303 Papers found (0.011 seconds)

Meyerowitz EM (2002) Science "Plants compared to animals: the broadest comparative study of development."

[

Science,

2002]

If the last common ancestor of plants and animals was unicellular, comparison of the developmental mechanisms of plants and animals would show that development was independently invented in each lineage. And if this is the case, comparison of plant and animal developmental processes would give us a truly comparative study of development, which comparisons merely among animals, or merely among plants, do not-because in each of these lineages, the fundamental mechanisms are similar by descent. Evidence from studies of developmental mechanisms in both kingdoms, and data from genome-sequencing projects, indicate that development evolved independently in the lineages leading to plants and to animals.

Kniss SL et al. (1998) Midwest Worm Meeting "A genetic analysis of C. elegans elongation using sma-1 suppressors"

Austin JA, Kniss SL

[

Midwest Worm Meeting,

1998]

During morphogenesis, the C. elegans embryo elongates four-fold from a ball of cells into a long, thin worm. This elongation is dependent on circumferentially oriented actin bundles which form in the embryonic epidermal cells and are thought to generate the contractile force required for elongation. SMA-1 is a cytoskeletal protein which is homologous to Drosophila Beta-H-spectrin and is required for normal elongation: in sma-1 mutants, the rate of elongation is decreased resulting in larvae that are shorter than wild-type at hatching. In many of the sma-1 mutants it is been found that the organization of the epidermal actin bundles is altered (V. Praitis, personal communication). The sma-1 alleles isolated in our lab vary in their effect on the rate of elongation suggesting that, in addition to its likely structural function, SMA-1 plays a role in regulation of elongation. To understand these functions of SMA-1, we have begun to isolate suppressors of sma-1 mutants. We have designed a simple genetic screen that takes advantage of the inability of sma-1 larvae to chemotax to food and have used this screen to isolate both dominant and recessive suppressors. We are currently examining the suppressed phenotypes and mapping the mutations. Characterization of our sma-1 suppressors will begin to answer questions regarding the role of the cytoskeleton during morphogenesis and the function of SMA-1 in the regulation of elongation. In addition, we hope to understand the relationship between SMA-1's role in body elongation and its role in morphogenesis of the pharynx and excretory cell canal.

Jiang, Hongbing et al. (2017) International Worm Meeting "Identification of an evolutionarily conserved pathway essential for virus infection using Orsay virus infection of C. elegans."

Sandoval, Luis, Leung, Christian, Wang, David, Chen, Kevin, Jiang, Hongbing

[

International Worm Meeting,

2017]

Many fundamental biological discoveries have been made in C. elegans. The discovery of Orsay virus has enabled studies of host-virus interactions in this model organism. To identify host factors critical for virus infection, we designed a forward genetic screen that utilizes a virally induced GFP reporter. Following chemical mutagenesis, two viro (virus induced reporter off) mutants that failed to express GFP were mapped to sid-3, a non-receptor tyrosine kinase, and B0280.13, an orthologue of human WASP (Wiskott-Aldrich Syndrome Protein). Both mutants yielded 5-6 logs lower Orsay RNA levels than the parent strain, which approximates residual input Orsay levels, demonstrating the essential roles of these genes. However, Orsay virus replication initiated from an endogenous transgene bypasses the requirements for both SID-3 and B0280.13 suggesting that these two genes function at an early step in the virus lifecycle. Strikingly, the human ortholog of SID-3, activated CDC42 associated kinase (ACK1/TNK2), is capable of phosphorylating human WASP, suggesting that B0280.13 may be a substrate for SID-3 in C. elegans. In support of this model, fluorescently tagged SID-3 and B0280.13 co-localized to the apical membrane of C. elegans intestinal cells. Further genetic ablation of TNK2 through CRISPR-Cas9 in two human cell lines, A549 (an adenocarcinomic human alveolar basal epithelial cell line) and Hap1 (a near haploid human cell line derived from myeloid leukemia patients), showed reduced infection by polio virus. Thus, an unbiased genetic screen in C. elegans identified critical roles in virus infection for evolutionarily conserved genes in a known human pathway.

Shu X et al. (2011) PLoS Biol "A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues, and organisms."

Ramko EB, Shu X, Jin Y, Qi Y, Lev-Ram V, Davidson MW, Deerinck TJ, Tsien RY, Ellisman MH

[

PLoS Biol,

2011]

Electron microscopy (EM) achieves the highest spatial resolution in protein localization, but specific protein EM labeling has lacked generally applicable genetically encoded tags for in situ visualization in cells and tissues. Here we introduce "miniSOG" (for mini Singlet Oxygen Generator), a fluorescent flavoprotein engineered from Arabidopsis phototropin 2. MiniSOG contains 106 amino acids, less than half the size of Green Fluorescent Protein. Illumination of miniSOG generates sufficient singlet oxygen to locally catalyze the polymerization of diaminobenzidine into an osmiophilic reaction product resolvable by EM. MiniSOG fusions to many well-characterized proteins localize correctly in mammalian cells, intact nematodes, and rodents, enabling correlated fluorescence and EM from large volumes of tissue after strong aldehyde fixation, without the need for exogenous ligands, probes, or destructive permeabilizing detergents. MiniSOG permits high quality ultrastructural preservation and 3-dimensional protein localization via electron tomography or serial section block face scanning electron microscopy. EM shows that miniSOG-tagged SynCAM1 is presynaptic in cultured cortical neurons, whereas miniSOG-tagged SynCAM2 is postsynaptic in culture and in intact mice. Thus SynCAM1 and SynCAM2 could be heterophilic partners. MiniSOG may do for EM what Green Fluorescent Protein did for fluorescence microscopy.

Santella A et al. (2022) Elife "Cross-modality synthesis of EM time series and live fluorescence imaging."

Kolotuev I, Santella A, Kizilyaprak C, Bao Z

[

Elife,

2022]

Analyses across imaging modalities allow the integration of complementary spatiotemporal information about brain development, structure and function. However, systematic atlasing across modalities is limited by challenges to effective image alignment. We combine highly spatially resolved electron microscopy (EM) and highly temporally resolved time-lapse fluorescence microscopy (FM) to examine the emergence of a complex nervous system in C. elegans embryogenesis. We generate an EM time series at four classic developmental stages and create a landmark-based co-optimization algorithm for cross-modality image alignment, which handles developmental heterochrony among datasets to achieve accurate single-cell level alignment. Synthesis based on the EM series and time-lapse FM series carrying different cell-specific markers reveals critical dynamic behaviors across scales of identifiable individual cells in the emergence of the primary neuropil, the nerve ring, as well as a major sensory organ, the amphid. Our study paves the way for systematic cross-modality data synthesis in C. elegans and demonstrates a powerful approach that may be applied broadly.

Christian Stigloher et al. (2009) European Worm Neurobiology Meeting "EM-TOMOGRAPHIC RECONSTRUCTION OF SYNAPTIC ULTRASTRUCTURE"

Christian Stigloher, Douglas Holmyard, Jean-Louis BESSEREAU, Mei ZHEN

[

European Worm Neurobiology Meeting,

2009]

The specific functionality of a synapse is crucially linked to the spatial organization of its ultrastructural components. To perform electron microscopy (EM) on synapses in a close-to-native status, we froze living C. elegans under high pressure, which results in instant immobilization of the synaptic structures in amorphic ice. After processing, samples can be observed by classical imaging or used for immuno-EM analyses. We have previously shown that UNC-10/Rim is localized at the active zone, next to the dense projections which are the organizing centers of presynaptic specializations at C. elegans synapses. Genetic disruption of unc-10 causes a redistribution of membrane-contacting vesicles out of the active zone (Weimer et al., 2006). Our spatial analysis of the effect of loss of UNC-10/Rim function has up to now been hampered by the low resolution of classical EM imaging in the z-axis. In order to produce a seamless 3D model of synaptic structures, we are adapting EM-tomography techniques for the use in the C. elegans model. EM-tomography yields high resolution three dimensional ultrastructural images in samples with up to several hundred nm thickness. Specifically, we can visualize structures emanating from dense projections and contacting synaptic vesicles and filamentous connections between synaptic vesicles. With the resulting high resolution 3D reconstructions of synaptic ultrastructure and processing of the resulting datasets with image segmentation software, we will be able to compare wild-type and mutant organization in a comprehensive manner. In particular, this might provide a better insight into the function of UNC-10/Rim. Moreover, our approach can easily be applied to the study of other candidate synaptic proteins such as SYD-2.

Markert SM et al. (2016) Neurophotonics "Filling the gap: adding super-resolution to array tomography for correlated ultrastructural and molecular identification of electrical synapses at the C. elegans connectome."

Mulcahy B, Britz S, Markert SM, Lang M, Witvliet D, Proppert S, Sauer M, Stigloher C, Bessereau JL, Zhen M

[

Neurophotonics,

2016]

Correlating molecular labeling at the ultrastructural level with high confidence remains challenging. Array tomography (AT) allows for a combination of fluorescence and electron microscopy (EM) to visualize subcellular protein localization on serial EM sections. Here, we describe an application for AT that combines near-native tissue preservation via high-pressure freezing and freeze substitution with super-resolution light microscopy and high-resolution scanning electron microscopy (SEM) analysis on the same section. We established protocols that combine SEM with structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). We devised a method for easy, precise, and unbiased correlation of EM images and super-resolution imaging data using endogenous cellular landmarks and freely available image processing software. We demonstrate that these methods allow us to identify and label gap junctions in Caenorhabditis elegans with precision and confidence, and imaging of even smaller structures is feasible. With the emergence of connectomics, these methods will allow us to fill in the gap-acquiring the correlated ultrastructural and molecular identity of electrical synapses.

Santella, Anthony et al. (2021) International Worm Meeting "An Electron Microscopy Pseudo Time Series of the C. elegans Embryo"

Bao, Zhirong, Kolotuev, Irina, Santella, Anthony

[

International Worm Meeting,

2021]

Advances in Electron Microscopy (EM) bring about the possibility of temporal analysis using staged samples over time as well as offering opportunities to gain insights by synthesizing EM with other imaging modalities. We present a pseudo time series of C. elegans embryonic development, four volumes covering around 2 hours particularly rich in development between 320 and 475 minutes post first cleavage. This period encompasses neurulation, neural organogenesis and neuropil formation key events that build most of the major structures of the nervous system as well as critical events for many other organ systems. We correlate this EM data with florescence data spanning the first eight hours of embryogenesis. Every cell in the florescence data is identifiable via lineaging. To correlate these data sets we develop a novel computational method for alignment of identities between data sets in the challenging presence of spatial and temporal variation. This approach involves co-optimization of spatial alignment and the structure of labeled data based on a model of dynamic anatomy in the form of an adjacency graph with expected variation. This model captures both variable elements and consistent spatial proximity relationships. We identify every cell in three of the four time points. Identity results are accurate, ranging from 72 to 78 percent correct when assessed against a large set of manual annotations based on position and morphology. This is better than any previously reported results for identifying all cells in an organism based on position alone. The resulting single cell level annotation allows efficient navigation of this large EM data set. We use the sequence to probe the interactions over time between different components within the nerve ring elucidating the relationship between the temporal and spatial location of initial outgrowths into the ring and ultimate structure. We also examine the interaction between cells during the formation of the amphid dendrite structure providing insight into the timing and succession of events at the inter and intra cellular level. Our observations only scratch the surface of the details available in the data set. We will provide the EM data with single-cell level annotation as a resource for the community.

Rahman MM et al. (2021) Bio Protoc "A Workflow for High-pressure Freezing and Freeze Substitution of the Caenorhabditis elegans Embryo for Ultrastructural Analysis by Conventional and Volume Electron Microscopy."

Chang IY, Narayan K, Rahman MM, Cohen-Fix O

[

Bio Protoc,

2021]

The free-living nematode Caenorhabditis elegans is a popular model system for studying developmental biology. Here we describe a detailed protocol to high-pressure freeze the C. elegans embryo (either ex vivo after dissection, or within the intact worm) followed by quick freeze substitution. Processed samples are suitable for ultrastructural analysis by conventional electron microscopy (EM) or newer volume EM (vEM) approaches such as Focused Ion Beam Scanning Electron Microscopy (FIB-SEM). The ultrastructure of cellular features such as the nuclear envelope, chromosomes, endoplasmic reticulum and mitochondria are well preserved after these experimental procedures and yield accurate 3D models for visualization and analysis ( Chang et al., 2020 ). This protocol was used in the 3D reconstruction of membranes and chromosomes after pronuclear meeting in the C. elegans zygote ( Rahman et al., 2020 ).

Clark SG et al. (1997) Proc Natl Acad Sci U S A "A dynamin GTPase mutation causes a rapid and reversible temperature-inducible locomotion defect in C. elegans."

Clark SG, Van der Bliek AM, Meyerowitz EM, Shurland D-L, Bargmann CI

[

Proc Natl Acad Sci U S A,

1997]

Drosophila shibire and its mammalian homologue dynamin regulate an early step in endocytosis. We identified a Caenorhabditis elegans dynamin gene, dyn-1, based upon hybridization to the Drosophila gene. The dyn-1 RNA transcripts are trans-spliced to the spliced leader 1 and undergo alternative splicing to code for either an 830- or 838-amino acid protein. These dyn-1 proteins are highly similar in amino acid sequence, structure, and size to the Drosophila and mammalian dynamins: they contain an N-terminal GTPase, a pleckstrin homology domain, and a C-terminal proline-rich domain. We isolated a recessive temperature-sensitive dyn-1 mutant containing an alteration within the GTPase domain that becomes uncoordinated when shifted to high temperature and that recovers when returned to lower temperatures, similar to D. shibire mutants. When maintained at higher temperatures, dyn-1 mutants become constipated, egg-laying defective, and produce progeny that die during embryogenesis. Using a dyn-1::lacZ gene fusion, a high level of dynamin expression was observed in motor neurons, intestine, and pharyngeal muscle. Our results suggest that dyn-1 function is required during development and for normal locomotion.

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