A glial K+/Cl− cotransporter modifies temperature‐evoked dynamics in Caenorhabditis elegans sensory neurons

K⁺/Cl^? cotransporters (KCCs) are known to be crucial in the control of neuronal electrochemical Cl^? gradient. However, the role of these proteins in glial cells remains largely unexplored despite a number of studies showing expression of KCC proteins in glial cells of many species. Here, we show that the Caenorhabditis elegans K⁺/Cl^? cotransporter KCC‐3 is expressed in glial‐like cells and regulates the thermosensory behavior through modifying temperature‐evoked activity of a thermosensory neuron. Mutations in the kcc‐3 gene were isolated from a genetic screen for mutants defective in thermotaxis. KCC‐3 is expressed and functions in the amphid sheath glia that ensheathes the AFD neuron, a major thermosensory neuron known to be required for thermotaxis. A genetic analysis indicated that the regulation of the thermosensory behavior by KCC‐3 is mediated through AFD, and we further show that KCC‐3 in the amphid sheath glia regulates the dynamics of the AFD activity. Our results show a novel mechanism by which the glial KCC‐3 protein non‐cell autonomously modifies the stimulus‐evoked activity of a sensory neuron and highlights the functional importance of glial KCC proteins in modulating the dynamics of a neural circuitry to control an animal behavior.     

Amphid defective mutant ofCaenorhabditis elegans

Studies are reported on a chemoreception mutant which arose in a mutator strain. The mutant sensory neurons do not stain with fluoresceine isothiocyanate (Dyf phenotype), hence the name,dyf-1, given to the gene it identifies. The gene maps on LGI, 0.4 map units fromdpy-5 on theunc-11 side. The response of mutant worms to various repellents has been studied and shown to be partially altered. Other chemoreception based behaviors are less affected. The cilia of the sensory neurons of the amphid are shorter than normal and the primary defect may be in the capacity of the sheath cells to secrete the matrix material that fills the space between cilia in the amphid channel. Progress toward the molecular cloning of the gene is also reported. Relevant results from other laboratories are briefly reviewed.     

Mutant sensory cilia in the nematode Caenorhabditis elegans

Eight classes of chemosensory neurons in C. elegans fill with fluorescein when living animals are placed in a dye solution. Fluorescein enters the neurons through their exposed sensory cilia. Mutations in 14 genes prevent dye uptake and disrupt chemosensory behaviors. Each of these genes affects the ultrastructure of the chemosensory cilia or their accessory cells. In each case, the cilia are shorter or less exposed than normal, suggesting that dye contact is the principal factor under selection. Ten genes affect many or all of the sensory cilia in the head. The daf-19 (m86) mutation eliminates all cilia, leaving only occasional centrioles in the dendrites. The cilia in che-13 (e1805), osm-1 (p808), osm-5 (p813), and osm-6 (p811) mutants have normal transition zones and severely shortened axonemes. Doublet-microtubules, attached to the membrane by Y links, assemble ectopically proximal to the cilia in these mutants. The amphid cilia in che-11 (e1810) are irregular in diameter and contain dark ground material in the middle of the axonemes. Certain mechanocilia are also affected. The amphid cilia in che-10 (e1809) apparently degenerate, leaving dendrites with bulb-shaped endings filled with dark ground material. The mechanocilia lack striated rootlets. Cilia defects have also been found in che-2, che-3, and daf-10 mutants. The osm-3 (p802) mutation specifically eliminates the distal segment of the amphid cilia. Mutations in three genes affect sensillar support cells. The che-12 (e1812) mutation eliminates matrix material normally secreted by the amphid sheath cell. The che-14 (e1960) mutation disrupts the joining of the amphid sheath and socket cells to form the receptor channel. A similar defect has been observed in daf-6 mutants. Four additional genes affect specific classes of ciliated sensory neurons. The mec-1 and mec-8 (e398) mutations disrupt the fasciculation of the amphid cilia. The cat-6 (e1861) mutation disrupts the tubular bodies of the CEP mechanocilia. A cryophilic thermotaxis mutant, ttx-1 (p767), lacks fingers on the AFD dendrite, suggesting this neuron is thermosensory.     

Two Heteromeric Kinesin Complexes in Chemosensory Neurons and Sensory Cilia of Caenorhabditis elegans

Chemosensation in the nervous system of the nematode Caenorhabditis elegans depends on sensory cilia, whose assembly and maintenance requires the transport of components such as axonemal proteins and signal transduction machinery to their site of incorporation into ciliary structures. Members of the heteromeric kinesin family of microtubule motors are prime candidates for playing key roles in these transport events. Here we describe the molecular characterization and partial purification of two heteromeric kinesin complexes from C. elegans, heterotrimeric CeKinesin-II and dimeric CeOsm-3. Transgenic worms expressing green fluorescent protein driven by endogenous heteromeric kinesin promoters reveal that both CeKinesin-II and CeOsm-3 are expressed in amphid, inner labial, and phasmid chemosensory neurons. Additionally, immunolocalization experiments on fixed worms show an intense concentration of CeKinesin-II and CeOsm-3 polypeptides in the ciliated endings of these chemosensory neurons and a punctate localization pattern in the corresponding cell bodies and dendrites. These results, together with the phenotypes of known mutants in the pathway of sensory ciliary assembly, suggest that CeKinesin-II and CeOsm-3 drive the transport of ciliary components required for sequential steps in the assembly of chemosensory cilia.     

Ultrastructure of aesthetasc innervation and external morphology of the lateral antennule setae of the spiny lobster Panulirus interruptus (Randall)

Summary Six types of setae and one type of cuticular depression were examined on the lateral antennule of the spiny lobster Panulirus interruptus using scanning electron microscopy. The organization and ultrastructure of the innervation of the most numerous setal type, the aesthetasc, were investigated using light-and transmission electron microscopy.Each aesthetasc is innervated by approximately 300 bipolar neurons whose sensory dendrites penetrate the hair and extend toward the tip, and whose axons project towards the central nervous system. The neuronal somata and two types of glia form a cluster within the antennular lumen. The inner sheath-cell somata encircle the dendritic tract distal to the sensory somata. These cells appear to extend distal processes which wrap the dendritic tract to the base of the aesthetasc. Elongate outer sheath cells are interposed between the glia-wrapped dendritic tract and the hypodermis which underlies the antennule cuticle. A continuous investment of neural lamella separates the hypodermis, the entire cluster of somata, and sensillar nerve from the antennule lumen. The organization of the neuronal somata and their association with outer and inner sheath cells in this marine species appear similar to those of crustaceans from freshwater and terrestrial habitats.     

Volume transmission in activity-dependent regulation of myelinating glia

The importance of neural impulse activity in regulating neuronal plasticity is widely appreciated; increasingly, it is becoming apparent that activity-dependent communication between neurons and glia is critical in regulating many aspects of nervous system development and plasticity. This communication takes place not only at the synapse, but also between premyelinating axons and glia, which form myelin in the PNS and CNS. Recent work indicates that neural impulse activity releases ATP and adenosine from non-synaptic regions of neurons, which activates purinergic receptors on myelinating glia. Acting through this receptor system, neural impulse activity can regulate gene expression, mitosis, differentiation, and myelination of Schwann cells (SCs) and oligodendrocytes, helping coordinate nervous system development with functional activity in the perinatal period. ATP and adenosine have opposite effects on differentiation of Schwann cells and oligodendrocytes, providing a possible explanation for the opposite effects of impulse activity reported on myelination in the CNS and PNS.     

Functional redundancy of the B9 proteins and nephrocystins in Caenorhabditis elegans ciliogenesis

Meckel-Gruber syndrome (MKS), nephronophthisis (NPHP), and Joubert syndrome (JBTS) are a group of heterogeneous cystic kidney disorders with partially overlapping loci. Many of the proteins associated with these diseases interact and localize to cilia and/or basal bodies. One of these proteins is MKS1, which is disrupted in some MKS patients and contains a B9 motif of unknown function that is found in two other mammalian proteins, B9D2 and B9D1. Caenorhabditis elegans also has three B9 proteins: XBX-7 (MKS1), TZA-1 (B9D2), and TZA-2 (B9D1). Herein, we report that the C. elegans B9 proteins form a complex that localizes to the base of cilia. Mutations in the B9 genes do not overtly affect cilia formation unless they are in combination with a mutation in nph-1 or nph-4, the homologues of human genes (NPHP1 and NPHP4, respectively) that are mutated in some NPHP patients. Our data indicate that the B9 proteins function redundantly with the nephrocystins to regulate the formation and/or maintenance of cilia and dendrites in the amphid and phasmid ciliated sensory neurons. Together, these data suggest that the human homologues of the novel B9 genes B9D2 and B9D1 will be strong candidate loci for pathologies in human MKS, NPHP, and JBTS.     

Functional modulation of IFT kinesins extends the sensory repertoire of ciliated neurons in Caenorhabditis elegans

The diversity of sensory cilia on Caenorhabditis elegans neurons allows the animal to detect a variety of sensory stimuli. Sensory cilia are assembled by intraflagellar transport (IFT) kinesins, which transport ciliary precursors, bound to IFT particles, along the ciliary axoneme for incorporation into ciliary structures. Using fluorescence microscopy of living animals and serial section electron microscopy of high pressure-frozen, freeze-substituted IFT motor mutants, we found that two IFT kinesins, homodimeric OSM-3 kinesin and heterotrimeric kinesin II, function in a partially redundant manner to build full-length amphid channel cilia but are completely redundant for building full-length amphid wing (AWC) cilia. This difference reflects cilia-specific differences in OSM-3 activity, which serves to extend distal singlets in channel cilia but not in AWC cilia, which lack such singlets. Moreover, AWC-specific chemotaxis assays reveal novel sensory functions for kinesin II in these wing cilia. We propose that kinesin II is a "canonical" IFT motor, whereas OSM-3 is an "accessory" IFT motor, and that subtle changes in the deployment or actions of these IFT kinesins can contribute to differences in cilia morphology, cilia function, and sensory perception.     

Caenorhabditis elegans ciliary protein NPHP-8, the homologue of human RPGRIP1L, is required for ciliogenesis and chemosensation

Nephronophthisis (NPHP) is the most frequent genetic cause of end-stage renal failure in children and young adults. NPHP8/RPGRIP1L is a novel ciliary gene that, when mutated, in addition to causing NPHP, also causes Joubert syndrome (JBTS) and Meckel syndrome (MKS). The exact function of NPHP8 and how defects in NPHP8 lead to human diseases are poorly understood. Here, we studied the Caenorhabditis elegans homolog nphp-8 (C09G5.8) and explored the possible function of NPHP-8 in ciliated sensory neurons. We determined the gene structure of nphp-8 through rapid amplification of cDNA ends (RACE) analysis and discovered an X-box motif that had been previously overlooked. Moreover, NPHP-8 co-localized with NPHP-4 at the transition zone at the base of cilia. Mutation of nphp-8 led to abnormal dye filling (Dyf) and shorter cilia lengths in a subset of ciliary neurons. In addition, chemotaxis to several volatile attractants was significantly impaired in nphp-8 mutants. Our data suggest that NPHP-8/RPGRIP1L plays an important role in cilia formation and cilia-mediated chemosensation in a cell type-specific manner.     

Radial glia and neural stem cells

During the last decade, the role of radial glia has been radically revisited. Rather than being considered a mere structural component serving to guide newborn neurons towards their final destinations, radial glia is now known to be the main source of neurons in several regions of the central nervous system, notably in the cerebral cortex. Radial glial cells differentiate from neuroepithelial progenitors at the beginning of neurogenesis and share with their ancestors the bipolar shape and the expression of some molecular markers. Radial glia, however, can be distinguished from neuroepithelial progenitors by the expression of astroglial markers. Clonal analyses showed that radial glia is a heterogeneous population, comprising both pluripotent and different lineage-restricted neural progenitors. At late-embryonic and postnatal stages, radial glial cells give rise to the neural stem cells responsible for adult neurogenesis. Embryonic pluripotent radial glia and adult neural stem cells may be clonally linked, thus representing a lineage displaying stem cell features in both the developing and mature central nervous system. This work was supported by AIRC (Associazione Italiana per la Ricerca sul Cancro) NUSUG grant (In vivo screening for genes implicated in glioma formation and development of new animal models of glial tumors) and by Fondazione CARIGE grant (Basi molecolari e cellulari dei gliomi: individuazione di marcatori diagnostici e di nuovi bersagli terapeutici).     

Neuronal control of lipid metabolism by STR‐2 G protein‐coupled receptor promotes longevity in Caenorhabditis elegans

The G protein‐coupled receptor (GPCR) encoding family of genes constitutes more than 6% of genes in Caenorhabditis elegans genome. GPCRs control behavior, innate immunity, chemotaxis, and food search behavior. Here, we show that C. elegans longevity is regulated by a chemosensory GPCR STR‐2, expressed in AWC and ASI amphid sensory neurons. STR‐2 function is required at temperatures of 20°C and higher on standard Escherichia coli OP50 diet. Under these conditions, this neuronal receptor also controls health span parameters and lipid droplet (LD) homeostasis in the intestine. We show that STR‐2 regulates expression of delta‐9 desaturases, fat‐5, fat‐6 and fat‐7, and of diacylglycerol acyltransferase dgat‐2. Rescue of the STR‐2 function in either AWC and ASI, or ASI sensory neurons alone, restores expression of fat‐5, dgat‐2 and restores LD stores and longevity. Rescue of stored fat levels of GPCR mutant animals to wild‐type levels, with low concentration of glucose, rescues its lifespan phenotype. In all, we show that neuronal STR‐2 GPCR facilitates control of neutral lipid levels and longevity in C. elegans.     

Counterbalance between BAG and URX neurons via guanylate cyclases controls lifespan homeostasis in C. elegans

Lifespan of C. elegans is affected by the nervous system; however, the underlying neural integration still remains unclear. In this work, we targeted an antagonistic neural system consisting of low‐oxygen sensing BAG neurons and high‐oxygen sensing URX neurons. While ablation of BAG neurons increases lifespan of C. elegans, ablation of URX neurons decreases lifespan. Genetic analysis revealed that BAG and URX neurons counterbalance each other via different guanylate cyclases (GCYs) to control lifespan balance. Lifespan‐modulating effects of GCYs in these neurons are independent of the actions from insulin/IGF‐1 signalling, germline signalling, sensory perception, or dietary restriction. Given the known gas‐sensing property of these neurons, we profiled that lifespan of C. elegans is promoted under moderately low oxygen (4–12%) or moderately high carbon dioxide (5%) but inhibited under high‐level oxygen (40%); however, these pro‐longevity and anti‐longevity effects are counteracted, respectively, by BAG and URX neurons via different GCYs. In conclusion, BAG and URX neurons work as a neural‐regulatory system to counterbalance each other via different GCYs to control lifespan homeostasis.     

Analysis of the flt-1 Gene Encoding a ECM Protein, Flectin, in Caenorhabditis elegans

Flectin is a new type of extracellular matrix protein and its function was suggested to provide a micro-environment of great elasticity. The C. elegans genome database revealed the presence of a flectin homologue, flt-1, which shows approximately 40% similarity (20% identity) to chick flectin. Here we propose a new gene structure for the flt-1 based on our experiments and the partial cDNA clones obtained from Y. Kohara and further suggest that the previous gene prediction is incorrect. FLT-1 is shown to be expressed in various neurons, hypodermal cells, distal tip cells and vulva epithelial cells. Immunostaining results with anti-FLT-1 antibody, further confirm the FLT-1 expression in vulva epithelial cells. The lipophilic dye, DiI, was used to identify the head neurons expressing GFP and results indicated that none of the head neurons expressing GFP are the 6 chemosensory neurons. In order to determine the function of flt-1 gene, RNA-mediated interference (RNAi) experiments were conducted.     

Expression of muscarinic acetylcholine receptors and choline acetyltransferase enzyme in cultured antennal sensory neurons and non‐neural cells of the developing moth Manduca sexta

Antennal sensory neurons of Manduca sexta emerge from epidermal cells that also give rise to sheath cells surrounding the peripheral parts of the neurons and to glial cells that enwrap the sensory axons in the antennal nerve. Reciprocal interactions between sensory neurons and glial cells are believed to aid in axon growth and guidance, but the exact nature of these interactions is not known. We investigated the possibility of cholinergic interactions in this process by locating muscarinic acetylcholine receptors (mAChRs) and choline acetyltransferase (ChAT) enzyme in cultured antennal sensory neurons and non‐neural cells. ChAT and mAChRs were present in the sensory neurons from the first day in culture. Therefore, the sensory neurons are probably cholinergic, as previously suggested, but they may also be controlled by ACh. In 7‐day‐old cultures a subgroup of small non‐neural cells with processes expressed ChAT activity, and in 14‐day‐old cultures non‐neural cells that formed lamellipodia and scaffoldlike structures on the culture substrate were labeled with ChAT antibody. mAChR activity was detected in similar non‐neural cells but only in areas surrounding the nuclei. In addition, mAChRs were found in flat lamellipodia and filopodia forming cells that were present in 1‐day‐old cultures and grew in size during the 2 week investigation period. These findings suggest muscarinic cholinergic interactions between the neural and non‐neural cells during the development of Manduca antenna. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2005     

STAM and Hrs down-regulate ciliary TRP receptors

Cilia are endowed with membrane receptors, channels, and signaling components whose localization and function must be tightly controlled. In primary cilia of mammalian kidney epithelia and sensory cilia of Caenorhabditis elegans neurons, polycystin-1 (PC1) and transient receptor polycystin-2 channel (TRPP2 or PC2), function together as a mechanosensory receptor-channel complex. Despite the importance of the polycystins in sensory transduction, the mechanisms that regulate polycystin activity and localization, or ciliary membrane receptors in general, remain poorly understood. We demonstrate that signal transduction adaptor molecule STAM-1A interacts with C. elegans LOV-1 (PC1), and that STAM functions with hepatocyte growth factor–regulated tyrosine kinase substrate (Hrs) on early endosomes to direct the LOV-1-PKD-2 complex for lysosomal degradation. In a stam-1 mutant, both LOV-1 and PKD-2 improperly accumulate at the ciliary base. Conversely, overexpression of STAM or Hrs promotes the removal of PKD-2 from cilia, culminating in sensory behavioral defects. These data reveal that the STAM-Hrs complex, which down-regulates ligand-activated growth factor receptors from the cell surface of yeast and mammalian cells, also regulates the localization and signaling of a ciliary PC1 receptor-TRPP2 complex.     

CNS midline cells influence the division and survival of lateral glia in the Drosophila nervous system

Central nervous system (CNS) midline cells are essential for identity determination and differentiation of neurons in the Drosophila nervous system. It is not clear, however, whether CNS midline cells are also involved in the development of lateral glial cells. The roles of CNS midline cells in lateral glia development were elucidated using general markers for lateral glia, such as glial cell missing and reverse polarity, and specific enhancer trap lines labeling the longitudinal, A, B, medial cell body, peripheral, and exit glia. We found that CNS midline cells were necessary for the proper expression of glial cell missing, reverse polarity, and other lateral glia markers only during the later stages of development, suggesting that they are not required for initial identity determination. Instead, CNS midline cells appear to be necessary for proper division and survival of lateral glia. CNS midline cells were also required for proper positioning of three exit glia at the junction of segmental and intersegmental nerves, as well as some peripheral glia along motor and sensory axon pathways. This study demonstrated that CNS midline cells are extrinsically required for the proper division, migration, and survival of various classes of lateral glia from the ventral neuroectoderm.     

Ultrastructure of the supporting cells in the chemoreceptor areas of the tentacles of Pomatias elegans (Müller) (mollusca,prosobranchia) and the ommatophore of Helix pomatia L. (Mollusca,Pulmonata)

The ultrastructure of the supporting cells in the chemoreceptor areas of the tentacles of Pomatias elegans and Helix pomatia is very similar. Complex apical structures are present, and the lateral plasma membrane exhibits three zones: (1) a zone of slight interdigitations; (2) a zone characterized by longitudinal plicae; (3) a zone of basal radiculae. The portions of the sensory cells located within the epithelial layer are accommodated in longitudinal grooves in the supporting cells. However, there are also differences. In Pomatias elegans the apical surface is differentiated into long microvilli that are sometimes dichotomously branched and invested by a surface coat along their entire length. Cytofilia and cilia of the sensory cells pass through this layer of microvilli and surface coat throughout its entire width. In Helix pomatia the supporting cells are somewhat smaller and the apical differentiation consists of candelabra-like protrusions, which are usually three times dichotomously branched. The final branchings, corresponding to microvilli, are called terminal twigs. They are covered by a surface coat, which forms a feltwork. The cytofilia and cilia of the sensory cells that intertwine among the protrusions are confined to the space below the terminal twigs, where they compose the spongy layer.     

Fine structure of a sensory organ in the arista of Drosophila melanogaster and some other dipterans

Summary The arista, a characteristic appendage of dipteran antennae, consists of 2 short segments at the base and a long distal shaft. A small sensory ganglion, from which arises the aristal nerve, is located proximally in the shaft. The fine structure of the aristal sensory organ was studied in detail in the fruitfly (Drosophila) and for comparison in the housefly (Musca) and the blowfly (Calliphora). In Drosophila, the aristal sense organ consists of 3 identical sensilla that terminate in the hemolymph space of the aristal shaft, and not in an external cuticular apparatus. Each sensillum comprises 2 bipolar neurons and 2 sheath cells; a third sheath cell envelops the somata of all six neurons of the ganglion. The neurons have long slender dendrites with the usual subdivision into an inner and an outer segment. One of the outer segments is highly lamellated and bears small particles (BOSS-structures) on the outside of its cell membrane; the other outer segment is unbranched and has a small diameter. The fine structure of the first dendrite is strongly reminiscent of thermoreceptors known from the antennae of other insects. These thermoreceptors are often coupled with hygroreceptors; however, we can only speculate whether the second dendrite of the aristal organ also has this function. Our present results argue against mechanoreceptive functions, as formerly postulated. The aristal sense organs in Musca and Calliphora are similar to those in Drosophila, but contain more sensilla (12 in Musca, 18 in Calliphora).     

Mechanisms underlying olfactory neuronal connectivity in Drosophila-the atonal lineage organizes the periphery while sensory neurons and glia pattern the olfactory lobe

Patterning of the antennal lobe of adult Drosophila occurs through a complex interaction between sensory neurons, glia, and central neurons of larval and adult origin. Neurons from the olfactory sense organs are organized into distinct fascicles lined by glial cells. The glia originate from one of the three types of sensory lineages-specified by the proneural gene atonal. Gain-of-function as well as loss-of-function analysis validates a role for cells of the Atonal lineage in the ordered fasciculation of sensory neurons. Upon entry of the antennal nerve to central regions, sensory neurons at first remain closely associated with central glia which lie around the periphery of the lobe anlage. Coincident with the arrival of sensory neurons into the brain, glial precursors undergo mitosis and neural precursors expressing Dachshund appear around the lobe. Sensory neurons and glial cells project into the lobe at around the same time and are likely to coordinate the correct localization of different glomeruli. The influence of sensory neurons on the development of the olfactory lobe could serve to match and lock peripheral and central properties important for the generation of olfactory behavior.