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Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology, Crete, Greece
ABSTRACT I. INTRODUCTION A. Emergence of Mechanotransduction 1. Mechanically gated ion channels in bacteria 2. Mechanically gated ion channels in archaea B. Regulatory Mechanotransduction C. Sensory Mechanotransduction II. DECIPHERING MECHANOTRANSDUCTION A. Theoretical Concepts for Mechanotransduction B. Molecular Characterization of Eukaryotic Mechanotransducers: Methodological Limitations C. Genetic Models III. MECHANOTRANSDUCTION IN CAENORHABDITIS ELEGANS A. Introducing C. elegans B. Gentle Body Touch Response 1. Neuroanatomy: the neuronal circuit for touch response 2. Development and differentiation of touch receptors 3. Genetics and molecular biology of the gentle body touch response: the mec genes A) REGULATORY/CELL-SPECIFICATION GENES. B) MECHANOSENSOR CORE STRUCTURAL COMPONENTS. C) PERIPHERAL ASSOCIATED PROTEINS. C. Proprioception: Regulation of Locomotion 1. Neuroanatomy: the neuronal circuit for locomotion 2. Genes involved in the regulation of locomotion A) UNC-8 AND DEL-1. B) UNC-1 AND UNC-24. C) UNC-105. D. The Nose Touch Responses 1. Head-on collision response 2. Foraging and the head withdrawal response E. Other Mechanosensitive Behaviors 1. The harsh touch response 2. The tap withdrawal reflex 3. Regulatory mechanotransduction in C. elegans IV. DEGENERINS: FROM NEURODEGENERATION TO MECHANOTRANSDUCTION A. Features of the DEG/ENaC Ion Channels B. Degenerin-Induced Cell Death C. An All-Purpose Model for the Mechanotransducer in C. elegans Touch Receptor Neurons, Motorneurons, and Muscle V. MECHANOTRANSDUCTION IN DROSOPHILA MELANOGASTER A. Drosophila Mechanosensory Organs B. Genetics of Mechanotransduction in Drosophila 1. Mechanosensory mutants 2. The nompC gene 3. nompA 4. Painless 5. Nanchung 6. Candidate DEG/ENaC mechanosensitive channels in Drosophila VI. SENSORY MECHANOTRANSDUCTION IN VERTEBRATES A. Zebrafish Mutants With Mechanosensory Defects 1. The zebrafish NompC B. Candidate Mammalian Mechanosensitive Ion Channels 1. {gamma}-ENaC 2. BNC1 3. ASIC3 (DRASIC) 4. TRPV4 VII. EMERGING THEMES VIII. CONCLUDING REMARKS AND FUTURE DIRECTIONS A. Open Issues B. Limitations of Current Methodologies C. Alternative/Complementary Approaches ACKNOWLEDGMENTS REFERENCES
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I. INTRODUCTION |
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All living organisms have developed highly specialized structures that are receptive to mechanical forces originating either from the surrounding environment or from within the organism itself. Among the most elaborate and greatly efficient, such structures are the mechanotransducers that are responsible for sensory awareness, for example, those facilitating touch, balance proprioception, and hearing (137, 152, 163, 404). In this article, we survey the current state of the art in the field of sensory mechanotransduction in Caenorhabditis elegans and highlight landmark discoveries in other model organisms, ranging from bacteria to mammals that have decisively shaped our understanding of the phenomenon of mechanotransduction. We further touch on the still missing pieces of the puzzle that impinge on the dominant concepts in the field and discuss potential experimental approaches that could help clarify sensitive open issues.
A. Emergence of Mechanotransduction
Perception of incident mechanical stimuli is critically important for interfacing with the physical world. Naturally, the mechanisms underlying the capability of living cells to receive and act in response to mechanical inputs are among the most ancient, implemented during evolution. Proteins with mechanosensitive properties are ubiquitously present in eubacteria, archaea, and eukarya and are postulated to have been an essential part of the physiology of the Last Universal Ancestor (137, 234, 237, 240, 276). The first mechanosensitive processes may have evolved as backup mechanisms for cell protection, e.g., to reduce intracellular pressure and membrane tension during osmotic swelling. Subsequent organismal diversification and specialization resulted in variable requirements for mechanotransduction in different organisms (304). Hence, evolutionary pressure has shaped a large repertoire of mechanotransducers, optimized for a great assortment of tasks that range from maintenance of intracellular osmotic balance and pressure to our impressive ability of hearing and discriminating sounds, and reading Braille code with our fingertips (152, 168).
Below, we briefly present mechanically gated ion channels from bacteria and archaea. Although these channels probably represent the simplest and more ancient forms of a mechanotransducer, they nevertheless comply with the same basic principles that dictate the function of mechanosensitive molecules and structures in all organisms.
1. Mechanically gated ion channels in bacteria
The best-characterized bacterial mechanosensitive channels are those of Escherichia coli, which are involved in cell turgor regulation. The primary role of this channel is osmoregulation or osmoprotection of bacterial cells (30, 249, 353). These mechanically gated channels can directly sense mechanical stretch on the membrane during severe osmotic challenges and can switch between open and closed conformations in response to bilayer tension (4, 25, 38, 40, 392). They have been studied in detail by patch-clamp methodologies in giant spheroplasts and in reconstituted membrane fractions (168, 278, 392). Based on their conductance and sensitivity to negative pressure applied to the patch-clamp pipette, three types of mechanosensitive channels (Msc) can be distinguished: MscM (M for mini conductance), MscS (S for small conductance), and MscL (L for large conductance). Another bacterial channel, MscK (previously called AefA and KefA), shares homology with MscS but contains additional domains at its amino terminus, which may be required for the allosteric regulation of this channel by ionic concentration (251, 287). MscL was the first identified and is the best-characterized of all mechanosensitive channels (39, 41, 277, 391).
Fractionation of E. coli membrane constituents by column chromatography and functional examination of the individual fractions for mechanically gated channel activity by patch-clamp electrophysiological recordings led to the identification of a membrane protein underlying the activity of MscL and the cloning of the corresponding gene (391, 393). The MscL secondary structure includes two 
-helical transmembrane domains (TM1 and TM2) connected by a periplasmic loop, with the amino and carboxy termini located in the cytoplasm (41, 392). The MscL ion channel is nonselective and exhibits very large conductivity, which is indicative of a large, water-filled pore, probably formed in the center of homomultimeric structures (91). The higher order structure of MscL is somewhat controversial. Cross-linking experiments indicated that MscL might form homohexamers (40, 41) or homopentamers (280, 394). Likewise, crystallographic studies have identified both homopentameric (Tb-MscL; from Mycobacterium tuberculosis) and homoexameric (Eco-MscL; from E. coli) structures (72, 356).
Molecular dissection of the MscL channel through mutational analysis of the mscL gene identified structural domains critical for function (39, 41, 277, 391, 477). Several models of MscL mechanosensitivity, based on structure-function relations, have been proposed (37, 168). The crystal structure of MscL from M. tuberculosis (Tb-MscL), at 3.5Å resolution, has provided a basis for molecular modeling of the channel gating mechanism (38, 72, 296, 305). At present, a combination of electrophysiological, biophysical, and structural studies in bacterial ion channels (25, 31, 33, 280, 311, 312, 378, 389, 390) support the following simplified model of the MscL gating. The channel is composed of five identical subunits arranged around a central pore. The closed pore is formed by the hydrophophic constriction of the first transmembrane (TM1) helices at the cytoplasmic end of the channel. The mobile amino termini probably serve to stabilize the open-channel conformation and may interfere with the passage of ions, whereas the carboxy termini possibly play a role in stabilizing the closed configuration of the channel. The periplasmic loop domains most likely function as elastic springs, resisting the opening of the channel by membrane tension (168, 276). Membrane tension entails the channel opening through an intermediate step that involves small movements in the first transmembrane helices (TM1). Subsequent concerted, massive rearrangements in both TM1 and TM2 of the channel subunits result in irislike expansion and opening of the channel (31, 311). The mobile amino termini probably serve to stabilize the open-channel conformation and may interfere with the passage of ions. The carboxy termini possibly play a role in stabilizing the closed configuration of the channel (276).
MscS and MscM are two additional mechanically gated channels in E. coli that show different pressure sensitivity, with MscM appearing less frequent in bacterial cells, compared with MscL and MscS, which are typically found in excess. MscS is relatively nonselective, displaying a slight preference for anions over cations, whereas MscM exhibits a slight preference for cations (30, 278, 393). Because all three channels can be activated by hyposmotic stress, this indicates that they are probably activated sequentially to provide a gradual efflux conduit (147, 168). Studies of the E. coli MscS channel structure demonstrated that it folds as heptamer with three transmembrane helices (TM1–3) in each subunit (22).
2. Mechanically gated ion channels in archaea
Two types of mechanically gated channels have been identified in the cell membranes of the halophilic archaeon Haloferax volcanii using the patch-clamp technique, MscA1 and MscA2 (246). Both show large conductance, but they differ in their distinct rectification properties, and similarly to the bacterial mechanically gated channels, both respond to bilayer tension and are blocked by submillimolar concentrations of gadolinium (Gd3+) (232, 246). In addition, mechanosensitive channels have been identified and cloned from two other archaeal species that occupy different environmental habitats: the thermophilic archaeon Thermoplasma volcanium and methanogenic archaeon Methanococcus jannashii. By reconstitution of detergent-solubilized membrane proteins onto liposomes and following their function in patch-clamp experiments, a 15-kDa membrane protein that forms a novel mechanosensitive channel, termed MscTA, was identified in T. volcanium (232). The T. volcanium channel protein exhibited properties typical of a mechanosensitive ion channel when heterologously expressed in E. coli and used for channel reconstitution experiments onto liposomes (234). Secondary structure analysis indicates that the MscTA channel has two -helical transmembrane domains, similarly to the bacterial MscL (232).
With the use of the TM1 transmembrane domain of Eco-MscL as a genetic probe, the hypothetical protein MJ0170 was identified in the M. jannashii genome, which shares sequence similarity with both the TM1 of Eco-MscL and the YggB protein underlying the activity of MscS in E. coli (234, 249). This suggests that the hybrid MscMJ protein might have evolved by gene duplication from an ancestral MscL-like gene (233). These studies demonstrate that archaeal mechanosensitive channels share structural and functional similarity with bacterial mechanosensitive channels. A common gating mechanism is at operation where mechanical force transmitted via the lipid bilayer triggers channel opening (232, 234). Even though no sequence homologs for MscL have been identified in eukaryotes, bacterial and archaeal mechanosensitive channels share intriguing similarities in basic structural motifs and membrane topology with eukaryotic channels of different function [e.g., the degenerin (DEG)/epithelial Na+ channel (ENaC) family of ion channels; see sect. IVA]. This indicates that common biophysical principles underlie the mechanosensitive properties of diverse classes of channels (168). Hence, mechanosensory transduction probably originated along with the appearance of the first life forms according to such biophysical principles. Phylogenetic analysis, which indicates that prokaryotic mechanosensitive channels derived from a common ancestral molecule resembling the bacterial MscL channel protein, provides support for this hypothesis (232, 240, 295). Moreover, a recent study suggests that the MscL channel shares a common evolutionary origin with the sensor module of eukaryotic mechano- and voltage-gated channels (243).
B. Regulatory Mechanotransduction
Mechanotransduction in living organisms can operationally be categorized as regulatory or sensory. Both cellular and organismal homeostasis often requires adjustment to mechanical forces generated by environmental sources or internal processes (168, 353). For example, osmotic balance, ion concentration homeostasis, cell volume and shape regulation, blood pressure, and turgor control all depend on appropriately responding to mechanical stretch or shearing forces (93, 112, 168, 353). Dedicated mechanotransducers in these paradigms serve as regulatory valves that initiate a cascade of events towards adjusting to or counteracting any substantial deviation from normal conditions. The requirement for regulatory mechanotransduction is probably as ancient as life itself. Cells constantly need to fight shearing and stretch forces they encounter, and the faculty of mechanotransduction was most likely decisive for the survival of the first cell. The universal occurrence of mechanotransduction capabilities in all living organisms argues for such early emergence of mechanotransducers (137, 233, 237). An alternative, plausible scenario is that mechanotransduction is the outcome of convergent evolution and evolved independently multiple times by the conversion of various types of ion channels into mechanotransducers (137).
C. Sensory Mechanotransduction
Sensory mechanotransduction or mechanosensation alerts the organism to mechanical inputs in the form of touch, pressure, stretch, sound, vibration, and acceleration (149, 152, 168, 353). Such stimuli provide vital awareness of the environment and information with regard to the organism's relative position and movement. This prowess is important in negotiating with the physical world and is based on highly adapted mechanotransducers that have evolved to optimally carry out the task. Sensory and regulatory mechanotransducers obey similar principles, and it is likely that the first derived from the second by refinement towards acquiring dedicated functions. In higher organism, specific neurons, the mechanoreceptors are equipped with a mechanotransducing apparatus and signal upon reception of a stimulus. Frequently these cells are implanted within accessory structures that serve to filter and amplify an incoming stimulus. For example, skin touch receptor neurons are occasionally associated with hair shafts, while hair cells of the inner ear are enclosed in elaborate anatomical structures that greatly facilitate capture and tunneling of sound wave energy (113, 134, 292, 317, 374, 375).
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II. DECIPHERING MECHANOTRANSDUCTION |
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A. Theoretical Concepts for Mechanotransduction
The nature of the triggering stimulus puts specific constraints on the gating properties of a mechanotransducer. Specifically, two physical parameters are of paramount importance: sensitivity and speed (88, 151, 152, 317). An efficient mechanotransducer is capable of detecting minute forces and responding within microseconds upon stimulation (195, 197). These requirements exclude slow mechanisms involving second messengers and ligands from gating sensory mechanotransducers (152, 196, 236, 354). Rather, mechanical force needs to be directly applied to the channel, reducing immediate response. An additional essential feature of a mechanotransducer is adaptation to background, constitutive mechanical forces. Adaptation bolsters the capacity to differentiate between weak signals and relatively large constant mechanical forces (196, 197, 211, 317).
Two general models have been proposed for gating mechanosensitive ion channels: the "lipid bilayer" and the "tethered channel" models (Fig. 1). In the lipid bilayer model, mechanosensitivity of ion channels inserted in lipid bilayers arises from the intimate association of the channel with the bilayer, which allows dilation, thinning, or changes in local membrane curvature, to directly shift the equilibrium between open and closed channel conformations (168, 273, 276, 322). Bilayer reconstitution experiments have provided unequivocal evidence for a bilayer model of mechanical gating for bacterial channels, and there is growing evidence that at least some eukaryotic channels are gated by tension developed in the bilayer (e.g., the SAT-CAT channel in Xenopus oocytes; Ref. 479). Another eukaryotic channel that retains mechanosensitivity when reconstituted into lipid bilayers is the epithelial Na+ channel (ENaC; Refs. 12, 200, 201). However, it should be noted that, when expressed in Xenopus oocytes, ENaC proteins do not form mechanosensitive channels, which casts doubts as to the physiological relevance of lipid-bilayer observations (13, 346).
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B. Molecular Characterization of Eukaryotic Mechanotransducers: Methodological Limitations
For years, mechanically gated ion channels tenaciously eluded cloning and molecular characterization efforts for reasons pertinent to their highly specific and efficient function. For example, owing to their high sensitivity, the relative density of skin touch receptors is exceedingly low; there are only 
17,000 such receptors in the finger and palm skin pad (105, 127, 152, 239). Also, in the specialized hair cells of the vertebrate inner ear, only a few hundred mechanosensitive ion channels may exist (47, 88, 195, 317, 379). These are prohibitive densities for biochemical isolation of the molecules involved. Moreover, there are no known reagents that will interact with these channels with high specificity and high affinity, thus further thwarting efforts at biochemical purification.
Second, mechanosensory channels are embedded and intertwined with materials that attach to the surrounding environment (152, 168). These contacts, while probably critical for function, make physical dissociation of the channel a challenge, and hinder studies in heterologous expression systems where such components and intricate associations are either lacking or are difficult to reproduce faithfully (140, 147, 152, 404). Apart from raising concerns about heterologous expression studies, the delicate surroundings of eukaryotic mechanosensitive channels also encumber in situ electrophysiological recordings. In sharp contrast to voltage- or ligand-gated ion channels, the very act of measurement using electrophysiological probes is likely to inadvertently disturb the structure and the properties of the mechanotransducing apparatus. Together with the subunits of the mechanically gated, core ion channel, the interacting molecules that provide the necessary gating tension need to be identified and included in potential mechanotransducer reconstitution attempts. Without these, it is highly likely that the core ion channel would not retain its native mechanosensitive characteristics.
The intrinsic difficulties of directly isolating and studying mechanosensitive macromolecular complexes and ion channels necessitate the development and utilization of genetic models for mechanotransduction. Indeed, genetic approaches have been exceptionally successful in identifying candidate mechanically gated ion channels and other components of mechanotransducing complexes. Pioneering genetic screens conducted by Sydney Brenner, Martin Chalfie, Marilyn Dew and John Sulston, using the nematode Caenorhabditis elegans, have allowed the detailed dissection of touch sensation in this simple organism (55, 58, 59, 64, 65, 395). Extensive follow-up studies have culminated in the identification of a plethora of proteins implicated in mechanotransduction and the formulation of an elegant model for a mechanosensory apparatus (15, 18, 58, 138, 402). Similarly motivated genetic approaches in the fly Drosophila melanogaster have resulted in the dissection of a different type of mechanotransducer (113–115, 205, 223, 438). Two vertebrate organisms have also contributed to our understanding of mechanotransduction, the fish Danio rerio (zebrafish) and the mouse (9, 106, 158, 300, 302, 326, 327). Remarkably, investigations in such distant organisms have converged to a limited number of mechanisms and many common components for the metazoan mechanotransducer (134, 152, 168, 171, 404). This conservation suggests that, while individual implementations of mechanosensitive structures may somewhat differ, a similar basic principle underlies mechanotransduction from nematodes to mammals (113, 115, 152, 404). Below, we elaborate on findings in C. elegans and describe work in Drosophila and vertebrates, aiming to portray the commonalities that epitomize this general principle.
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III. MECHANOTRANSDUCTION IN CAENORHABDITIS ELEGANS |
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C. elegans is a small (1 mm) soil-dwelling, free-living nematode worm (46). In the laboratory, animals feed on an E. coli diet and complete a reproductive life cycle in 2.5 days at 25°C, progressing from fertilized embryos through four larval stages to become egg-laying adults, which then live for 2 wk (see Fig. 2; Refs. 46, 231, 470). Because C. elegans can reproduce by self-fertilization, it is possible to raise genetically identical populations that do not undergo inbreeding depression. While the dominant sexual form is the hermaphrodite (genotype: XX), males (genotype X0) can also be propagated and used to construct strains carrying multiple mutations (46). Under adverse conditions such as starvation, overcrowding, or high temperature, larvae can enter an alternative life stage called the dauer (enduring) larva, during which animals move but do not feed (53, 119). The dauer larva is a "nonaging" developmental form that survives for weeks or even months (153, 222). When a dauer larva encounters favorable environmental conditions, it reenters the life cycle at the fourth larval stage, progresses into adulthood to reproduce, and then completes the final week or so of its life span.
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20,000 predicted open reading frames (ORFs) have been subjected to expression profiling under numerous conditions using microarray technology (42, 208, 228, 229, 339). Detailed gene expression and protein-protein interaction maps have been developed and are publicly available (44, 95, 279, 435; http://www.wormbase.org).
C. elegans is also particularly amenable to reverse genetics studies. Investigators can take advantage of the wealth of genome data available to perform "reverse genetics," directly knocking out genes (116, 261, 476). A novel method of generating mutant phenocopies, called double-stranded RNA-mediated interference (dsRNAi), enables probable loss-of-function phenotypes to be rapidly evaluated (126, 198, 213, 410, 420, 421). A comprehensive RNAi approach to knock down expression of each of the 20,000 ORFs was recently published (212a), and examination of all genes on chromosomes I and III as well as of those expressed in the ovary has already been published (16, 129, 154, 266, 314). DNA manipulated in vitro can be microinjected or bombarded back into animals for functional assays (125, 288, 324). Vectors are available for identification of transformants, cell-specific expression, and generation of fusions to marker genes such as E. coli 
-galactosidase and the jellyfish green fluorescent protein (GFP) so that individual cells can be visualized in stained or living animals (57, 69, 124, 291).
The laboratory assay for the gentle body touch response involves a mild stroke of the animal with an eyelash hair attached to a toothpick, transversely to the anterior-posterior body axis (64, 65, 395). Forces exerted during the stimulation, which can be sensed by wild-type animals, are in the range of 10–20 µN (137, 157). When no response is observed, animals are prodded with a thin platinum wire to confirm that they are touch insensitive rather than paralyzed (gentle-touch insensitive animals typically still respond to a strong stimulus, the harsh touch response, with forces in excess of 100 µN) (65, 70, 103, 157, 469). Depending on the part of the body touched, animals will either accelerate or initiate forward movement (when stimulated at the posterior or the tail), or reverse and move backwards (when stimulated at the anterior part of the body). Hermaphrodite, male, juvenile (except L1), and dauer animals respond identically to touch. The response is adaptive: repetitive stimulation leads to short periods of insensitivity (267, 335, 458).
1. Neuroanatomy: the neuronal circuit for touch response
The neuronal morphologies, chemical synapses, and gap junctions of all C. elegans neurons have been described by means of reconstruction from serial section electron micrographs (443, 455, 456; http://www.wormatlas.org). The significance of identified synaptic relationships has been tested using laser microsurgery. The touch reflex of the mature animal involves 6 touch receptor neurons, 5 pairs of interneurons, and 69 motorneurons (55, 65). The 6 touch receptor neurons were originally designated as the microtubule cells because of distinctive bundles of 15-protofilament (tubulin dimmer filaments) microtubules that fill their processes (ALML/R, anterior lateral microtubule cell, left/right; AVM, anterior ventral microtubule cell; PLML/R, posterior lateral microtubule cell, left/right; PVM, posterior ventral microtubule cell; Refs. 55, 60, 65–67). All six cells are dispensable for the viability of the organism. Apart from insensitivity to gentle body touch, laser ablation of all six neurons does not result in any additional adverse effects (56, 65).
Two fields, anterior and posterior, of touch sensitivity are defined by the arrangement of the six touch receptor neurons along the body axis (Fig. 4; Ref. 65). All the touch receptor neuron cell bodies have anteriorly directed processes. ALM processes are embedded in the hypodermis, extend along the cuticle, and form a short branch into the circumpharyngeal nerve ring (Fig. 4; Refs. 59, 68). Most of the synapses of the ALM cells, including coupling via gap junctions to the AVM cell, are formed on this branch. In addition to a long anteriorly directed process, the PLM cells have a short posteriorly directed process (65, 456). Similarly to ALM, the anteriorly directed process is embedded in the hypodermis and runs close to the cuticle. The PLM process turns and enters the ventral cord near the vulva. PLML does not make it around the hypodermal ridge and has no synapses. PLMR runs along the neuropile and makes direct interneuron synapses (65, 456). The two postembryonic touch cells have single processes that enter the ventral nerve cord and run anteriorly at its extreme ventral edge. AVM branches at its anterior end. The branch enters the nerve ring where it forms synapses with the ALM cells and other neurons (Fig. 4). PVM does not branch or enter the nerve ring (68, 455, 456). Touch receptors also are uniquely surrounded by an osmiophillic extracellular material referred to as the mantle. The amount of mantle varies along the length of the process. Periodic darkly staining patches are often seen in the cuticle (65, 456). These patches resemble those seen under muscle cells, and it has been speculated that they may represent touch cell attachment sites (Fig. 5; Refs. 103, 402). Several lines of evidence support that touch cell processes are mechanosensory organs (103). First, the touch cell processes lack synaptic specializations, and hence are likely to be dendritic (68, 456). Second, the touch cell processes are embedded in the hypodermis adjacent to the cuticle, positioning expected to facilitate detection of mechanical stimuli (55, 65). Third, the position of the processes along the body axis correlates with the sensory field of the touch cell (65, 103).
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In newly hatched larvae, the processes of touch cells lie between the lateral hypodermis and the adjacent muscle quadrant, while at 12 h they are engulfed by the hypodermis (55, 65). All touch receptors except PVM have a short synaptic branch, which is the site of most synapses to other neurons. The ALMs, AVM, and PVM also make synaptic connections to other neurons on their receptor processes. Interestingly, the induction of synaptic branching appears to depend on the position of the cell body (68). If the PVM cell is situated more anteriorly as in mab-5 mutant animals, a branch forms (64, 174, 358). mab-5 encodes a homeodomain protein, related to Drosophila antennapedia, that functions at the posterior part of the body to determine cell fate and migration (discussed in section IIIB2). The displaced cell can mediate a weak touch response. This indicates that PVM has the potential to branch if situated in the appropriate environment. However, it is not clear if in the mab-5 genetic background, PVM adopts an anterior touch cell fate (358).
Bundles of darkly staining large-diameter mictotubules distinguish the touch receptor neurons (66, 456). Cross-bridges between microtubules of a bundle are observed in micrographs obtained with electron microscopy and may increase the structural integrity of the bundle. These microtubules are unique to the nematode touch receptor neurons and contain 15-protofilament microtubules, a unique feature of these six cells (67). In most eukaryotic cells, - and -tubulin coassemble into 13-protofilament microtubules, whereas the vast majority of microtubules in C. elegans cells have 11 protofilaments (67, 456). In normal touch receptors, 11-protofilament microtubules typical of most other cells in this nematode are occasionally observed. If the 15-protofilament microtubules are eliminated by mutation, the number of 11-protofilament microtubules in the touch cell processes increases (55, 60, 66, 135, 360). Normally there are 450 15-protofilament microtubules in a touch cell and none or very few 11-protofilament microtubules; when 15-protofilament microtubules are absent, 100 11-protofilament microtubules fill the cell process. Fifteen-protofilament microtubules are differentially sensitive to colchicine. At low concentrations, 15-protofilament microtubules and touch sensitivity are disrupted, but other behaviors and microtubules in other cells are unaffected (66). Colchicine also has the potential to block the production of the 11-protofilament microtubules to a lesser extent (67). At hatching, lateral microtubule cells contain very few microtubules, which are short (2.5 µm). The array is not yet continuous as it is in adults. Still, these larvae are touch sensitive. By 12 h, microtubules have increased in number and length. At 12–36 h posthatching, numbers increase but length remains constant, whereas after 36–48 h, they increase in number and length to adult level. After 48 h, the microtubule number still increases, but the length is constant (67). Overall, process length increases 2.4-fold, while total length of microtubules increases 5 times (66).
Microtubules may provide a rigid intracellular "point of reference," against which a touch stimulus could exert mechanical force to the mechanotransducing apparatus (103, 402). This function of the microtubule network is further discussed in section IVB. Microtubules also appear to play a role in process outgrowth, since processes are lacking in cells that have been treated with colchicine and benomyl (benomyl interferes with the 11-protofilament microtubules that take over in the absence of 15-protofilament microtubules; Ref. 102). Continuity of the microtubules does not appear necessary for axonal outgrowth (60, 371). Examination of serial section electron micrographs revealed that the 15-protofilament microtubules do not span the entire length of the touch receptor process. The processes are 400–500 µm long, whereas the microtubules are 10–20 µm long (443, 456). Mature processes are filled with overlapping bundles of 15-protofilament microtubules. The average microtubule length varies with cell type, with lateral cell processes containing more microtubules than the ventral cell processes (66, 456). Such short microtubules may facilitate sliding relative to each other, which would be required to accommodate changes in cell length that is likely to accompany the sinusoidal motion of the animal. Microtubules have a distinct polarity: the distal end is found on the outside of the microtubule bundle and the proximal end is preferentially found within the bundle (66, 456). The distal end is distinguished by its diffuse ending, which is a diffuse patch of stained material with a diameter up to twice that of the microtubules. Proximal ends often have a filled appearance. Intriguingly, the diffusely stained structure of the distal end often appears to associate with the plasma membrane (66, 456).
There is no significant synaptic input to the touch receptor neurons. The branches of the ALM cells make gap junctions with AVM in the nerve ring. PVM is connected to AVM by a gap junction in the ventral cord. PLML and PLMR are not coupled to each other or to other microtubule cells (65, 268). The major synaptic output of touch receptor neurons is channeled to interneurons that connect to motorneurons controlling body wall muscle contractions (Fig. 6). Four pairs of interneurons synapse onto both motorneurons and touch receptor neurons, as well as onto each other, and are thus candidates for function in the touch circuit: AVA, AVB, PVC, and AVD (65, 455, 456). Initially ALM cells work independently via AVD. When AVM is added to the circuit, it couples the anterior cells into a circuit. AVM makes gap junctions to AVD and chemical synapses to AVB (65, 268). There is a reciprocal pattern of chemical synapses formed between interneurons and sensory neurons. Such an arrangement may serve to reinforce the potency of the signal while quenching an inappropriate response (103). For example, PVC makes a gap junction to the posterior touch receptors, and at the same time, this interneuron forms a chemical synapse to anterior touch receptors (Fig. 6). The chemical synapse might negatively regulate other neuronal input, thus preventing inadvertent forward locomotion upon stimulation at the anterior part of the body. When PVC is ablated, posterior touch response is eliminated, while ablation of AVD abolishes anterior touch response (65). In both cases, gap junctions to the touch receptor neurons are involved in relaying the signal (65, 268). These neurons modify movement but are not essential for normal locomotion. Hence, they are principally engaged in mediating the touch response. Contrary to PVC and AVD, ablation of AVA and AVB results in touch sensitive but uncoordinated animals (65). Therefore, these two interneurons are primarily involved in regulating normal locomotion. The posterior touch system is more complex. Although PLMR makes direct synapses to interneurons AVA and AVD, PLML does not. However, both tail touch receptors form gap junctions with a pair of cells, LUAR and LUAL. LUAL/R in turn form chemical synapses with AVM and AVD and gap junctions with PVR (65, 103). Because of the complexity of these synaptic relationships, it has not been possible to confirm experimentally the role of the LUA cells in the touch response circuit. Nevertheless, this connectivity plan adds to the asymmetry of the touch circuit; the anterior touch neurons form chemical synapses anterior to the ventral cord, while the LUA interneurons make synapses posterior to the ventral cord.
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2. Development and differentiation of touch receptors
Four of the six touch receptors (ALML/R and PLML/R) are born during embryogenesis. Two additional cells, the AVM and the PVM, arise from identical lineages postembryonically (55, 396; Fig. 3). These touch receptors are situated sublaterally toward ventral on the anterior right and posterior left, respectively. The positions of postembryonic touch receptors AVM and PVM are determined by the migrations of their precursor cells QR and QL, correspondingly (55, 64, 396). The Q neuroblasts are born at symmetrical positions (left and right) in lateral epidermis, 1 h before hatching. While they start from the same position, shortly after hatching the precursor to AVM (QR) moves anteriorly and the precursor to PVM (QL) moves slightly posterior (64, 180). AVM arises 10 h after hatching and forms functional connections via its branch 20 h later. ALM neurons reach midbody positions by extended migration in the embryo. They migrate posteriorly, and migrations are completed before the general elongation of the embryo (55, 64, 397). Defective ALM migrations would leave the region between ALM and the ends of the PLM axons without mechanosensory innervations. Touch neurons situated in different positions differ in branching, connectivity, and function. Neuronal branching patterns and process trajectories appear dependent in correct cell positioning, but the length of neuron process outgrowth and ultrastructural features are not affected (55, 68).
Even though ALM, PLM, and AVM/PVM cells are derived from three distinct lineages via different patterns of cell divisions, the six touch receptor neurons express nearly identical terminally differentiated features (55, 456). The common touch receptor fate is specified by the concerted action of several groups of genes. For example, genes that function in the execution of cell lineages that generate the touch receptors, the specification of touch cell fate, the restricted expression of touch cell structural genes, the precursor cell migration, and touch cell process outgrowth have been identified in various genetic screens (59, 60, 64, 104, 108). Here, we briefly describe the key players.
The activities of at least three regulatory proteins are required in the final steps of the cell lineages that generate some or all of the touch receptor neurons. Lin-14 acts as a genetic switch that controls the choice to execute the sublineage that generates the postembryonic touch cells AVM/PVM (191, 293). Lin-32, a transcription factor related to Drosophila aschete-scute of the basic helix-loop helix family, is needed for the generation of precursors for the PLM and the AVM/PVM cells (108, 293, 323, 367, 481). The unc-86 POU homeodomain protein and the MEC-3 LIM homeodomain protein are essential for directing appropriate differentiation of precursors that generate all six touch receptor neurons (discussed in detail in section IIIB3; Refs. 27, 122, 123, 252). LIN-14, LIN-32, MEC-3, and UNC-86 also appear to be required for the function of differentiated touch receptor neurons (108, 111, 293). In addition, EGL-5 and MAB-5, which are homeodomain proteins, related to the Drosophila Abdominal-B and antennapedia, respectively, determine posterior cell fate and migration and are needed for touch sensitivity in the tail (77, 367). mab-5 expression is necessary and sufficient to make the QL (the progenitor of PVM) neuroblast distinct from QR (the progenitor of AVM; Ref. 174). mab-5 affects migration of the QL cell, but not the ability of descendents to differentiate to mechanosensory neurons. In the mab-5(e1239) mutant, QL is situated more anteriorly than in wild type (174, 358). Consequently, the resulting PVM cell adopts an anterior touch cell fate similar to AVM; it branches and becomes capable of mediating a weak touch response akin to AVM (103). Mutations in mig-1, which encodes a frizzled-related, G protein-coupled wnt receptor, also affect QL migration (174, 448).
Another important aspect of specification of touch cell fate is the action of regulatory genes that restrict expression of touch cell-specific genes (103, 293). egl-44 and egl-46 turn off expression of touch receptor neuron-specific genes in the FLP neurons, pag-3 turns off their expression in the lineage sisters of the ALM cells, the BDUs, and sem-4 prevents mec-3 expression in the tail PHC neurons (293). Moreover, two genes involved in the regulation and execution of programmed cell death, ced-3 and ced-4, help restrict the number of cells expressing touch cell-specific features. The activity of ced-3 and ced-4 ensures the elimination of cells destined to die within the lineages that give rise to the six touch receptors (191, 293). In their absence, extra cells with touch neuron-specific characteristics emerge, located near the PLM and AVM and PVM cells. Additional spurious touch cells are also occasionally seen in unc-40 (extra ALM and PLM), unc-73 (duplicated ALML, AVM, PVM and PLM), and vab-8 mutants (382, 448, 462).
An assortment of >25 genes affects axonal guidance and/or positioning of the touch receptors (59, 371). Here, we epigrammatically catalogue the genes involved and defects observed in mutant animals. Mutations in unc-13, unc-14, unc-33, unc-44, unc-51, unc-53, unc-59, unc-69, unc-71, unc-73, unc-75, and unc-85 cause the most pronounced defects in touch cell morphology, axonal outgrowth, and positioning (abnormal varicosities, arborizations, and growth in abnormal directions). Mutations in 13 others, unc-1, unc-3, unc-5, unc-6, unc-27, unc-30, unc-34, unc-40, unc-55, unc-61, unc-62, unc-98, and unc-104, result in less severe defects. Specifically, unc-34, unc-76, and unc-71 are considered necessary for normal longitudinal axon elongation and fasciculation (180). unc-14, unc-33, unc-44, and unc-73 affect multiple aspects of axonogenesis, namely, circumferential growth, elongation fasciculation, and axonal ultrastructure (181). unc-53 and unc-73 are particularly involved in the proper guidance of PLMs; PLM outgrowth is affected in mutant animals, and neurons veer abnormally into the ventral nerve cord (183, 448). unc-59 and unc-85 affect cytokinesis, and mutant animals show more than one axonal process (191). Ventral growth of AVM is affected by mutations in unc-1, unc-5, unc-6, unc-27, unc-34, unc-51, unc-55, unc-59, and unc-61 (181, 289). unc-6, unc-13, unc-33, unc-44, unc-51, unc-61, unc-71, unc-73, and unc-98 mutants show abnormal PLM processes among other defects (181, 306, 382).
The picture that emerges is that the concerted activity of genes forming a complex pattern of regulatory relationships, specifies touch cell fate, restricts this fate to the appropriate cells, and ensures their proper development, anatomic integrity, and fine structure. We further elaborate on the genes that function within the touch receptors themselves to provide mechanosensory characteristics in the following section.
3. Genetics and molecular biology of the gentle body touch response: the mec genes
To identify molecules dedicated to touch transduction, Brenner, Chalfie, Dew, Sulston, and colleagues (59, 60, 64, 108, 162, 193, 395) mounted a tour-de-force exercise of forward genetics, to isolate gentle body touch-insensitive nematode mutants. Briefly, populations of wild-type, touch-sensitive animals were mutagenized, and touch-insensitive individuals were sought among their descendants by stroking with an eyelash hair and prodding with a platinum wire (58). During the course of this very tedious screening process, over 417 mutations in 17 different genes, randomly distributed in all six chromosomes of C. elegans, were isolated (103, 402). By design, the screen yields mutations in genes that are fairly specific for normal gentle body touch perception. For example, gene mutations with pleotropic effects that result in lethality or uncoordinated and paralyzed phenotypes would have been missed. In addition to being touch insensitive, mec mutants tend to be lethargic when grown normally in the presence of ample food (103). Reduced spontaneous movement is probably due to their inability to sense microvibrations in their environment, interaction with external objects, or stretch produced by the locomotory movements themselves. However, when starved or during mating they move as well as wild type. The 17 genes isolated are designated as the mec genes for their "mechanosensory abnormal" phenotype (see Table 1). It is likely that the screen has reached statistical saturation, since multiple alleles have been isolated for each gene (103, 190). Corroborating the high specificity of the screen, while most of the alleles generated cause complete touch insensitivity, only a few other abnormalities accompany the mutants (103). As expected, certain types of mutations are missing from the mec collection. For example, mutations that specifically disrupt cell migrations and process outgrowth were not isolated. Thus genes that dictate aspects of process extension and precursor cell positioning, which affect the patterning of the nervous system in general rather than being touch cell specific, have not been identified with the screen. Furthermore, genes that are required specifically for the function of the anterior or posterior touch cells also have not been identified (59, 103). Hence, despite the fact that various touch neurons have distinct morphologies and positions, they appear to operate under the same set of instructions.
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A) REGULATORY/CELL-SPECIFICATION GENES. mec genes that control the expression of other mec genes or genes specific to the touch receptor neurons belong to this category. We also discuss here mec genes encoding putative regulators or modifiers of the activity of the touch receptor mechanotransducer. The key member of
