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Hall, David H. et al. (2009) International Worm Meeting "Electron tomography: a new tool for exploring C. elegans cellular anatomy."

Fisher, Kevin, Nguyen, Ken C.Q., Hall, David H., Crocker, Chris, Derr, KD, Rice, William J., Politi, Kristin A., Gunther, Leslie

[

International Worm Meeting,

2009]

C. elegans has been studied intensively using serial section reconstruction for several decades, so that every cell type is now known in considerable detail. Nevertheless, limitations of the serial section technique (notably the poor resolution of detail within the depth of each 50 nm "thin" section) have made it difficult to model the shapes of fine details in many organelles, such as the cristae of a mitochondrion, the canaliculi of the excretory canal, membrane ruffles in many cell types, or the constituents at a chemical synapse. High pressure freeze fixation and freeze substitution (1) are a required element in preserving smaller structures that generally escaped our notice in conventional TEM imaging. Modern electron microscopes using higher energy electrons now offer much better resolution by collecting multiple images in a tilting series through comparatively thick sections (150 nm) using the SerialEM program (2). The Protomo software package (3) is used to compute a 3D tomographic reconstruction that offers the same level of detail in any dimension (roughly 2 nm resolution). Annotation and modeling is done using IMOD (4) and/or Amira. This permits us to take a new look at many familiar objects in the anatomy of C. elegans, to identify missing parts of the whole anatomy, and potentially, to detect smaller anatomical defects in a variety of mutant backgrounds. Here we will introduce the tomographic procedure, and share finished 3D models of some typical intracellular organelles at high resolution. Supported by NIH RR 12596 to DHH. 1.Weimer, R.M. (2006) Methods Mol. Biol. 351: 203-221. 2.Mastronarde, D.N. (2005) J. Struct. Biol. 152: 36-51 3.Winkler, H. and Taylor, K.A. (2006) Ultramicroscopy 106: 240-254. 4.Kremer, J.R., Mastronarde, D.N. and McIntosh, J.R. (1996) J. Struct. Biol. 116: 71-76.

David H Hall et al. (2001) International C. elegans Meeting "New! Improved! Fixation protocols for TEM"

David H Hall, Tylon Stephney, Gloria Stephney, Ya Chen, Frank Macaluso

[

International C. elegans Meeting,

2001]

We continue to test alternate methods for preparing worms for transmission electron microscopy. We will describe new protocols, and will demonstrate what makes them better [or different] in comparison to previous methods (Hall, 1995). We still like simple immersion fixation and chopping open the animals by knife blade, and have made minor changes in the starting solutions to get optimum results. For early larval stages, which have never fixed well by immersion, and which are too little to chop open easily, we have adapted a new microwave protocol which gives very good results on intact worms. The resulting fixation looks equivalent to our immersion preparations of adults. Microwave fixation is proving very useful in the analysis of arrested animals from RNAi preparations, and should be excellent for looking at late embryos or dauers. Fast freezing methods offer a quite different approach, and the quality of tissue preservation can be superb. Both metal mirror freezing and high pressure freezing can produce excellent results, and they are achieving wider use over the past few years (Mohler et al., 1998; Rappleye et al., 1999). The inherent contrast after freeze substitution is often much greater, in part because the primary fixation contains only osmium, or a combination of osmium and aldehyde together. These methods allow much more rapid fixation. We can capture more "life-like" views of biological events in action, particularly for events such as vesicle fusions at the plasma membrane. Delicate cytoskeletal elements such as microtubules are also well preserved. We continue to try new combinations of fixatives and solvents to improve the appearance of nerve processes and synapses by fast freezing. Kent McDonald has been very helpful in suggesting improvements to these protocols. Laserhole fixations of embryos are technically rather difficult to accomplish, but can facilitate the passage of fixatives and embedding resins through the eggshell. We are continuing to use the protocol worked out by Carolyn Norris. See our website for details.

David H Hall et al. (2003) International Worm Meeting "Synapses in decline: age-related deterioration in neuronal anatomy"

David H Hall, Peter J Schmeissner, Laura A Herndon, Gloria Stephney, Monica Driscoll

[

International Worm Meeting,

2003]

As nematodes age, various tissue-specific changes occur that coincide with the deterioration of the animal. We characterized the general appearance and the locomotory behavior of wild type adults over time and found that we could distinguish three classes (A, B and C) of behavioral types in the same-age population. By following individuals throughout their lifetime, we determined that decline is progressive. However, we also established that individual time of onset and rate of decline are strikingly variable suggesting aging in C. elegans has a stochastic component. We have used GFP-tagged proteins that highlight the morphologies of specific tissues, cells and subcellular structures in conjunction with electron microscopy to give a detailed histological description of aging in C. elegans. Initial studies indicate that the nervous system remains remarkably intact in old animals while other tissues, such as muscle and intestine, undergo dramatic deterioration (Herndon et al., 2002). While the nervous system appears intact in aged nematoes, we are now trying to determine whether the neurons undergo more modest changes during the aging process. Measures of synapse numbers using snb-1::GFP demonstrate that they remain relatively constant over the adult lifespan. Similar studies of the GABAergic receptor found them present in both neuromuscular junctions and in neurons of old nematodes. However, electron microscopy studies in 18-day-old adults indicate that synaptic vesicle number decreases as the animals progress from A to B to C class. Although synapses can still be recognized, neuronal processes are markedly thinner, their plasma membranes more electron dense, and pre-synaptic densities at active zones are smaller. The terminal volume at individual synapses is reduced, contains fewer vesicles, and has fewer docked vesicles per active zone. Changes are evident when class A animals are compared to young adults, but the decline is more severe in all respects in B and C class animals. Thus, the age-related decline of the nervous system is subtle, with virtually no cell loss and little loss in synapse number. However, the reduction in the pool of releasable synaptic vesicles may play a significant role in the behavioral changes of B and C class animals.

David H Hall et al. (2003) International Worm Meeting "Wormatlas: A comprehensive online reference of C. elegans anatomy"

David H Hall, Zeynep F Altun

[

International Worm Meeting,

2003]

David H Hall et al. (2002) West Coast Worm Meeting "Wormatlas: A web-based behavioral and structural atlas of C. elegans"

Thomas Boulin, Zeynep F Altun, Michael Zhang, Maurice Volaski, Constantinos Polydorou, David H Hall

[

West Coast Worm Meeting,

2002]

David H Hall et al. (2002) Mid-west Worm Meeting "Wormatlas: A web-based behavioral and structural atlas of C. elegans"

David H Hall, Zeynep F Altun, Constantinos Polydorou, Thomas Boulin, Maurice Volaski, Michael Zhang

[

Mid-west Worm Meeting,

2002]

We have recently launched the prototype of Wormatlas (www.wormatlas.org). This atlas is designed to serve the scientific community with the main goal of bringing all the anatomical information pertinent to C. elegans within one readily accessible and easy to use web site. By creating extensive links to the WormBase as well as the C. elegansWWW server, we are aiming to provide users with seamless links between these databases. We hope to create the most comprehensive and complete online anatomy atlas for any genetic model organism. Wormatlas is designed to have two main sections, Index and Guides, with multiple chapters within each section. The Index section will contain the Handbook, Slidable Worm, Literature Archive, Cell Identifications, Neuron Data, Glossary, and Methods. The main goal of the Handbook is to provide a relatively simplified, image-supported and curated information about the general and specific anatomy of C. elegans.The images included in the Handbook will be annotated scanning and transmission electron (TEM) micrographs, computer-drawn images as well as DIC and fluorescent micrographs. The Slidable Worm is designed to provide 600-1200 annotated and nonannotated versions of TEM cross-sections of the animal available for viewing by the users with the help of a newly designed JAVA applet interface. The images will come from the original images from the MRC/LMB archive, from the MIT archive (courtesy of E. Hartwieg and R. H. Horvitz), from our Caltech/AECOM archives, and possibly others. The Literature Archive will provide on-line copies of landmark articles and treatises about the anatomy of the nematode. These HTML format articles include multiple web links to other sites in Wormatlas and WormBase to strengthen their interactivity for the structures mentioned. The Glossary aims to provide a comprehensive list of all nomenclature used to describe any cell structure in the nematode. Cell Identification and Neuron Data are planned to provide enough detail on features of single cells, esp. neurons, to aid researchers in recognizing and studying individual cells by their 3D shapes and positions, comparing TEM, DIC and GFP information, and by providing links to curated data on their gene expression patterns. Finally, Anatomical Methods will provide an up to date summary of the different modalities that are currently used in cell identification and tissue pathology studies. This website is designed to accommodate the vast amount of structural, behavioral and gene expression data that has appeared since publication of The Mind of a Worm in a dynamic and easily updatable medium. This curated information can be viewed in individual neuron pages as well as neuron data appendices. In the future, we want to develop interactive user interfaces to visualize behavioral circuitries and perhaps neurophysiology information as they become available.

David H Hall et al. (2002) East Coast Worm Meeting "Wormatlas: A web-based behavioral and structural atlas of C. elegans"

David H Hall, Maurice Volaski, Michael Zhang, Constantinos Polydorou, Thomas Boulin, Zeynep F Altun

[

East Coast Worm Meeting,

2002]

Silva, Malan et al. (2011) International Worm Meeting "Doublets' well kept secret: the sister singlets."

O'Hagan, Robert, Silva, Malan, Barr, Maureen, Nguyen, Ken, Hall, David H.

[

International Worm Meeting,

2011]

Cilia are cellular antennae; in humans, defects in ciliary form or function give rise to ciliopathies. C. elegans sensory organs, or sensilla, consist of morphologically and functionally diverse arrays of cilia that mediate sensation. The length and shape of cilia depends on axonemal microtubule (MT) architecture. In the amphid channel cilia, the ciliary axoneme is anchored to the membrane by a region known as the transition zone (TZ). In the TZ, MT doublets are made of a 13 protofilament A-tubule and an attached 11 protofilament B-tubule. MT doublets are anchored to the membrane with "Y" links of unknown composition. Beyond the TZ, amphid channel cilia display two distinct zones classified using MT architecture. The middle segment consists of nine MT doublets that lack 'Y' links. The distal segment is composed entirely of MT singlets. Not all C. elegans cilia share this axonemal structure. For example, we have shown that cilia of the male cephalic neurons (CEMs) are composed of singlet MTs (1). We used high pressure freeze fixation and electron tomography to observe CEM cilia ultrastructure. By comparing cephalic and amphid channel cilia, we hope to understand at the molecular level how axonemes are specialized in form and function. We focused on transition from MT doublets to singlets and traced individual A- and B-tubules at the doublet-to-singlet transition. We found that the A- and B-tubules in middle segment doublets in CEM cilia separate into two sister singlets in the distal segment. These sister singlets are continuous to the distal segment of the cilium and spatially diverge from each other. Additionally we found that as compared to TZs of amphid neurons, CEM TZs span a longer distance. We are interested in determining how this spatial and structural disposition of the CEM MTs occurs, and how it affects ciliary motors. We are currently testing the hypothesis that tubulin post-translational modifications contribute to MT arrangement and stability in C. elegans sensory cilia (see Abstract by O'Hagan et al). 1. A. R. Jauregui, K. C. Q. Nguyen, D. H. Hall, M. M. Barr, The JCB 180, 973-988 (2008).

Kim, Grace S et al. (2009) International Worm Meeting "unc-3 is necessary for axon pioneering and guidance in C. elegans."

Hall, David H., White, John G., Xu, Meng, Kim, Grace S

[

International Worm Meeting,

2009]

Genetic and phenotypic studies of unc-3 mutants have shown that unc-3 is required for proper organization of ventral cord processes (1,2). The defasciculation and wiring defects seen in unc-3 mutants have been attributed to disruption of pathfinding in the pioneer neurons during embryonic development. We have used Elegance, a 3D reconstruction software program developed at AECOM (3), to analyze the deformed morphology of the unc-3 (e151) ventral cord to unprecedented detail at the EM level. Both the e151/e151 and e151/Df animals display severe axon guidance errors that affect some pioneers (AVKs) and some motor neurons more strongly than the command interneurons. These results support the essential role of the unc-3 gene in axon pioneering and guidance. We present the pattern of severe axon guidance errors in terms of the mispositioning of specific axons, their deviance from their normal neighborhoods, and their synaptic wiring defects. This study, along with molecular genetic studies identifying the unc-3 gene product as a CeO/E transcription factor, should guide future studies of specific axon guidance cues at the molecular level and provide further insight into the role of the mammalian O/E family members. This study used annotated unc-3 TEM datasets received from John White (MRC/LMB and U. Wisconsin) that are now part of the MRC archive in the Hall lab. This work was supported by NIH RR12596 (to DHH) and an AECOM summer research fellowship (to GSK). 1.Wightman, B., Baran R. and Garriga, G. (1997) Development 124: 2571-2580. 2.Prasad, B.C. et al. (1998) Development 125: 1561-1568. 3.Emmons, S.W. et al. (2009) at this meeting.

Semaya, Emily S. et al. (2017) International Worm Meeting "Abnormal ER morphology revealed through ultrastructural analysis of C. elegans atx-2 mutants."

Semaya, Emily S., Gnazzo, Megan M., Skop, Ahna R., Hall, David H.

[

International Worm Meeting,

2017]

ATX-2, a conserved RNA binding protein, is the C. elegans ortholog of the human RNA binding protein Ataxin-2. Loss of Ataxin-2 leads to the late-onset neurodegenerative disease spinocerebellar ataxia type-2 (SCA2) and is associated with an increased risk for amyotrophic lateral sclerosis (ALS). To define the cellular role of ATX-2, we sought to determine the ultrastructure of ATX-2-depleted embryos and adults. ATX-2 is necessary for embryonic patterning in C. elegans (Kiehl et al., 2000) and recent work from the Skop and Song labs has shown cell division defects in atx-2 mutants (Stubenvoll et al., 2016; Gnazzo et al., 2016). To examine the cellular defects in the early embryo, we characterized atx-2 ts mutant animals (WM210 (atx-2(ne4297));(Gnazzo et al., 2016)) at high resolution. Transmission electron microscopy (TEM) of ultra-thin sections revealed that ATX-2-depleted animals exhibit fertilization and egg-laying defects. Both unfertilized oocytes and defective embryos are present in the uterus of ATX-2 depleted animals. Mutant embryos also exhibit defects in nuclear division, in which daughter cells are multinucleated. Ataxin-2 localizes to the endoplasmic reticulum (ER) in humans (van de Loo et al., 2009), yet the dynamics of this association during cell division are unclear. Preliminary data from the Skop lab suggests a role for ATX-2 in maintaining proper ER morphology, yet the ultrastructure was unknown. Using TEM of ultra-thin sections, we found that ATX-2 depleted embryos have fragmented rough ER, suggesting that ATX-2 is necessary for maintaining proper rough ER morphology during mitosis. Given the human phenotypes, ultrastructural studies of ER morphology in neurons and glia of ATX-2-depleted adult animals is currently underway. This research is supported by an NSF grant MCB-1158003 (Skop Lab) and an NIH grant OD010943 (Hall Lab).

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