Controlled Microfluidics to Examine Growth-Factor induced Migration of Neural Progenitors in the Drosophila Visual System (original) (raw)
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Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc
Journal of Visualized Experiments, 2009
Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons. While recent progress has been made to describe the live migration of glial cells in the developing pupal wing (1), studies of Drosophila glial cell migration have typically involved the examination of fixed tissue. Live microscopic analysis of motile cells offers the ability to examine cellular behavior throughout the migratory process, including determining the rate of and changes in direction of growth. Paired with use of genetic tools, live imaging can be used to determine more precise roles for specific genes in the process of development. Previous work by Silies et al. (2) has described the migration of glia originating from the optic stalk, a structure that connects the developing eye and brain, into the eye imaginal disc in fixed tissue. Here we outline a protocol for examining the live migration of glial cells into the Drosophila eye imaginal disc. We take advantage of a Drosophila line that expresses GFP in developing glia to follow glial cell progression in wild type and in mutant animals. Protocol Part 1: Pre-experimental setup. Part 2: Dissection of the Drosophila eye-brain complex. Part 3: Mounting the eye imaginal disc in a magnetic culture chamber.
Cell adhesion & migration, 2016
Central nervous system (CNS) cells cultured in vitro as neuroclusters are useful models of tissue regeneration and disease progression. However, the role of cluster formation and collective migration of these neuroclusters to external stimuli has been largely unstudied in vitro. Here, three distinct CNS cell types, medulloblastoma (MB), medulloblastoma-derived glial progenitor cells (MGPC), and retinal progenitor cells (RPC), were examined with respect to cluster formation and migration in response to Stromal-Derived Growth Factor (SDF-1). A microfluidic platform was used to distinguish collective migration of neuroclusters from that of individual cells in response to controlled concentration profiles of SDF-1. Cell lines were also compared with respect to expression of CXCR4, the receptor for SDF-1, and the gap junction protein Connexin 43 (Cx43). All cell types spontaneously formed clusters and expressed both CXCR4 and Cx43. RPC clusters exhibited collective chemotactic migration ...
Low Concentration Microenvironments Enhance the Migration of Neonatal Cells of Glial Lineage
Cellular and molecular bioengineering, 2012
Glial tumors have demonstrated abilities to sustain growth via recruitment of glial progenitor cells (GPCs), which is believed to be driven by chemotactic cues. Previous studies have illustrated that mouse GPCs of different genetic backgrounds are able to replicate the dispersion pattern seen in the human disease. How GPCs with genetic backgrounds transformed by tumor paracrine signaling respond to extracellular cues via migration is largely unexplored, and remains a limiting factor in utilizing GPCs as therapeutic targets. In this study, we utilized a microfluidic device to examine the chemotaxis of three genetically-altered mouse GPC populations towards tumor conditioned media, as well as towards three growth factors known to initiate the chemotaxis of cells excised from glial tumors: Hepatocyte Growth Factor (HGF), Platelet-Derived Growth Factor-BB (PDGF-BB), and Transforming Growth Factor-α (TGF-α). Our results illustrate that GPC types studied exhibited chemoattraction and chem...
Imaging in vivo Neuronal Transport in Genetic Model Organisms Using Microfluidic Devices
Traffic, 2011
Microfluidic devices have been developed for imaging behavior and various cellular processes in Caenorhabditis elegans, but not subcellular processes requiring high spatial resolution. In neurons, essential processes such as axonal, dendritic, intraflagellar and other long-distance transport can be studied by acquiring fast time-lapse images of green fluorescent protein (GFP)-tagged moving cargo. We have achieved two important goals in such in vivo studies namely, imaging several transport processes in unanesthetized intact animals and imaging very early developmental stages. We describe a microfluidic device for immobilizing C. elegans and Drosophila larvae that allows imaging without anesthetics or dissection. We observed that for certain neuronal cargoes in C. elegans, anesthetics have significant and sometimes unexpected effects on the flux. Further, imaging the transport of certain cargo in early developmental stages was possible only in the microfluidic device. Using our device we observed an increase in anterograde synaptic vesicle transport during development corresponding with synaptic growth. We also imaged Q neuroblast divisions and mitochondrial transport during early developmental stages of C. elegans and Drosophila, respectively. Our simple microfluidic device offers a useful means to image high-resolution subcellular processes in C. elegans and Drosophila and can be readily adapted to other transparent or translucent organisms.
Axon Guidance Studies Using a Microfluidics-Based Chemotropic Gradient Generator
Methods in molecular biology (Clifton, N.J.), 2016
Microfluidics can be used to generate flow-driven gradients of chemotropic guidance cues with precisely controlled steepnesses for indefinite lengths of time. Neuronal cells grown in the presence of these gradients can be studied for their response to the effects exerted by the cues. Here we describe a polydimethylsiloxane (PDMS) microfluidics chamber capable of producing linear gradients of soluble factors, stable for at least 18 h, suitable for axon guidance studies. Using this device we demonstrate turning of superior cervical ganglion axons by gradients of nerve growth factor (NGF). The chamber produces robust gradients, is inexpensive to mass produce, can be mounted on a tissue culture dish or glass coverslip for long term time-lapse microscopy imaging, and is suitable for immunostaining.
Small, 2014
The density of integrin-binding ligands in an extracellular matrix (ECM) is known to regulate cell migration speed by imposing a balance of traction forces between the leading and trailing edges of the cell, but the effect of cell-adhesive ligands on neurite chemoattraction is not well understood. We present a platform that combines gradient-generating microfluidic devices with threedimensional (3D) protein-engineered hydrogels to study the effect of RGD ligand density on neurite pathfinding from chick dorsal root ganglia-derived spheroids. Spheroids are encapsulated in elastin-like polypeptide (ELP) hydrogels presenting either 3.2 or 1.6 mM RGD ligands and exposed to a microfluidic gradient of nerve growth factor (NGF). While the higher ligand density matrix enhanced neurite initiation and persistence of neurite outgrowth, the lower ligand density matrix significantly improved neurite pathfinding and increased the frequency of growth cone turning up the NGF gradient. The apparent trade-off between neurite extension and neurite guidance is reminiscent of the well-known parabolic relationship between cell adhesion and migration speed, implying that a similar matrix-mediated balance of forces regulate neurite elongation and growth cone turning. These results have implications in the design of engineered materials for in vitro models of neural tissue and in vivo nerve guidance channels.
In vitro analysis of neuron-glial cell interactions during cellular migration
European Biophysics Journal, 2002
We used time-lapse microscopy to study the in vitro migration of neuronal cells from developing chick ciliary ganglion. These cells, when dissociated and cultured in a chemically defined medium, are able to migrate and to associate into clusters. We focused our attention on the study of the distribution of neuronal velocity components. Quantitative analysis of cell trajectories allowed us to demonstrate that, in many cells, velocities are well described by the Langevin equation, when deterministic components of the forces acting on the cells are taken into account. We also have shown that the majority of neurons whose movement is not purely random migrate in association with glial cells. We conclude that glial cells, by guiding neurons during migration, play an important role in the cell organization in vitro.
Biomedical Microdevices, 2007
A method for assembling Drosophila embryos in a microfluidic device was developed for studies of thermal perturbation of early embryonic development. Environmental perturbation is a complimentary method to injection of membrane-impermeable macromolecules for assaying genetic function and investigating robustness in complex biochemical networks. The development of a high throughput method for perturbing embryos would facilitate the isolation and mapping of signaling pathways. We immobilize Drosophila embryos inside a microfluidic device on minimal potential-energy wells created through surface modification, and thermally perturb these embryos using binary laminar flows of warm and cold solutions. We self-assemble embryos onto oil adhesive pads with an alcohol surfactant carrier fluid (detachment: 0.1 mL/min), and when the surfactant is removed, the embryo-oil adhesion increases to ∼25 mL/min flow rates, which allows for high velocities required for sharp gradients of thermal binary flows. The microfluidic thermal profile was numerically characterized by simulation and experimentally characterized by fluorescence thermometry. The effects of thermal perturbation were observed to induce abnormal morphogenetic movements in live embryos by using time-lapse differential interference contrast (DIC) microscopy.