows the transition from migrating leaders to assembled followers to be quantitatively resolved for the first time in vivo. Using live reporters and a novel fluorescent protein timer approach, we investigate changes in cell-cell adhesion underlying this transition by monitoring cadherin receptor localisation and stability. This reveals that while cadherin 2 is expressed across the entire tissue, functional apical junctions are first assembled in the transition zone and become progressively more stable across the leader-follower axis of the tissue. Perturbation experiments demonstrate that the formation of these apical adherens junctions requires dynamic microtubules. However, once stabilised, adherens junction maintenance is microtubule independent. Combined, these data identify a mechanism for regulating leader-to-follower transitions within migrating collectives, based on the relocation and stabilisation of cadherins, and reveal a key role for dynamic microtubules in this process.
Subtle changes in motif positioning cause tissue-specific effects on robustness of an enhancer's activity.
Erceg, J., Saunders, T.E., Girardot, C., Devos, D.P., Hufnagel, L. & Furlong, E.E.
PLoS Genet. 2014 Jan;10(1):e1004060. doi: 10.1371/journal.pgen.1004060. Epub 2014Jan 2.
Deciphering the specific contribution of individual motifs within cis-regulatory modules (CRMs) is crucial to understanding how gene expression is regulated and how this process is affected by sequence variation. But despite vast improvements in the ability to identify where transcription factors (TFs) bind throughout the genome, we are limited in our ability to relate information on motif occupancy to function from sequence alone. Here, we engineered 63 synthetic CRMs to systematically assess the relationship between variation in the content and spacing of motifs within CRMs to CRM activity during development using Drosophila transgenic embryos. In over half the cases, very simple elements containing only one or two types of TF binding motifs were capable of driving specific spatio-temporal patterns during development. Different motif organizations provide different degrees of robustness to enhancer activity, ranging from binary on-off responses to more subtle effects including embryo-to-embryo and within-embryo variation. By quantifying the effects of subtle changes in motif organization, we were able to model biophysical rules that explain CRM behavior and may contribute to the spatial positioning of CRM activity in vivo. For the same enhancer, the effects of small differences in motif positions varied in developmentally related tissues, suggesting that gene expression may be more susceptible to sequence variation in one tissue compared to another. This result has important implications for human eQTL studies in which many associated mutations are found in cis-regulatory regions, though the mechanism for how they affect tissue-specific gene expression is often not understood.
Mechanical stress inference for two dimensional cell arrays.
Chiou, K.K., Hufnagel, L. & Shraiman, B.I.
PLoS Comput Biol. 2012;8(5):e1002512. doi: 10.1371/journal.pcbi.1002512. Epub2012 May 17.
Many morphogenetic processes involve mechanical rearrangements of epithelial tissues that are driven by precisely regulated cytoskeletal forces and cell adhesion. The mechanical state of the cell and intercellular adhesion are not only the targets of regulation, but are themselves the likely signals that coordinate developmental process. Yet, because it is difficult to directly measure mechanical stress in vivo on sub-cellular scale, little is understood about the role of mechanics in development. Here we present an alternative approach which takes advantage of the recent progress in live imaging of morphogenetic processes and uses computational analysis of high resolution images of epithelial tissues to infer relative magnitude of forces acting within and between cells. We model intracellular stress in terms of bulk pressure and interfacial tension, allowing these parameters to vary from cell to cell and from interface to interface. Assuming that epithelial cell layers are close to mechanical equilibrium, we use the observed geometry of the two dimensional cell array to infer interfacial tensions and intracellular pressures. Here we present the mathematical formulation of the proposed Mechanical Inverse method and apply it to the analysis of epithelial cell layers observed at the onset of ventral furrow formation in the Drosophila embryo and in the process of hair-cell determination in the avian cochlea. The analysis reveals mechanical anisotropy in the former process and mechanical heterogeneity, correlated with cell differentiation, in the latter process. The proposed method opens a way for quantitative and detailed experimental tests of models of cell and tissue mechanics.
Collective and single cell behavior in epithelial contact inhibition.
Puliafito, A., Hufnagel, L., Neveu, P., Streichan, S., Sigal, A., Fygenson, D.K. & Shraiman, B.I.
Proc Natl Acad Sci U S A. 2012 Jan 17;109(3):739-44. doi:10.1073/pnas.1007809109. Epub 2012 Jan 6.
Control of cell proliferation is a fundamental aspect of tissue physiology central to morphogenesis, wound healing, and cancer. Although many of the molecular genetic factors are now known, the system level regulation of growth is still poorly understood. A simple form of inhibition of cell proliferation is encountered in vitro in normally differentiating epithelial cell cultures and is known as "contact inhibition." The study presented here provides a quantitative characterization of contact inhibition dynamics on tissue-wide and single cell levels. Using long-term tracking of cultured Madin-Darby canine kidney cells we demonstrate that inhibition of cell division in a confluent monolayer follows inhibition of cell motility and sets in when mechanical constraint on local expansion causes divisions to reduce cell area. We quantify cell motility and cell cycle statistics in the low density confluent regime and their change across the transition to epithelial morphology which occurs with increasing cell density. We then study the dynamics of cell area distribution arising through reductive division, determine the average mitotic rate as a function of cell size, and demonstrate that complete arrest of mitosis occurs when cell area falls below a critical value. We also present a simple computational model of growth mechanics which captures all aspects of the observed behavior. Our measurements and analysis show that contact inhibition is a consequence of mechanical interaction and constraint rather than interfacial contact alone, and define quantitative phenotypes that can guide future studies of molecular mechanisms underlying contact inhibition.
Multiview light-sheet microscope for rapid in toto imaging.
Krzic, U., Gunther, S., Saunders, T.E., Streichan, S.J. & Hufnagel, L.
Nat Methods. 2012 Jun 3;9(7):730-3. doi: 10.1038/nmeth.2064.
We present a multiview selective-plane illumination microscope (MuVi-SPIM), comprising two detection and illumination objective lenses, that allows rapid in toto fluorescence imaging of biological specimens with subcellular resolution. The fixed geometrical arrangement of the imaging branches enables multiview data fusion in real time. The high speed of MuVi-SPIM allows faithful tracking of nuclei and cell shape changes, which we demonstrate through in toto imaging of the embryonic development of Drosophila melanogaster.
Light Sheet Fluorescence Microscopy (SPIM) and laser excitation in orange for imaging of live organisms.
Uros Krzic, Stefan Günther, Hufnagel, L., Dag von Gegerfelt, Håkan Karlsson, Elizabeth Illy, and Jonas Hellström.
Photonik International, no. 1, pp. 4143, Nov. 2011
Quantitative fluorescence imaging of protein diffusion and interaction in living cells.
Capoulade, J., Wachsmuth, M., Hufnagel, L. & Knop, M.
Nat Biotechnol. 2011 Aug 7;29(9):835-9. doi: 10.1038/nbt.1928.
Diffusion processes and local dynamic equilibria inside cells lead to nonuniform spatial distributions of molecules, which are essential for processes such as nuclear organization and signaling in cell division, differentiation and migration. To understand these mechanisms, spatially resolved quantitative measurements of protein abundance, mobilities and interactions are needed, but current methods have limited capabilities to study dynamic parameters. Here we describe a microscope based on light-sheet illumination that allows massively parallel fluorescence correlation spectroscopy (FCS) measurements and use it to visualize the diffusion and interactions of proteins in mammalian cells and in isolated fly tissue. Imaging the mobility of heterochromatin protein HP1alpha (ref. 4) in cell nuclei we could provide high-resolution diffusion maps that reveal euchromatin areas with heterochromatin-like HP1alpha-chromatin interactions. We expect that FCS imaging will become a useful method for the precise characterization of cellular reaction-diffusion processes.
Collective cell migration guided by dynamically maintained gradients.
Streichan, S.J., Valentin, G., Gilmour, D. & Hufnagel, L.
Phys Biol. 2011 Aug;8(4):045004. doi: 10.1088/1478-3975/8/4/045004. Epub 2011 Jul12.
How cell collectives move and deposit subunits within a developing embryo is a question of outstanding interest. In many cases, a chemotactic mechanism is employed, where cells move up or down a previously generated attractive or repulsive gradient of signalling molecules. Recent studies revealed the existence of systems with isotropic chemoattractant expression in the lateral line primordium of zebrafish. Here we propose a mechanism for a cell collective, which actively modulates an isotropically expressed ligand and encodes an initial symmetry breaking in its velocity. We derive a closed solution for the velocity and identify an optimal length that maximizes the tissues' velocity. A length dependent polar gradient is identified, its use for pro-neuromast deposition is shown by simulations and a critical time for cell deposition is derived. Experiments to verify this model are suggested.
Lichtscheiben‐Fluoreszenzmikroskopie (SPIM) und Laser‐Anregung in
orange zur Abbildung lebender Organismen.
Uros Krzic, Stefan Günther, Hufnagel, L., Dag von Gegerfelt, Håkan Karlsson, Elizabeth Illy, and Jonas Hellström.
BioPhotonik, no. 1, Apr. 2011.
On the mechanism of wing size determination in fly development.
Hufnagel, L., Teleman, A.A., Rouault, H., Cohen, S.M. & Shraiman, B.I.
Proc Natl Acad Sci U S A. 2007 Mar 6;104(10):3835-40. Epub 2007 Feb 28.
A fundamental and unresolved problem in animal development is the question of how a growing tissue knows when it has achieved its correct final size. A widely held view suggests that this process is controlled by morphogen gradients, which adapt to tissue size and become flatter as tissue grows, leading eventually to growth arrest. Here, we present evidence that the decapentaplegic (Dpp) morphogen distribution in the developing Drosophila wing imaginal disk does not adapt to disk size. We measure the distribution of a functional Dpp-GFP transgene and the Dpp signal transduced by phospho-Mad and show that the characteristic length scale of the Dpp profile remains approximately constant during growth. This finding suggests an alternative scenario of size determination, where disk size is determined relative to the fixed morphogen distribution by a certain threshold level of morphogen required for growth. We propose that when disk boundary reaches the threshold the arrest of cell proliferation throughout the disk is induced by mechanical stress in the tissue. Mechanical stress is expected to arise from the nonuniformity of morphogen distribution that drives growth. This stress, through a negative feedback on growth, can compensate for the nonuniformity of morphogen, achieving uniform growth with the rate that vanishes when disk boundary reaches the threshold. The mechanism is demonstrated through computer simulations of a tissue growth model that identifies the key assumptions and testable predictions. This analysis provides an alternative hypothesis for the size determination process. Novel experimental approaches will be needed to test this model.
Front propagation in reaction-superdiffusion dynamics: Taming Levy flights with fluctuations.
Brockmann, D. & Hufnagel, L.
Physical Review Letters 2007 98(17) 178301
We investigate front propagation in a reacting particle system in which particles perform scale-free random walks known as Levy flights. The system is described by a fractional generalization of a reaction-diffusion equation. We focus on the effects of fluctuations caused by a finite number of particles per volume. We show that, in spite of superdiffusive particle dispersion and contrary to mean-field theoretical predictions, wave fronts propagate at constant velocities, even for very large particle numbers. We show that the asymptotic velocity scales with the particle number and obtain the scaling exponent.
On the role of glypicans in the process of morphogen gradient formation.
Hufnagel, L., Kreuger, J., Cohen, S.M. & Shraiman, B.I.
Dev Biol. 2006 Dec 15;300(2):512-22. Epub 2006 Sep 19.
Glypicans are cell surface molecules that influence signaling and gradient formation of secreted morphogens and growth factors. Several distinct functions have been ascribed to glypicans including acting as co-receptors for signaling proteins. Recent data show that glypicans are also necessary for morphogen propagation in the tissue. In the present study, a model describing the interaction of a morphogen with glypicans is formulated, analyzed and compared with measurements of the effect of glypican Dally-like (Dlp) overexpression on Wingless (Wg) morphogen signaling in Drosophila melanogaster wing imaginal discs. The model explains the opposing effect that Dlp overexpression has on Wg signaling in the distal and proximal regions of the disc and makes a number of quantitative predictions for further experiments. In particular, our model suggests that Dlp acts by allowing Wg to diffuse on cell surface while protecting it from loss and degradation, and that Dlp rather than acting as Wg co-receptor competes with receptors for morphogen binding.
The scaling laws of human travel.
Brockmann, D., Hufnagel, L. & Geisel, T.
Nature. 2006 Jan 26;439(7075):462-5.
The dynamic spatial redistribution of individuals is a key driving force of various spatiotemporal phenomena on geographical scales. It can synchronize populations of interacting species, stabilize them, and diversify gene pools. Human travel, for example, is responsible for the geographical spread of human infectious disease. In the light of increasing international trade, intensified human mobility and the imminent threat of an influenza A epidemic, the knowledge of dynamical and statistical properties of human travel is of fundamental importance. Despite its crucial role, a quantitative assessment of these properties on geographical scales remains elusive, and the assumption that humans disperse diffusively still prevails in models. Here we report on a solid and quantitative assessment of human travelling statistics by analysing the circulation of bank notes in the United States. Using a comprehensive data set of over a million individual displacements, we find that dispersal is anomalous in two ways. First, the distribution of travelling distances decays as a power law, indicating that trajectories of bank notes are reminiscent of scale-free random walks known as Levy flights. Second, the probability of remaining in a small, spatially confined region for a time T is dominated by algebraically long tails that attenuate the superdiffusive spread. We show that human travelling behaviour can be described mathematically on many spatiotemporal scales by a two-parameter continuous-time random walk model to a surprising accuracy, and conclude that human travel on geographical scales is an ambivalent and effectively superdiffusive process.
Dynamics of Modern Epidemics.
Brockmann, D., Hufnagel, L. & Geisel, T.
In 'SARS: A Case Study in Emerging Infections', McLean, A., May, R., Pattison, J. & Weiss, R. (eds), 2005, Oxford University Press
Forecast and control of epidemics in a globalized world.
Hufnagel, L., Brockmann, D. & Geisel, T.
Proc Natl Acad Sci U S A. 2004 Oct 19;101(42):15124-9. Epub 2004 Oct 11.
The rapid worldwide spread of severe acute respiratory syndrome demonstrated the potential threat an infectious disease poses in a closely interconnected and interdependent world. Here we introduce a probabilistic model that describes the worldwide spread of infectious diseases and demonstrate that a forecast of the geographical spread of epidemics is indeed possible. This model combines a stochastic local infection dynamics among individuals with stochastic transport in a worldwide network, taking into account national and international civil aviation traffic. Our simulations of the severe acute respiratory syndrome outbreak are in surprisingly good agreement with published case reports. We show that the high degree of predictability is caused by the strong heterogeneity of the network. Our model can be used to predict the worldwide spread of future infectious diseases and to identify endangered regions in advance. The performance of different control strategies is analyzed, and our simulations show that a quick and focused reaction is essential to inhibiting the global spread of epidemics.