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Apical extracellular matrix (aECM) constitutes the interface between every tissue and the outside world. It is patterned into diverse tissue-specific structures through unknown mechanisms. Here, we show that a male-specific genetic switch in a singleC. elegansglial cell patterns the aECM into a ∼200 nm pore, allowing a male sensory neuron to access the environment. We find that this glial sex difference is controlled by factors shared with neurons (mab-3, lep-2, lep-5) as well as previously unidentified regulators whose effects may be glia-specific (nfya-1, bed-3, jmjd-3.1). The switch results in male-specific expression of a Hedgehog-related protein, GRL-18, that we discover localizes to transient nanoscale rings at sites of aECM pore formation. Blocking male-specific gene expression in glia prevents pore formation, whereas forcing male-specific expression induces an ectopic pore. Thus, a switch in gene expression in a single cell is necessary and sufficient to pattern aECM into a specific structure.

Neurons and epithelia are viewed as fundamentally different cell types, yet some sensory neurons exhibit hallmarks of epithelial cells. For example, they use tight junctions to form a diffusion barrier continuous with the skin or other epithelia and they exhibit bona fide apical-basal polarity, with an outward-facing apical surface that is biochemically and functionally distinct from their inward-facing basolateral surface. Yet they are unmistakeably neurons with axon-dendrite polarity. Examples include olfactory receptor neurons and photoreceptors. In this review, I highlight how viewing these neurons as specialized epithelial cells informs our understanding of their development and raises intriguing questions about the establishment of apical-basal and axon-dendrite polarity.

Cells are highly organized machines with functionally specialized compartments. For example, membrane proteins are localized to axons or dendrites in neurons and to apical or basolateral surfaces in epithelial cells. Interestingly, many sensory cells—including vertebrate photoreceptors and olfactory neurons—exhibit both neuronal and epithelial features. Here, we show that Caenorhabditis elegans amphid neurons simultaneously exhibit axon-dendrite sorting like a neuron and apical-basolateral sorting like an epithelial cell. The distal ∼5–10 µm of the dendrite is apical, while the remainder of the dendrite, soma, and axon are basolateral. To determine how proteins are sorted among these compartments, we studied the localization of the conserved adhesion molecule SAX-7/L1CAM. Using minimal synthetic transmembrane proteins, we found that the 91-aa cytoplasmic tail of SAX-7 is necessary and sufficient to direct basolateral localization. Basolateral localization can be fully recapitulated using either of 2 short (10-aa or 19-aa) tail sequences that, respectively, resemble dileucine and Tyr-based motifs known to mediate sorting in mammalian epithelia. The Tyr-based motif is conserved in human L1CAM but had not previously been assigned a function. Disrupting key residues in either sequence leads to apical localization, while “improving” them to match epithelial sorting motifs leads to axon-only localization. Indeed, changing only 2 residues in a short motif is sufficient to redirect the protein between apical, basolateral, and axonal localization. Our results demonstrate that axon-dendrite and apical-basolateral sorting pathways can coexist in a single cell, and suggest that subtle changes to short sequence motifs are sufficient to redirect proteins between these pathways.

Glia shape the development and function of the C. elegans nervous system, especially its sense organs and central neuropil (nerve ring). Cell-type-specific promoters allow investigators to label or manipulate individual glial cell types, and therefore provide a key tool for deciphering glial function. In this technical resource, we compare the specificity, brightness, and consistency of cell-type-specific promoters for C. elegans glia. We identify a set of promoters for the study of seven glial cell types (F16F9.3, amphid and phasmid sheath glia; F11C7.2, amphid sheath glia only; grl-2, amphid and phasmid socket glia; hlh-17, cephalic (CEP) sheath glia; and grl-18, inner labial (IL) socket glia) as well as a pan-glial promoter (mir-228). We compare these promoters to promoters that are expressed more variably in combinations of glial cell types (delm-1 and itx-1). We note that the expression of some promoters depends on external conditions or the internal state of the organism, such as developmental stage, suggesting glial plasticity. Finally, we demonstrate an approach for prospectively identifying cell-type-specific glial promoters using existing single-cell sequencing data, and we use this approach to identify two novel promoters specific to IL socket glia (col-53 and col-177).

The extracellular matrix (ECM) guides and constrains the shape of the nervous system. InC. elegans, DIG-1 is a giant ECM component that is required for fasciculation of sensory dendrites during development and for maintenance of axon positions throughout life. We identified four novel alleles ofdig-1in three independent screens for mutants affecting disparate aspects of neuronal and glial morphogenesis. First, we find that disruption of DIG-1 causes fragmentation of the amphid sheath glial cell in larvae and young adults. Second, it causes severing of the BAG sensory dendrite from its terminus at the nose tip, apparently due to breakage of the dendrite as animals reach adulthood. Third, it causes embryonic defects in dendrite fasciculation in inner labial (IL2) sensory neurons, as previously reported, as well as rare defects in IL2 dendrite extension that are enhanced by loss of the apical ECM component DYF-7, suggesting that apical and basolateral ECM contribute separately to dendrite extension. Our results highlight novel roles for DIG-1 in maintaining the cellular integrity of neurons and glia, possibly by creating a barrier between structures in the nervous system.

During convergent differentiation, multiple developmental lineages produce a highly similar or identical cell type. However, few molecular players that drive convergent differentiation are known. Here, we show that the C. elegans Forkhead transcription factor UNC-130 is required in only one of three convergent lineages that produce the same glial cell type. UNC-130 acts transiently as a repressor in progenitors and newly-born terminal cells to allow the proper specification of cells related by lineage rather than by cell type or function. Specification defects correlate with UNC-130:DNA binding, and UNC-130 can be functionally replaced by its human homolog, the neural crest lineage determinant FoxD3. We propose that, in contrast to terminal selectors that activate cell-type specific transcriptional programs in terminally differentiating cells, UNC-130 acts early and specifically in one convergent lineage to produce a cell type that also arises from molecularly distinct progenitors in other lineages.

Dendrites develop elaborate morphologies in concert with surrounding glia, but the molecules that coordinate dendrite and glial morphogenesis are mostly unknown.C. elegansoffers a powerful model for identifying such factors. Previous work in this system examined dendrites and glia that develop within epithelia, similar to mammalian sense organs. Here, we focus on the neurons BAG and URX, which are not part of an epithelium but instead form membranous attachments to a single glial cell at the nose, reminiscent of dendrite-glia contacts in the mammalian brain. We show that these dendrites develop by retrograde extension, in which the nascent dendrite endings anchor to the presumptive nose and then extend by stretch during embryo elongation. Using forward genetic screens, we find that dendrite development requires the adhesion protein SAX-7/L1CAM and the cytoplasmic protein GRDN-1/CCDC88C to anchor dendrite endings at the nose. SAX-7 acts in neurons and glia, while GRDN-1 acts in glia to non-autonomously promote dendrite extension. Thus, this work shows how glial factors can help to shape dendrites, and identifies a novel molecular mechanism for dendrite growth by retrograde extension.

Neuronal activity often leads to alterations in gene expression and cellular architecture. The nematode Caenorhabditis elegans, owing to its compact translucent nervous system, is a powerful system in which to study conserved aspects of the development and plasticity of neuronal morphology. Here we focus on one pair of sensory neurons, termed URX, which the worm uses to sense and avoid high levels of environmental oxygen. Previous studies have reported that the URX neuron pair has variable branched endings at its dendritic sensory tip. By controlling oxygen levels and analyzing mutants, we found that these microtubule-rich branched endings grow over time as a consequence of neuronal activity in adulthood. We also find that the growth of these branches correlates with an increase in cellular sensitivity to particular ranges of oxygen that is observable in the behavior of older worms. Given the strengths of C. elegans as a model organism, URX may serve as a potent system for uncovering genes and mechanisms involved in activity-dependent morphological changes in neurons and possible adaptive changes in the aging nervous system.

Neuronal activity often leads to alterations in gene expression and cellular architecture. The nematode Caenorhabditis elegans, owing to its compact translucent nervous system, is a powerful system in which to study conserved aspects of the development and plasticity of neuronal morphology. Here we focus on one sensory neuron in the worm, termed URX, which senses oxygen and signals tonically proportional to environmental oxygen. Previous studies have reported that URX has variable branched endings at its dendritic sensory tip. By controlling oxygen levels and analyzing mutants, we found that these branched endings grow over time as a consequence of neuronal activity. Furthermore, we observed that the branches contain microtubules, but do not appear to harbor the guanylyl cyclase GCY-35, a central component of the oxygen sensory transduction pathway. Interestingly, we found that although URX dendritic tips grow branches in response to long-term activity, the degree of branch elaboration does not correlate with oxygen sensitivity at the cellular or the behavioral level. Given the strengths of C. elegans as a model organism, URX may serve as a potent system for uncovering genes and mechanisms involved in activity-dependent morphological changes in neurons.

To sense the outside world, some neurons protrude across epithelia, the cellular barriers that line every surface of our bodies. To study the morphogenesis of such neurons, we examined theC. elegansamphid, in which dendrites protrude through a glial channel at the nose. During development, amphid dendrites extend by attaching to the nose via DYF-7, a type of protein typically found in epithelial apical ECM. Here, we show that amphid neurons and glia exhibit epithelial properties, including tight junctions and apical-basal polarity, and develop in a manner resembling other epithelia. We find that DYF-7 is a fibril-forming apical ECM component that prevents rupture of the tube-shaped glial channel, reminiscent of roles for apical ECM in other narrow epithelial tubes. We also identify a role for FRM-2, a homolog of EPBL15/moe/Yurt which promote epithelial integrity in other systems. Finally, we show that other environmentally-exposed neurons share a requirement for DYF-7. Together, our results suggest that these neurons and glia can be viewed as part of an epithelium continuous with the skin, and are shaped by mechanisms shared with other epithelia.

Biological systems are organized into well-ordered structures and can evolve new patterns when perturbed. To identify principles underlying biological order, we turned toC. elegansfor its simple anatomy and powerful genetics. We developed a method to quantify the arrangement of three dendrites in the main sensory nerve bundle, and found that they exhibit a stereotyped arrangement throughout larval growth. Dendrite order does not require prominent features including sensory cilia and glial junctions. In contrast, loss of the cell adhesion molecule (CAM) CDH-4/Fat-like cadherin causes dendrites to be ordered randomly, despite remaining bundled. Loss of the CAMs PTP-3/LAR or SAX-7/L1CAM causes dendrites to adopt an altered order, which becomes increasingly random as animals grow. Misexpression of SAX-7 leads to subtle but reproducible changes in dendrite order. Our results suggest that differential expression of CAMs allows dendrites to self-organize into a stereotyped arrangement that readily gives rise to new patterns when perturbed.

Neurons develop elaborate morphologies that provide a model for understanding cellular architecture. By studyingC. eleganssensory dendrites, we previously identified genes that act to promote the extension of ciliated sensory dendrites during embryogenesis. Interestingly, the nonciliated dendrite of the oxygen-sensing neuron URX is not affected by these genes, suggesting it develops through a distinct mechanism. Here, we use a visual forward genetic screen to identify mutants that affect URX dendrite morphogenesis. We find that disruption of the MAP kinase MAPK-15 or the βH-spectrin SMA-1 causes a phenotype opposite to what we had seen before: dendrites extend normally during embryogenesis but begin to overgrow as the animals reach adulthood, ultimately extending up to 150% of their normal length. SMA-1 is broadly expressed and acts non-cell-autonomously, while MAPK-15 is expressed in many sensory neurons including URX and acts cell-autonomously. MAPK-15 acts at the time of overgrowth, localizes at the dendrite ending, and requires its kinase activity, suggesting it acts locally in time and space to constrain dendrite growth. Finally, we find that the oxygen-sensing guanylate cyclase GCY-35, which normally localizes at the dendrite ending, is localized throughout the overgrown region, and that overgrowth can be suppressed by overexpressing GCY-35 or by genetically mimicking elevated cGMP signaling. These results suggest that overgrowth may correspond to expansion of a sensory compartment at the dendrite ending, reminiscent of the remodeling of sensory cilia or dendritic spines. Thus, in contrast to established pathways that promote dendrite growth during early development, our results reveal a distinct mechanism that constrains dendrite growth throughout the life of the animal, possibly by controlling the size of a sensory compartment at the dendrite ending.AUTHOR SUMMARYLewis Carroll’s Alice told the Caterpillar, “Being so many different sizes in a day is very confusing.” Like Alice, the cells of our bodies face a problem in size control – they must become the right size and remain that way throughout the life of the organism. This problem is especially relevant for nerve cells (neurons), as the lengths of their elaborate dendrites determine the connections they can make. To learn how neurons control their size, we turned not to a Caterpillar but to a worm: the microscopic nematodeC. elegans, in which single neurons can be easily visualized and the length of each dendrite is highly predictable across individuals. We focused on a single dendrite, that of the oxygen-sensing neuron URX, and we identified two genes that control its length. When these genes are disrupted, the dendrite develops correctly in embryos but then, like Alice, grows too much, eventually extending up to 1.5x its normal length. Thus, in contrast to known pathways that promote the initial growth of a dendrite early in development, our results help to explain how a neuron maintains its dendrite at a consistent length throughout the animal’s life.

Coordination of neurite morphogenesis with surrounding tissues is crucial to the establishment of neural circuits, but the underlying cellular and molecular mechanisms remain poorly understood. We show that neurons in a C. elegans sensory organ, called the amphid, undergo a collective dendrite extension to initiate formation of the sensory nerve. The amphid neurons first assemble into a multicellular rosette. The vertex of the rosette, which becomes the dendrite tips, is attached to the anteriorly migrating epidermis and carried to the sensory depression, extruding the dendrites away from the neuronal cell bodies. Multiple adhesion molecules including DYF-7, SAX-7, HMR-1 and DLG-1 function redundantly in rosette-to-epidermis attachment. PAR-6 is localized to the rosette vertex and dendrite tips, and promotes DYF-7 localization and dendrite extension. Our results suggest a collective mechanism of neurite extension that is distinct from the classical pioneer-follower model and highlight the role of mechanical cues from surrounding tissues in shaping neurites.

Biological systems are organized into well-ordered structures and can evolve new patterns when perturbed. To identify principles underlying biological order, we turned to C. elegans for its simple anatomy and powerful genetics. We developed a method to quantify the arrangement of three dendrites in the main sensory nerve bundle, and found that they exhibit a stereotyped arrangement throughout larval growth. Dendrite order does not require prominent features including sensory cilia and glial junctions. In contrast, loss of the cell adhesion molecule (CAM) CDH-4/Fat-like cadherin causes dendrites to be ordered randomly, despite remaining bundled. Loss of the CAMs PTP-3/LAR or SAX-7/L1CAM causes dendrites to adopt an altered order, which becomes increasingly random as animals grow. Misexpression of SAX-7 leads to subtle but reproducible changes in dendrite order. Our results suggest that combinations of CAMs allow dendrites to self-organize into a stereotyped arrangement and can produce altered patterns when perturbed.

A major goal in the study of human diseases is to assign functions to genes or genetic variants. The model organism Caenorhabditis elegans provides a powerful tool because homologs of many human genes are identifiable, and large collections of genetic vectors and mutant strains are available. However, the delivery of such vector libraries into mutant strains remains a long-standing experimental bottleneck for phenotypic analysis. Here, we present a computer-assisted microinjection platform to streamline the production of transgenic C. elegans with multiple vectors for deep phenotyping. Briefly, animals are immobilized in a temperature-sensitive hydrogel using a standard multiwell platform. Microinjections are then performed under control of an automated microscope using precision robotics driven by customized computer vision algorithms. We demonstrate utility by phenotyping the morphology of 12 neuronal classes in six mutant backgrounds using combinations of neuron-type-specific fluorescent reporters. This technology can industrialize the assignment of in vivo gene function by enabling large-scale transgenic engineering.

Glia constitute a major, understudied population of cells in the nervous system. Currently, it is appreciated that these cells exhibit vast morphological, functional, and molecular diversity, but our understanding of glial biology is limited. Some key unanswered questions include how glial diversity is generated during development and what functions distinct glial subtypes serve in the mature nervous system. The nematode Caenorhabditis elegans contains a defined set of glia, which have clear morphological and molecular differences, and thus provides a simplified model for understanding glial diversity. In addition, recent experiments suggest that the molecular mechanisms underlying the generation of glial diversity in C. elegans are conserved with those in mammals. In this review, we summarize the surprising diversity of glial subtypes present in this simple organism, and highlight current thinking about what roles they perform in the nervous system. We emphasize how genetic approaches may be used to identify the mechanistic origins of glial diversity, which is key to understanding how glia function in health and disease. WIREs Dev Biol 2015, 4:151–160. doi: 10.1002/wdev.171 For further resources related to this article, please visit the WIREs website. Conflict of interest: The authors have declared no conflicts of interest for this article.

Black Redhorse (Moxostoma duquesnei) larval and juvenile habitat was characterized in the Grand River, Ontario from June to September 2007–2012. Similar to adult Black Redhorse and their congeners, larval Black Redhorse were most likely to be located in clean, clear, stable runs with low to moderate flow, over pebble, gravel and cobble substrate, mixed with sand. Areas (n = 22) where 0+ Black Redhorse were observed and collected were 1.4 ± 0.2 m from shore, with a mean water temperature of 22.0 ± 0.5 °C, mean depth of 0.20 ± 0.02 m and mean water velocity of 0.12 ± 0.05 m/s. Larval and juvenile Black Redhorse occupied riffles, runs, pools and backwater areas; however, there was a strong preference for runs. Juvenile Black Redhorse moved upstream in the early evening and at night to overwintering areas in the Grand River in November when water temperature approached 5 °C. The persistence of Black Redhorse populations in the Grand River may be related to the presence of groundwater, which provides refuge from extreme temperature and poor water quality during the summer.

Primary cilia are ubiquitous sensory organelles that mediate diverse signaling pathways. Cilia position on the cell surface is determined by the location of the basal body (BB) that templates the cilium. The mechanisms that regulate BB positioning in the context of ciliogenesis are largely unknown. Here we show that the conserved signaling and scaffolding protein Girdin localizes to the proximal regions of centrioles and regulates BB positioning and ciliogenesis in Caenorhabditis elegans sensory neurons and human RPE-1 cells. Girdin depletion alters localization of the intercentriolar linker and ciliary rootlet component rootletin, and rootletin knockdown in RPE-1 cells mimics Girdin-dependent phenotypes. C. elegans Girdin also regulates localization of the apical junction component AJM-1, suggesting that in nematodes Girdin may position BBs via rootletin- and AJM-1-dependent anchoring to the cytoskeleton and plasma membrane, respectively. Together, our results describe a conserved role for Girdin in BB positioning and ciliogenesis.

A dendrite grows by sprouting filopodia, some of which mature into stable dendrite branches that bear synapses and sprout filopodia of their own. Recent work has shown that a filopodium begins deciding to become a stable branch within one minute of contacting a presynaptic partner, but what triggers this decision remains unknown. We consider the evidence for three possible triggers: activity of neurotransmitter receptors, signaling through adhesion proteins, and heightened membrane tension as the filopodium attempts to retract but is held in place by adhesive contacts with the target. Of these, membrane tension-induced signaling is especially appealing, as it would serve as a general reporter of attachment, independent of which specific adhesion molecules are used.

Simple cell-cell interactions can give rise to complex cellular patterns. For example, neurons of the same type can interact to create a complex patchwork of non-overlapping dendrite arbors, a pattern known as dendrite tiling. Dendrite tiling often involves mutual repulsion between neighboring neurons. While dendrite tiling is found across nervous systems, the nematode Caenorhabditis elegans has a relatively simple nervous system with few opportunities for tiling. Here, we show that genetic duplication of a single neuron, PVD, is sufficient to create dendrite tiling among the resulting ectopic neurons. We use laser ablation to show that this tiling is mediated by mutual repulsion between neighbors. Furthermore, we find that tiling requires a repulsion signal (UNC-6/Netrin and its receptors UNC-40/DCC and UNC-5) that normally patterns the PVD dendrite arbor. These results demonstrate that an apparently complex cellular pattern can emerge in a simple nervous system merely by increasing neuron number.

During development, biomechanical forces contour the body and provide shape to internal organs. Using genetic and molecular approaches in combination with a FRET-based tension sensor, we characterized a pulling force exerted by the elongating pharynx (foregut) on the anterior epidermis during C. elegans embryogenesis. Resistance of the epidermis to this force and to actomyosin-based circumferential constricting forces is mediated by FBN-1, a ZP domain protein related to vertebrate fibrillins. fbn-1 was required specifically within the epidermis and FBN-1 was expressed in epidermal cells and secreted to the apical surface as a putative component of the embryonic sheath. Tiling array studies indicated that fbn-1 mRNA processing requires the conserved alternative splicing factor MEC-8/RBPMS. The conserved SYM-3/FAM102A and SYM-4/WDR44 proteins, which are linked to protein trafficking, function as additional components of this network. Our studies demonstrate the importance of the apical extracellular matrix in preventing mechanical deformation of the epidermis during development. DOI: http://dx.doi.org/10.7554/eLife.06565.001, eLife digest For an animal embryo to develop, its cells must organize themselves into tissues and organs. For example, skin and the lining of internal organs—such as the lungs and gut—are made from cells called epithelial cells, which are tightly linked to form flat sheets. In a microscopic worm called Caenorhabditis elegans, the outermost layer of epithelial cells (called the epidermis) forms over the surface of the embryo early on in embryonic development. Shortly afterwards, the embryonic epidermis experiences powerful contractions along the surface of the embryo. The force generated by these contractions converts the embryo from an oval shape to a roughly cylindrical form. These contractions also squeeze the internal tissues and organs, which correspondingly elongate along with the epidermis. It has been known for decades that such ‘mechanical’ forces are important for the normal development of embryos. However, it remains poorly understood how these forces generate tissues and organs of the proper shape—partly because it is difficult to measure forces in living embryos. It is also not clear how the mechanical properties of specific tissues are controlled. Now, Kelley, Yochem, Krieg et al. have analyzed the development of C. elegans' embryos and discovered a novel mechanical interplay between the feeding organ (called the pharynx) and the worm's epidermis. The experiments involved studying several mutant worms that perturb epidermal contractions and disrupt the attachment of the pharynx to the epidermis. These studies suggested that the pharynx exerts a strong inward pulling force on the epidermis during development. Using recently developed methods, Kelley, Yochem, Krieg et al. then measured mechanical forces within intact worm embryos and demonstrated that greater forces were experienced in cells that were being pulled by the pharynx. Kelley, Yochem, Krieg et al. further analyzed how the epidermis normally resists this pulling force from the pharynx and implicated a protein called FBN-1. This worm protein is structurally related to a human protein that is affected in people with a disorder called Marfan Syndrome. Worm embryos without the FBN-1 protein become severely deformed because they are unable to withstand mechanical forces at the epidermis. FBN-1 is normally synthesized and then transported to the outside of the worm embryo by epidermal cells, where it is thought to assemble into a meshwork of long fibers. This provides a strong scaffold that attaches to the epidermis to prevent the epidermis from undergoing excessive deformation while it experiences mechanical forces. The work of Kelley, Yochem, Krieg et al. provides an opportunity to understand how FBN-1 and other fiber-forming proteins are produced and transported to the cell surface. Moreover, these findings may have implications for human diseases and birth defects that result from an inability of tissues to respond appropriately to mechanical forces. DOI: http://dx.doi.org/10.7554/eLife.06565.002

Glia constitute a major, understudied population of cells in the nervous system. Currently, it is appreciated that these cells exhibit vast morphological, functional, and molecular diversity, but our understanding of glial biology is limited. Some key unanswered questions include how glial diversity is generated during development and what functions distinct glial subtypes serve in the mature nervous system. The nematode Caenorhabditis elegans contains a defined set of glia, which have clear morphological and molecular differences, and thus provides a simplified model for understanding glial diversity. In addition, recent experiments suggest that the molecular mechanisms underlying the generation of glial diversity in C. elegans are conserved with those in mammals. In this review, we summarize the surprising diversity of glial subtypes present in this simple organism, and highlight current thinking about what roles they perform in the nervous system. We emphasize how genetic approaches may be used to identify the mechanistic origins of glial diversity, which is key to understanding how glia function in health and disease. For further resources related to this article, please visit the WIREs website.The authors have declared no conflicts of interest for this article.

The ciliated receptive endings of sensory cells and the dendrites of other neurons are shaped by adhesive interactions, many of which depend on machinery also present in epithelia. Sensory cells are shaped by interactions with support cells through adhesion junctions via the Crumbs complex, tight junction components such as claudins, as well as interactions with apical extracellular matrix composed of zona pellucida domain proteins. Neuronal dendrites are shaped by adhesion machinery that includes cadherins, catenins, afadin, L1CAM, CHL1, Sidekicks, Contactin and Caspr, many of which are shared with epithelia. This review highlights this shared machinery, and suggests that mechanisms of epithelial morphogenesis may thus provide a guide to understanding dendrite morphogenesis.

The regulatory protein calmodulin is a major mediator of calcium-induced changes in cellular activity. To analyze the roles of calmodulin in an intact animal, we have generated a calmodulin null mutation in Drosophila melanogaster. Maternal calmodulin supports calmodulin null individuals throughout embryogenesis, but they die within 2 days of hatching as first instar larvae. We have detected two pronounced behavioral abnormalities specific to the loss of calmodulin in these larvae. Swinging of the head and anterior body, which occurs in the presence of food, is three times more frequent in the null animals. More strikingly, most locomotion in calmodulin null larvae is spontaneous backward movement. This is in marked contrast to the wild-type situation where backward locomotion is seen only as a stimulus-elicited avoidance response. Our finding of spontaneous avoidance behavior has striking similarities to the enhanced avoidance responses produced by some calmodulin mutations in Paramecium. Thus our results suggest evolutionary conservation of a role for calmodulin in membrane excitability and linked behavioral responses.

Under mating conditions, yeast cells adopt a characteristic pear-shaped morphology, called a “shmoo,” as they project a cell extension toward their mating partners. Mating partners make contact at their shmoo tips, dissolve the intervening cell wall, and fuse their plasma membranes. We identified mutations in ERG4 , encoding the enzyme that catalyzes the last step of ergosterol biosynthesis, that impair both shmoo formation and cell fusion. Upon pheromone treatment, erg4 Δ mutants polarized growth, lipids, and proteins involved in mating but did not form properly shaped shmoos and fused with low efficiency. Supplementation with ergosterol partially suppressed the shmooing defect but not the cell fusion defect. By contrast, removal of the Erg4 substrate ergosta-5,7,22,24(28)-tetraenol, which accumulates in erg4 Δ mutant cells and contains an extra double bond in the aliphatic chain of the sterol, restored both shmooing and cell fusion to wild-type levels. Thus, a two-atom change in the aliphatic moiety of ergosterol is sufficient to obstruct cell shape remodeling and cell fusion.

The nematode Caenorhabditis elegans has a simple nervous system with glia restricted primarily to sensory organs. Some of the activities that would be provided by glia in the mammalian nervous system are either absent or provided by non-glial cell types in C. elegans, with only a select set of mammalian glial activities being similarly provided by specialized glial cells in this animal. These observations suggest that ancestral roles of glia may be to modulate neuronal morphology and neuronal sensitivity in sensory organs.

A relatively short highway construction season exists on the Canadian prairies and typically the paving season extends from May to October. Highway engineers and contractors are continually confronted with unfavorable weather conditions during the early spring and latter parts of the fall paving season. During these periods of cooler weather, the decision as to whether or not to allow paving to continue or begin becomes very difficult, since freshly laid pavements cool quickly and proper compaction is difficult to attain. The final density obtained in a pavement depends critically on its initial temperature and cooling rate. This study researches the subject of pavement cooling and investigates the feasibility of cold-weather paving. The results of this study illustrate the significance of the factors affecting pavement cooling and provide information for improving the paving specifications used by Saskatchewan Highways and Transportation. This study also shows that the short paving season which exists in Saskatchewan can be extended under certain conditions. Extension of the construction season into colder weather will provide financial and various other benefits, not only to Saskatchewan Highways and Transportation but also to paving contractors and the general public. Key words: pavement cooling, pavement temperature, computer simulation, compaction, asphalt concrete.

The molecular machines that mediate cell fusion are unknown. Previously, we identified a multispanning transmembrane protein, Prm1 (pheromone-regulated membrane protein 1), that acts during yeast mating (Heiman, M.G., and P. Walter. 2000. J. Cell Biol. 151:719–730). Without Prm1, a substantial fraction of mating pairs arrest with their plasma membranes tightly apposed yet unfused. In this study, we show that lack of the Golgi-resident protease Kex2 strongly enhances the cell fusion defect of Prm1-deficient mating pairs and causes a mild fusion defect in otherwise wild-type mating pairs. Lack of the Kex1 protease but not the Ste13 protease results in similar defects. Δkex2 and Δkex1 fusion defects were suppressed by osmotic support, a trait shared with mutants defective in cell wall remodeling. In contrast, other cell wall mutants do not enhance the Δprm1 fusion defect. Electron microscopy of Δkex2-derived mating pairs revealed novel extracellular blebs at presumptive sites of fusion. Kex2 and Kex1 may promote cell fusion by proteolytically processing substrates that act in parallel to Prm1 as an alternative fusion machine, as cell wall components, or both.

Cell fusion occurs throughout development, from fertilization to organogenesis. The molecular mechanisms driving plasma membrane fusion in these processes remain unknown. While yeast mating offers an excellent model system in which to study cell fusion, all genes previously shown to regulate the process act at or before cell wall breakdown; i.e., well before the two plasma membranes have come in contact. Using a new strategy in which genomic data is used to predict which genes may possess a given function, we identified PRM1, a gene that is selectively expressed during mating and that encodes a multispanning transmembrane protein. Prm1p localizes to sites of cell-cell contact where fusion occurs. In matings between Deltaprm1 mutants, a large fraction of cells initiate zygote formation and degrade the cell wall separating mating partners but then fail to fuse. Electron microscopic analysis reveals that the two plasma membranes in these mating pairs are tightly apposed, remaining separated only by a uniform gap of approximately 8 nm. Thus, the phenotype of Deltaprm1 mutants defines a new step in the mating reaction in which membranes are juxtaposed, possibly through a defined adherence junction, yet remain unfused. This phenotype suggests a role for Prm1p in plasma membrane fusion.

This paper reports evidence for a possible "chromosome 13 syndrome," which includes panic disorder, kidney or bladder problems, serious headaches, thyroid problems (usually hypothyroid), and/or mitral valve prolapse (MVP). In the course of a genetic linkage study of panic disorder, we noted these medical conditions in individual family members. (We were blind to family relationships and marker data.) We hypothesized that there may exist a subgroup of panic families with these medical conditions, which for simplicity we called it the "syndrome." Subsequently we reclassified the families as with or without the "syndrome" and extended the phenotype for analysis to include the above medical conditions. All these classifications were also done before the analysis and blind to marker data. We then examined our linkage results, looking for significant differences between families with and without the "syndrome" (using several definitions of the "syndrome")-i.e., testing for genetic heterogeneity. When the families with and without bladder/kidney problems were separated from each other, one marker-D13S779 (ATA26D07)-yielded a lod score of over 3 in the families with bladder/kidney problems. This lod score went up to 4.2 in these families when we diagnosed any individual with any one of the "syndrome" conditions as affected. These results were statistically significant even after applying an extremely overconservative Bonferroni correction for multiple tests. We present these results in order to alert other investigators working on panic disorder, for replication. If replicated, one may hypothesize that a candidate gene for the syndrome should be expressed in CNS, kidney, gut, thyroid, etc. We also noted that two independent studies report recent linkage findings between schizophrenia and the same region on chromosome 13. No connection between schizophrenia and panic disorder has ever been reported. Finally, we suggest that genetic studies of psychiatric disorders might prove more fruitful if phenotypes were expanded to include possible manifestations of the disorder in medical (non-mental) symptoms. Am. J. Med. Genet.(Neuropsychiatr. Genet.) 96:24-35, 2000.

Calmodulin (CAM) is recognized as a major intermediary in intracellular calcium signaling, but as yet little is known of its role in developmental and behavioral processes. We have generated and studied mutations to the endogenous Cam gene of Drosophila melanogaster that change single amino acids within the protein coding region. One of these mutations produces a striking pupal lethal phenotype involving failure of head eversion. Various mutant combinations produce specific patterns of ectopic wing vein formation or melanotic scabs on the cuticle. Anaphase chromosome bridging is also seen as a maternal effect during the early embryonic nuclear divisions. In addition, specific behavioral defects such as poor climbing and flightlessness are detected among these mutants. Comparisons with other Drosophila mutant phenotypes suggests potential CAM targets that may mediate these developmental and behavioral effects, and analysis of the CAM crystal structure suggests the structural consequences of the individual mutations.

A 3-year-old boy of German descent suffered from two episodes of Streptococcus pneumoniae meningitis within 2 months. One month previously, the first skin lesion of Kaposi sarcoma (KS) had been observed behind his right ear. During the following 2 years KS disseminated not only mucocutaneously but also to visceral organs. Immunological evaluation revealed severe lymphocytopenia with reduced helper/suppressor T-cell ratio and impaired humoral immune response to pneumococci. Extensive laboratory tests gave no evidence for known immunocompromising infections. However, recently described DNA sequences from a Kaposi sarcoma-associated herpesvirus (KSHV) could be identified within skin tissue. As chemotherapy failed to stop tumour progression the patient was referred for bone marrow transplantation. Eighteen months later the KS is in remission and the patient in a good general condition. Conclusion The case supports the hypothesis that KSHV is involved in the aetiology of KS. Bone marrow transplantation is possibly a therapeutic option for KS in patients with immunodeficiency not related to human immunodeficiency virus infection.

Objective: To identify common genetic variants associated with Gilles de la Tourette Syndrome (GTS). Background GTS is a highly heritable, neurodevelopmental disorder, though discovery of definitive GTS susceptibility genes has been challenging. Here, we report the first genome-wide association study (GWAS) of GTS in 1496 cases and 5249 unselected, ancestry-matched controls. Design/Methods: A multi-center case-control study was conducted with subjects of general European ancestry (EU), two European-derived population isolates, Ashkenazi Jews (AJ) and French Canadians (FC), and two closely-related Latin American population isolates from Antioquia, Colombia (ANT) and the Central Valley of Costa Rica (CVCR). Diagnoses were made using DSM-IV-TR criteria. 908 EU cases, all population-isolate cases, 298 EU controls and all FC and ANT controls were genotyped on the Illumina Hap610 platform. An additional 148 EU cases were genotyped on the IlluminaHap370, and remaining EU and AJ controls were previously genotyped on Illumina Hap550K and Hap1M arrays. Quality control (QC) was conducted in PLINK with specific focus on removing SNPs with differential performance across genotyping platforms. Ancestry-stratified case-control analyses were performed in PLINK using logistic regression. Case-weighted meta-analyses were conducted in METAL. Results: and Conclusions: In the primary meta-analysis of European-derived samples (EU, AJ, FC), no loci surpassed the threshold for genome-wide significance (p -8 ). However, the top 535 loci (p COL27A1 gene on chromosome 9 (rs7868992) with a genome-wide significant p-value =2.9 x 10 -8 . Additional neurodevelopmental genes of interest among the top signals included SLITRK6 , CNTN1 , and SEMA3G . Conclusions: Replication in additional samples is underway to validate the findings and to identify additional GTS susceptibility loci. Supported by: A grant from the Judah Foundation, NIH grants NS40024 to DLP and the Tourette Syndrome Association International Consortium for Genetics, NIH grant NS16648 and a grant from the Tourette Syndrome Association to DLP, NIH grant NS037484 to NBF, NIH grant NS043538 to AR-L, and an American Academy of Neurology Foundation grant and NIH grant MH085057 to JMS. Disclosure: Dr. Scharf has nothing to disclose. Dr. Yu has nothing to disclose. Dr. Mathews has nothing to disclose. Dr. Neale has nothing to disclose. Dr. Stewart has nothing to disclose. Dr. Fagerness has nothing to disclose. Dr. Scharf has nothing to disclose. Dr. Scharf has nothing to disclose. Dr. Scharf has nothing to disclose. Dr. Osiecki has nothing to disclose. Dr. Illmann has nothing to disclose. Dr. Cath has nothing to disclose. Dr. King has nothing to disclose. Dr. Dion has received personal compensation for activities with Biovail Pharma, Pfizer and Eli Lilly. Dr. Sandor has received personal compensation for activities with Psyadon, Shire, Solway, UCB Pharma, Janssen, Eli Lilly, Pfizer, and Prestwick. Dr. Barr has nothing to disclose. Dr. Budman has received research support from Psyadon Pharmaceuticals and Otsuka Pharmaceuticals. Dr. Lyon has nothing to disclose. Dr. Hoekstra has received personal compensation for activities with Desitin, Eli Lilly & Company and Shire. Dr. Singer has nothing to disclose. Dr. Jankovic has received personal compensation for activities with Allergan, Inc., Chelsea Therapeutics, Serono Inc., Merz Pharma, Lundbeck Research USA, Inc, Teva Neuroscience as a consultant. Dr. Jankovic has received personal compensation in an editorial capacity for Medlink: Neurology in Clinical Practice. Dr. Jankovic has received research support from Allergan, Inc, Allon Therapeutics, Ceregene, Inc., Chelsea Therapeutics; Diana Helis Henry Medical Research Foundation, Serono Inc., Huntington9s Disease Society of America, Huntington Study Group, Impax Pharmaceuticals, Ipsen Limited, Lundbeck Research USA, Inc. Dr. Gilbert has received personal compensation in an editorial capacity for Pediatrics Review and Education Program R11. Dr. Gilbert has received research support from Psyadon Pharmaceuticals and Otsuka Pharmaceuticals. Dr. Hoekstra has received personal compensation for activities with Desitin, Eli Lilly & Company and Shire. Dr. Heiman has nothing to disclose. Dr. Tischfield has nothing to disclose. Dr. State has nothing to disclose. Dr. Robertson has nothing to disclose. Dr. Kurlan has nothing to disclose. Dr. Ophoff has nothing to disclose. Dr. Gibbs has nothing to disclose. Dr. Cookson has nothing to disclose. Dr. Hardy has nothing to disclose. Dr. Singleton has nothing to disclose. Dr. Rodriguez has nothing to disclose. Dr. Rouleau has nothing to disclose. Dr. Heutink has nothing to disclose. Dr. Oostra has nothing to disclose. Dr. McMahon has nothing to disclose. Dr. Freimer has nothing to disclose. Dr. COX has nothing to disclose. Dr. Pauls has nothing to disclose.

Neurons are the pedestals of human consciousness, yet from a cell biological view they are little more than specialized epithelia. Indeed, as highlighted in the “Neuronal Development and Degeneration” Minisymposium, neurons inhabit conventional cell biology at an extreme: in their diversity of cell types, the specificity of their cell–cell junctions and cell–cell signaling, their degree of asymmetric polarization, their rate and volume of membrane traffic, the size and stability of their cytoskeletal assemblies, and their metabolic activity and ion flux. This extreme lifestyle requires extreme upkeep and, although neurons can be extreme in their longevity, if any of the above processes are compromised then neurodegenerative diseases can ensue. This session highlighted what can be learned by studying cell biology at the extremes, beginning with neuronal development and ending in studies of neurodegenerative disease.

At the CERN SPS we have measured a deep inelastic μ± cross section asymmetry at momentum transfers Q2 ranging from 15 to 180GeV2. The result is in quantitative agreement with the WS/GIM standard electroweak model and allows to determine the muon neutral current couplings.

New data is presented on the ratios of structure functions F 2 ( x , Q 2 ) measured in deep inelastic muon scattering with deuterium, nitrogen, and iron targets. The existence of nuclear effects at large Q 2 is confirmed with improved systematic accuracy. The ratio F 2 Fe ( x ) F 2 D 2 ( x ) covers the range 0.20 ⩽ x ⩽ 0.70 and is in agreement with earlier measurements. The ratio F 2 N 2 ( x )/ F 2 D 2 ( x ) is measured over the range 0.08 ⩽ x ⩽ 0.70 and is compatible with unity below x = 0.3.

The polarization of theM2 muon beam at the CERN SPS is studied at 220/200, 260/240, 300/280 GeV/c π+/μ+ momenta by using a lead-glass telescope and a simple trigger system to measure the positron spectrum from muon decay. A direct measurement of the polarization difference between the 220/200 GeV/c beam (forward decays, average polarization ∼−0.8) and the 300/200 GeV/c beam (backward decays, average polarization ∼+0.1) is also presented.

A 50 m long magnetized iron torus enclosing a 40 m long target provides the luminosity and acceptance necessary for the study of deep inelastic muon scattering at high Q 2 . The construction and performance of this spectrometer and the associated trigger are described. Details of the data acquisition system and data analysis are also given.

The high mass μ + μ − pairs produced by 280 GeV μ + on a carbon target are studied in a search for the Y production. The high mass continuum in the region 2–18 GeV is interpreted in terms of QED pair production and of μ pairs originating from the decay of hidden and open charm particles as well as of hadrons ( π , K) from deep inelastic interactions. The upper limit for the upsilon production by muons is found to be, at the 90% confidence level, σ γ ·(γ→μ + μ − ) −39 cm 2 /nucleon.

The interaction length of a particle can be measured directly by studying the attenuat ion of a particle beam in a material. The number of particles which have not interacted after a depth z of absorber is given by .N(z) = .N0 exp [-z/k], .N o being the incoming flux. In an unseparated negative high-energy beam, pions predominate, and the number of particles which have not suffered an interaction is given by

The lateral development of hadronic and electromagnetic showers has been measured in an iron-scintillator calorimeter equipped with 2 cm wide hodoscopes at 20, 40 and 86 GeV c−1.

Positrons from muon decay in the M2 beam line at the CERN SPS have been identified using a 34 r.l. lead-glass detector operated in coincidence with a synchrotron radiation tag.

The energy-loss spectrum of high energy muons after traversal of a 10 meter iron degrader has been measured with an accuracy of better than 1%. Data taken at energies between 50 and 120 GeV are in agreement with predictions from a Monte Carlo program based on the most recent theoretical descriptions of all relevant processes. The stopping power calculated by this program deviates up to several percent from earlier calculations.

Deep inelastic scattering cross sections have been measured with the CERN SPS muon beam at incident energies of 120 and 200 GeV. Approximately 10000 events at each energy used to obtain the structure function F/sub 2/(x, Q/sup 2/) in the kinematic region 0.3

SummaryCells are devices whose structures delimit function. For example, in the nervous system, neuronal and glial shapes dictate paths of information flow. To understand how cells acquire their shapes, we examined the formation of a sense organ in C. elegans. Using time-lapse imaging, we found that sensory dendrites form by stationary anchoring of dendritic tips during cell-body migration. A genetic screen identified DEX-1 and DYF-7, extracellular proteins required for dendritic tip anchoring, which act cooperatively at the time and place of anchoring. DEX-1 and DYF-7 contain, respectively, zonadhesin and zona pellucida domains, and DYF-7 self-associates into multimers important for anchoring. Thus, unlike other dendrites, amphid dendritic tips are positioned by DEX-1 and DYF-7 without the need for long-range guidance cues. In sequence and function, DEX-1 and DYF-7 resemble tectorins, which anchor stereocilia in the inner ear, suggesting that a sensory dendrite anchor may have evolved into part of a mechanosensor.