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[
International C. elegans Meeting,
1997]
We have developed an improved version of John White's 4dimensional microscope which permits a 3-dimensional time lapse analysis of embryogenesis. A new software, "Biocell", permits a rapid and thorough analysis of the embryonic lineages as well as the reconstruction of various embryonic stages. The analysis is stored in the form of an annotated lineage. Embryonic stages and cell movements can be viewed as 3-D models in which cells can be coloured to better visualize the relative position of cells or groups of cells. The models can be rotated to provide different views of the embryo as well as to allow an easy comparison of different embryos. These features should also facilitate the identification of cells in immunostainings or in situ hybridizations. We used the new program to create a set of data (cell cleavages, cell positions at various time points during development) for the reconstruction of embryonic stages of normal embryos. We observe an unexpected variability in the timing of cleavages, cell positions, cell-cell contacts and of cell movements in normal embryos. The analysis reveals a regional organisation of the lineage.
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[
International Worm Meeting,
2009]
Small sample spatial in vivo imaging techniques such as confocal microscopy, micro MRT (mMRT), Selective Plane Illumination Microscopy (SPIM), or contrast enhanced techniques, such as DICM (Differential Interference Contrast Microscopy) are common tools for imaging fluorescent expression in the nematode Caenorhabditis elegans. However, these methods have limited capacity for high resolution, rapid, whole body 3D microscopic imaging and/or imaging of multiple contrast agents. The recently developed approach of Optical Projection Tomography (OPT) enables 3D visualization of whole specimens up to several millimetres in sizes as it has already been shown in zebra fish, chick and mouse embryos. This is achieved by applying a filtered back projection algorithm on images taken from equidistant angles of a rotating specimen with magnification dependent resolution, down to 1-5 mm. We applied a modified OPT setup for 3D imaging of C. elegans expressing GFP in specific neurons. We demonstrate that this novel technique allows rapid acquisition of whole-animal fluorescent expression patterns in the nematode with high accuracy. OPT visualization can be easily adapted to image multiple tissues and cell types, with a variety of chromophores, that allow multi-colour projections, in the nematode.
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[
European Worm Neurobiology Meeting,
2009]
The nematode Caenorhabditis elegans is widely used for the analysis of nervous system function. Changes in worm behaviour caused by genetic mutations or neuronal ablations can be observed and recorded with the low cost, feature-rich single worm tracker, Worm Tracker 2.0, which we have developed together with the Sternberg lab. This work describes the Worm Analysis Toolbox 1.0, a method developed to analyze video data collected by Worm Tracker 2.0 and to extract a wide variety of biologically relevant features. The current release allows the calculation of morphological features, including worm length, width, fatness, head and tail colouration and locomotion parameters such as velocity, flex, bending frequency, track amplitude, track wavelength and reconstruction of the path of the worm. Together, Worm Tracker 2.0 and Worm Analysis Toolbox 1.0 establish a powerful phenotyping system, capable of comparing data collected by different laboratories. We are using this system to start the generation of a seed C. elegans phenotypic database that will be used to explore the relative similarities of mutant phenotypes and detect behavioural differences under multiple experimental conditions. Worm Tracker 2.0 and Worm Analysis Toolbox 1.0 are both available online at
http://www.mrc-lmb.cam.ac.uk/wormtracker/ .
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[
European Worm Meeting,
2008]
Small sample spatial in vivo imaging techniques such as confocal. microscopy, micro MRT (microMRT), Selective Plane Illumination Microscopy. (SPIM) or contrast enhanced techniques such as DICM (Differential. Interference Contrast Microscopy) are common tools for imaging fluorescent. expression in the nematode Caenorhabditis elegans. However, these methods. have limited capacity for high resolution, rapid, whole body 3D microscopic. imaging and/or imaging of multiple contrast agents. The recently developed. approach of Optical Projection Tomography (OPT) enables 3D visualization of. whole specimens up to several millimetres in sizes as it has already been. shown in zebra fish, chick and mouse embryos. This is achieved by applying. a filtered back projection algorithm on images taken from equidistant. angles of a rotating specimen with magnification dependent resolution, down. to 1-5 microm. We present a modified OPT setup for 3D imaging of GFP expressing. neuronal cells in C. elegans. We demonstrate that this novel technique. allows rapid acquisition of whole-animal fluorescent expression patterns in. the nematode with high accuracy. OPT visualization can easily be adapted to. image multiple tissues and cell types, with a variety of chromophores, that. allow multi-colour projections, in the nematode.
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[
International Worm Meeting,
2011]
Invertebrate axons and those of the mammalian peripheral nervous system are able to regenerate in the adult. Functional neuronal recovery following injury arises when severed axons reconnect with their targets. In C. elegans, following laser axotomy, the regrowing axon still attached to cell body (proximal) is able to regrow and reconnect with its separated distal segment through unknown mechanisms. Using the mechanosensory neurons ALM and PLM as a model system, we have found that reconnection of separated axon fragments during regeneration occurs through a mechanism of axonal fusion, which prevents Wallerian degeneration of the distal fragment. Through electron microscopy analysis and imaging with the photoconvertible fluorescent protein Kaede, we show that the fusion process re-establishes membrane continuity and repristinates anterograde and retrograde cytoplasmic diffusion. Through the use of dual colour labeling of adjacent axonal pairs, we also provide evidence that axonal fusion occurs with a remarkable level of accuracy, with the proximal re-growing axon specifically reconnecting with its own separated distal fragment. Finally, from a candidate screening approach, we have identified several molecules as being necessary for successful regeneration and specifically involved in the process of axonal fusion.
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[
International Worm Meeting,
2011]
Optogenetic technologies use light to gain endogenous control of defined cells or tissues in a non-invasive manner. Neuronal activity can be manipulated at the millisecond timescale by expressing the light-activated depolarizing cation channel channelrhodopsin-2 (ChR-2) and subsequent illumination with blue light (1). In contrast, yellow light-triggered inhibition of neuronal activity can be achieved by activation of the hyperpolarizing halorhodopsin (NpHR) (2). However, only few successful attempts were undertaken to inhibit C. elegans neurons using NpHR, probably due to the insufficient trafficking to the plasma membrane, and thus need for high expression levels, or the limited hyperpolarizing power of this Cl- channel. An extensive screen of type I microbial opsins from archaebacteria, bacteria, plants and fungi recently revealed powerful outward directed proton pumps as valuable alternative hyperpolarisers (3). As cells and extracellular fluid are strongly buffered, shuffling protons across the membrane is not expected to cause any appreciable pH changes. The yellow-green light-sensitive archaerhodopsin-3 (Arch) from Halorubrum sodomense appears to be significantly more powerful than NpHR. Another proton pump from the fungus Leptosphaeria maculans, Mac, enables neuronal silencing by green-blue light. This opens the possibility to inhibit different neuronal populations, depending on the illumination wavelengths used. Here we present the use of these outward-directed proton pumps as potent circuit breakers in C. elegans. Electrophysiological recordings on dissected muscle cells allowed us to quantify the outward current evoked by either NpHR, Arch and Mac. As Mac can be stimulated using blue light, we can activate a subset of neurons using ChR2 while Mac could be used to simultaneously inhibit downstream neurons, using the same colour of light. Alternatively, illumination of predefined body segments with different colours of light using an integrated LCD projector as light source (4) allows using the more potent Arch (maximal hyperpolarizing power with green light) for neuronal inhibition and simultaneous ChR2-induced activation of other cells with blue light. A few examples for circuit dissection with either bacteriorhodopsin will be presented at the meeting. (1) G. Nagel et al., Curr. Biol. 15, 2279 (2005); (2) F. Zhang et al., Nature 446, 633 (2007); (3) B. Y. Chow et al., Nature 463, 98 (2010); (4) J. N. Stirman et al., Nat. Meth. 8, 153 (2011).
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[
West Coast Worm Meeting,
1996]
We have developed an improved version of John White's 4-dimensional microscope which permits a 3-dimensional time lapse analysis of embryogenesis. A new software program, Biocell, permits a rapid and thorough analysis of the embryonic lineages as well as the reconstruction of various embryonic stages. The analysis is stored in the form of an annotated lineage. Embryonic stages and cell movements can be viewed as 3-D models in which cells can be coloured to better visualize the relative position of cells or, groups of cells. The models can be rotated to provide different views of the embryo as well as to allow easy comparison of different embryos. This feature should also faciliate identification of cells in immunostainings or in situ hybridizations. We used the new program to create a set of data (cell positions at various time points during development) for the reconstruction of embryonic stages of wild type embryos and the analysis of cell migrations in the embryo. Analysis of this data set reveals the dynamics of cell migrations in the embryo, e.g. in the establishment of left-right asymmetry, or during the migration of muscle precursors to form the four muscle qudrants. In addition, analysis of the development of several wild type embryos shows that the timing of cleavages and cell movements (migrations) are quite variable although the assignment of cell fates appears absolutely invariant.
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[
East Asia Worm Meeting,
2010]
Invertebrate axons and those of the mammalian peripheral nervous system are able to regenerate in the adult. Functional recovery takes place when a damaged axon regains connection with its target tissues. In C. elegans, following laser axotomy, the regrowing axon still attached to cell body (proximal) is able to reconnect with its separated distal segment through unknown mechanisms. Using the mechanosensory neurons ALM and PLM as a model system, we have found that during axonal regeneration reconnection between the proximal and distal axonal fragments occurs through a mechanism of axonal fusion, with reestablishment of cytoplasmic and membrane continuity. We found that when axonal fusion does not occur the distal fragment inevitably undergoes Wallerian degeneration and the original axonal tract cannot be restored. Through the use of dual colour labeling of adjacent axonal pairs, we found a high level of specific recognition occurring between a proximal re-growing axon and its own separated distal fragment, revealing possible cross talk between the two processes. Finally, from a candidate mutant approach, we have identified a molecule with homology to a human protein implicated in axonal degeneration, as being necessary for successful regeneration and specifically involved in the process of axonal fusion. We anticipate that a similar mechanism of axonal regeneration could be exploited to improve the outcome of axonal regeneration following injury in mammalian systems.
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[
Neuronal Development, Synaptic Function and Behavior, Madison, WI,
2010]
Invertebrate axons and those of the mammalian peripheral nervous system are able to regenerate in the adult. Functional recovery takes place when a damaged axon regains connection with its target tissues. In C. elegans, following laser axotomy, the regrowing axon still attached to cell body (proximal) is able to reconnect with its separated distal segment through unknown mechanisms. Using the mechanosensory neurons ALM and PLM as a model system, we have found that during axonal regeneration reconnection between the proximal and distal axonal fragments occurs through a mechanism of axonal fusion, with reestablishment of cytoplasmic and membrane continuity. We found that when axonal fusion does not occur the distal fragment inevitably undergoes Wallerian degeneration and the original axonal tract cannot be restored. Through the use of dual colour labeling of adjacent axonal pairs, we found a high level of specific recognition occurring between a proximal re-growing axon and its own separated distal fragment, revealing possible cross talk between the two processes. Finally, from a candidate mutant approach, we have identified a molecule with homology to a human protein implicated in axonal degeneration, as being necessary for successful regeneration and specifically involved in the process of axonal fusion. We anticipate that a similar mechanism of axonal regeneration could be exploited to improve the outcome of axonal regeneration following injury in mammalian systems.
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[
International Worm Meeting,
2005]
The smooth sinusoidal movements of C. elegans are effected by 95 obliquely striated bodywall muscle cells. There are also non-striated single-sarcomere muscles; the pharyngeal, intestinal, anal and sex-specific muscles. Force generated by the myofilament lattice of bodywall muscle is transmitted to the external cuticle through the attachment structures of the dense bodies, M-lines and fibrous organelles. Attachment structures are also present in the single-sarcomere muscle cells. Additional components of the lattice and associated attachment structures may be identified by investigating the subcellular localisation of endogenous proteins fused to GFP. The twin resources of the Promoterome and ORFeome are being used to construct Promoter::ORF::GFP fusions in a high-throughput manner using Multisite Gateway recombinational cloning. Transgenic lines are generated using these constructs to study the expression patterns of ORF::GFP fusion proteins in vivo. The genes selected for inclusion in this project were chosen because their respective promoters had been shown previously to drive expression of reporter genes in muscle cells. Including ORFs within Promoter::ORF::GFP constructs may reveal the subcellular localisation of the native protein. The genes W05F2.4, F07C3.4 and F02A9.3 were used to establish procedures and expression patterns will be presented. Relative localisation of the fusion proteins with well-known components of muscle structures will be investigated using reporter genes of differing colours. This would also facilitate visualisation of the various components of the contractile apparatus and associated attachment structures assembling throughout development. After initially identifying novel components, techniques such as RNAi and yeast two-hybrid experiments may be employed to clarify the roles and interactions of these proteins.