Distinct roles for BAI1 and TIM-4 in the engulfment of dying neurons by microglia.
Mazaheri, F.*, Breus, O.*, Durdu, S., Haas, P., Wittbrodt, J., Gilmour, D. & Peri, F.
Nat Commun. 2014 Jun 5;5:4046. doi: 10.1038/ncomms5046.
* these authors contributed equally.
The removal of dying neurons by microglia has a key role during both development and in several diseases. To date, little is known about the cellular and molecular processes underlying neuronal engulfment in the brain. Here we took a live imaging approach to quantify neuronal cell death progression in embryonic zebrafish brains and studied the response of microglia. We show that microglia engulf dying neurons by extending cellular branches that form phagosomes at their tips. At the molecular level we found that microglia lacking the phosphatidylserine receptors BAI1 and TIM-4, are able to recognize the apoptotic targets but display distinct clearance defects. Indeed, BAI1 controls the formation of phagosomes around dying neurons and cargo transport, whereas TIM-4 is required for phagosome stabilization. Using this single-cell resolution approach we established that it is the combined activity of BAI1 and TIM-4 that allows microglia to remove dying neurons.
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.
Directional tissue migration through a self-generated chemokine gradient.
Dona, E., Barry, J.D., Valentin, G., Quirin, C., Khmelinskii, A., Kunze, A., Durdu, S., Newton, L.R., Fernandez-Minan, A., Huber, W., Knop, M. & Gilmour, D.
Nature. 2013 Nov 14;503(7475):285-9. doi: 10.1038/nature12635. Epub 2013 Sep 25.
The directed migration of cell collectives is a driving force of embryogenesis. The predominant view in the field is that cells in embryos navigate along pre-patterned chemoattractant gradients. One hypothetical way to free migrating collectives from the requirement of long-range gradients would be through the self-generation of local gradients that travel with them, a strategy that potentially allows self-determined directionality. However, a lack of tools for the visualization of endogenous guidance cues has prevented the demonstration of such self-generated gradients in vivo. Here we define the in vivo dynamics of one key guidance molecule, the chemokine Cxcl12a, by applying a fluorescent timer approach to measure ligand-triggered receptor turnover in living animals. Using the zebrafish lateral line primordium as a model, we show that migrating cell collectives can self-generate gradients of chemokine activity across their length via polarized receptor-mediated internalization. Finally, by engineering an external source of the atypical receptor Cxcr7 that moves with the primordium, we show that a self-generated gradient mechanism is sufficient to direct robust collective migration. This study thus provides, to our knowledge, the first in vivo proof for self-directed tissue migration through local shaping of an extracellular cue and provides a framework for investigating self-directed migration in many other contexts including cancer invasion.
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.
EMT 2.0: shaping epithelia through collective migration.
Revenu, C. & Gilmour, D.
Curr Opin Genet Dev. 2009 Aug;19(4):338-42. Epub 2009 May 20.
Epithelial-mesenchymal transitions (EMTs) drive epithelial remodelling by converting cohesive, stable epithelial layers into individual, motile mesenchymal cells. It is now becoming clear that, from being an all-or-nothing switch, EMT can be applied in a fine-tuned manner to allow the efficient migration of cohesive epithelia that maintain their internal organisation. Recent work suggests that such collective motility involves a complex balance between epithelial and mesenchyme-like cell states that is driven by internal and external cues. Although this cohesive mode requires more complex control than single cell migration, it creates opportunities in term of tissue guidance and shaping that are starting to be unravelled.
Collective cell migration in morphogenesis, regeneration and cancer.
Friedl, P. & Gilmour, D.
Nat Rev Mol Cell Biol. 2009 Jul;10(7):445-57.
The collective migration of cells as a cohesive group is a hallmark of the tissue remodelling events that underlie embryonic morphogenesis, wound repair and cancer invasion. In such migration, cells move as sheets, strands, clusters or ducts rather than individually, and use similar actin- and myosin-mediated protrusions and guidance by extrinsic chemotactic and mechanical cues as used by single migratory cells. However, cadherin-based junctions between cells additionally maintain 'supracellular' properties, such as collective polarization, force generation, decision making and, eventually, complex tissue organization. Comparing different types of collective migration at the molecular and cellular level reveals a common mechanistic theme between developmental and cancer research.
A systematic analysis of Tinman function reveals Eya and JAK-STAT signaling as essential regulators of muscle development.
Liu, Y.H., Jakobsen, J.S., Valentin, G., Amarantos, I., Gilmour, D.T. & Furlong, E.E.
Dev Cell. 2009 Feb;16(2):280-91.
Nk-2 proteins are essential developmental regulators from flies to humans. In Drosophila, the family member tinman is the major regulator of cell fate within the dorsal mesoderm, including heart, visceral, and dorsal somatic muscle. To decipher Tinman's direct regulatory role, we performed a time course of ChIP-on-chip experiments, revealing a more prominent role in somatic muscle specification than previously anticipated. Through the combination of transgenic enhancer-reporter assays, colocalization studies, and phenotypic analyses, we uncovered two additional factors within this myogenic network: by activating eyes absent, Tinman's regulatory network extends beyond developmental stages and tissues where it is expressed; by regulating stat92E expression, Tinman modulates the transcriptional readout of JAK/STAT signaling. We show that this pathway is essential for somatic muscle development in Drosophila and for myotome morphogenesis in zebrafish. Taken together, these data uncover a conserved requirement for JAK/STAT signaling and an important component of the transcriptional network driving myogenesis.
Dynamic Fgf signaling couples morphogenesis and migration in the zebrafish lateral line primordium.
Lecaudey, V., Cakan-Akdogan, G., Norton, W.H. & Gilmour, D.
Development. 2008 Aug;135(16):2695-705. Epub 2008 Jul 3.
The collective migration of cells in the form of cohesive tissues is a hallmark of both morphogenesis and repair. The extrinsic cues that direct these complex migrations usually act by regulating the dynamics of a specific subset of cells, those at the leading edge. Given that normally the function of tissue migration is to lay down multicellular structures, such as branched epithelial networks or sensory organs, it is surprising how little is known about the mechanisms that organize cells behind the leading edge. Cells of the zebrafish lateral line primordium switch from mesenchyme-like leader cells to epithelial rosettes that develop into mechanosensory organs. Here, we show that this transition is regulated by an Fgf signaling circuit that is active within the migrating primordium. Point sources of Fgf ligand drive surrounding cells towards a ;non-leader' fate by increasing their epithelial character, a prerequisite for rosette formation. We demonstrate that the dynamic expression of Fgf ligands determines the spatiotemporal pattern of epithelialization underlying sensory organ formation in the lateral line. Furthermore, this work uncovers a surprising link between internal tissue organization and collective migration.
Polarising migrating tissues during organogenesis
Gilmour, D.; Haas, P; Cakan, G; Lecaucley, V; Colombelli, J; Stelzer, E.H.K.
Developmental Biology 2008/07/15 319(2) 488-488
In migrating cells, the Golgi complex and the position of the centrosome depend on geometrical constraints of the substratum.
Pouthas, F., Girard, P., Lecaudey, V., Ly, T.B., Gilmour, D., Boulin, C., Pepperkok, R. & Reynaud, E.G.
J Cell Sci. 2008 Jul 15;121(Pt 14):2406-14. Epub 2008 Jun 24.
Although cells migrate in a constrained 3D environment in vivo, in-vitro studies have mainly focused on the analysis of cells moving on 2D substrates. Under such conditions, the Golgi complex is always located towards the leading edge of the cell, suggesting that it is involved in the directional movement. However, several lines of evidence indicate that this location can vary depending on the cell type, the environment or the developmental processes. We have used micro contact printing (microCP) to study the migration of cells that have a geometrically constrained shape within a polarized phenotype. Cells migrating on micropatterned lines of fibronectin are polarized and migrate in the same direction. Under such conditions, the Golgi complex and the centrosome are located behind the nucleus. In addition, the Golgi complex is often displaced several micrometres away from the nucleus. Finally, we used the zebrafish lateral line primordium as an in-vivo model of cells migrating in a constrained environment and observe a similar localization of both the Golgi and the centrosome in the leading cells. We propose that the positioning of the Golgi complex and the centrosome depends on the geometrical constraints applied to the cell rather than on a precise migratory function in the leading region.
The chemokine SDF1a coordinates tissue migration through the spatially restricted activation of Cxcr7 and Cxcr4b.
Valentin, G., Haas, P. & Gilmour, D.
Curr Biol. 2007 Jun 19;17(12):1026-31.
Tissue migration is a collective behavior that plays a key role in the formation of many organ systems [1-3]. Although tissue movements are guided by extrinsic cues, in many contexts, their receptors need to be active only at the leading edge to ensure morphogenesis [4-8]. This has led to the prevalent view that extrinsic signals exert their influence by controlling a small number of leader cells. The zebrafish lateral-line primordium is a cohesive cohort of over 100 cells that is guided through CXCR4-SDF1 signaling [9-11]. Recent work has shown that Cxcr4b activity is only required in cells at the very tip, raising the question of what controls cell behavior within trailing regions . Here, we present the first mutant in zebrafish SDF1a/CXCL12a and show, surprisingly, that the resultant phenotype is stronger than a null mutation in its cognate receptor, Cxcr4b, indicating the involvement of other SDF1a receptors. A candidate approach identified Cxcr7/RDC1, whose expression is restricted to cells behind the leading edge. Morpholino knockdown of Cxcr7 leads to a novel phenotype in which the migration of trailing cells is specifically affected, causing tissue stretching, a defect rescued by the reintroduction of wild-type cells specifically at the back of the primordium. Finally, we present evidence that Cxcr4b and Cxcr7 act independently to regulate group migration. We provide the first example where a single extrinsic guidance cue, SDF1a, directly controls the migration of both leading and trailing edges of a tissue through the activation of two independent receptors, CXCR4b and CXCR7.
Pushing and pulling migrating tissues in vivo with GPCRs.
Gilmour, D.; Haas, P; Cakan, G; Lecaudey, V; Valentin, G; Colombelli, J; Stelzer, E.H.K.
FASEB Journal 2007/04 21(5) A3-A3
Chemokine signaling mediates self-organizing tissue migration in the zebrafish lateral line.
Haas, P. & Gilmour, D.
Dev Cell. 2006 May;10(5):673-80.
The shape of most complex organ systems arises from the directed migration of cohesive groups of cells. Here, we dissect the role of the chemokine guidance receptor Cxcr4b in regulating the collective migration of one such cohesive tissue, the zebrafish lateral line primordium. Using in vivo imaging, we show that the shape and organization of the primordium is surprisingly labile, and that internal cell movements are uncoordinated in embryos with reduced Cxcr4b signaling. Genetic mosaic experiments reveal that single cxcr4b mutant cells can migrate in a directional manner when placed in wild-type primordia, but that they are specifically excluded from the leading edge. Moreover, a remarkably small number of SDF1a-responsive cells are able to organize an entire cxcr4b mutant primordium to restore migration and organogenesis in the lateral line. These results reveal a role for chemokine signaling in mediating the self-organizing migration of tissues during morphogenesis.
Organizing moving groups during morphogenesis.
Lecaudey, V. & Gilmour, D.
Curr Opin Cell Biol. 2006 Feb;18(1):102-7. Epub 2005 Dec 13.
The directed migration of cells drives the formation of many complex organ systems. Although in this morphogenetic context cells display a strong preference for migrating in organized, cohesive groups, little is known about the mechanisms that coordinate their movements. Recent studies on several model systems have begun to dissect the organization of these migrating tissues in vivo and have shown that cell guidance is mediated by a combination of chemical and mechanical cues.
Towing of sensory axons by their migrating target cells in vivo.
Gilmour, D., Knaut, H., Maischein, H.M. & Nusslein-Volhard, C.
Nat Neurosci 2004 May;7(5):491-2. Epub 2004 Apr 18.
Many pathfinding axons must locate target fields that are themselves positioned by active migration. A hypothetical method for ensuring that these migrations are coordinated is towing, whereby the extension of axons is entirely dependent on the migration of their target cells. Here we combine genetics and time-lapse imaging in the zebrafish to show that towing by migrating cells is a bona fide mechanism for guiding pathfinding axons in vivo.
Migration and function of a glial subtype in the vertebrate peripheral nervous system.
Gilmour, D.T., Maischein, H.M. & Nusslein-Volhard, C.
Neuron 2002 May 16;34(4):577-88.
Glia-axon interactions are essential for the development and function of the nervous system. We combine in vivo imaging and genetics to address the mechanism by which the migration of these cells is coordinated during embryonic development. Using stable transgenic lines, we have followed the migration of one subset of glial cells and their target axons in living zebrafish embryos. These cells coalesce at an early stage and remain coupled throughout migration, with axons apparently pathfinding for glia. Mutant analysis demonstrates that axons provide instructive cues that are sufficient to control glial guidance. Furthermore, mutations in the transcription factor Sox10/cls uncouple the migration of axons and glia. Finally, genetic ablation of this glial subtype reveals an essential role in nerve fasciculation.
Manipulating gene expression in the zebrafish.
Gilmour, D.T., Jessen, J.R. & Lin, S.
In "Zebrafish, a practical approach" (2002) Nusslein-Volhard, C. & Dahm, R. (eds.), Oxford: OUP. pp. 121-143