Impaired mechanical, heat, and cold nociception in a murine model of genetic TACE/ADAM17 knockdown.

TNF-α-converting enzyme, a member of the ADAM (A disintegrin and metalloproteinase) protease family and also known as ADAM17, regulates inflammation and regeneration in health and disease. ADAM17 targets are involved in pain development and hypersensitivity in animal models of inflammatory and neuropathic pain. However, the role of ADAM17 in the pain pathway is largely unknown. Therefore, we used the hypomorphic ADAM17 (ADAM17ex/ex) mouse model to investigate the importance of ADAM17 in nociceptive behavior, morphology, and function of primary afferent nociceptors. ADAM17ex/ex mice were hyposensitive to noxious stimulation, showing elevated mechanical thresholds as well as impaired heat and cold sensitivity. Despite these differences, skin thickness and innervation were comparable to controls. Although dorsal root ganglia of ADAM17ex/ex mice exhibited normal morphology of peptidergic and nonpeptidergic neurons, a small but significant reduction in the number of isolectin ß-4-positive neurons was observed. Functional electrical properties of unmyelinated nociceptors showed differences in resting membrane potential, afterhyperpolarization, and firing patterns in specific subpopulations of sensory neurons in ADAM17ex/ex mice. However, spinal cord morphology and microglia activity in ADAM17ex/ex mice were not altered. Our data suggest that ADAM17 contributes to the processing of painful stimuli, with a complex mode of action orchestrating the function of neurons along the pain pathway.-Quarta, S., Mitric, M., Kalpachidou, T., Mair, N., Schiefermeier-Mach, N., Andratsch, M., Qi, Y., Langeslag, M., Malsch, P., Rose-John, S., Kress, M. Impaired mechanical, heat, and cold nociception in a murine model of genetic TACE/ADAM17 knockdown.

PACAP and its role in primary headaches.

Pituitary adenylate cyclase-activating peptide (PACAP) is a neuropeptide implicated in a wide range of functions, such as nociception and in primary headaches. Regarding its localization, PACAP has been observed in the sensory trigeminal ganglion (TG), in the parasympathetic sphenopalatine (SPG) and otic ganglia (OTG), and in the brainstem trigeminocervical complex. Immunohistochemistry has shown PACAP-38 in numerous cell bodies of SPG/OTG, co-stored with vasoactive intestinal peptide (VIP), nitric oxide synthase (NOS) and, to a minor degree, with choline acetyltransferase. PACAP has in addition been found in a subpopulation of calcitonin gene-related peptide (CGRP)-immunoreactive cells in the trigeminal system. The PACAP/VIP receptors (PAC1, VPAC1, and VPAC2) are present in sensory neurons and in vascular smooth muscle related to the trigeminovascular system. It is postulated that PACAP is involved in nociception. In support, abolishment of PACAP synthesis or reception leads to diminished pain responses, whereas systemic PACAP-38 infusion triggers pain behavior in animals and delayed migraine-like attacks in migraine patients without marked vasodilatory effects. In addition, increased plasma levels have been documented in acute migraine attacks and in cluster headache, in accordance with findings in experimental models of trigeminal activation. This suggest that the activation of the trigeminal system may result in elevated venous levels of PACAP, a change that can be reduced when headache is treated. The data presented in this review indicate that PACAP and its receptors may be promising targets for migraine therapeutics.

α-Conotoxin Vc1.1 inhibits human dorsal root ganglion neuroexcitability and mouse colonic nociception via GABAB receptors.

OBJECTIVE: α-Conotoxin Vc1.1 is a small disulfide-bonded peptide from the venom of the marine cone snail Conus victoriae. Vc1.1 has antinociceptive actions in animal models of neuropathic pain, but its applicability to inhibiting human dorsal root ganglion (DRG) neuroexcitability and reducing chronic visceral pain (CVP) is unknown. DESIGN: We determined the inhibitory actions of Vc1.1 on human DRG neurons and on mouse colonic sensory afferents in healthy and chronic visceral hypersensitivity (CVH) states. In mice, visceral nociception was assessed by neuronal activation within the spinal cord in response to noxious colorectal distension (CRD). Quantitative-reverse-transcription-PCR, single-cell-reverse-transcription-PCR and immunohistochemistry determined γ-aminobutyric acid receptor B (GABABR) and voltage-gated calcium channel (CaV2.2, CaV2.3) expression in human and mouse DRG neurons. RESULTS: Vc1.1 reduced the excitability of human DRG neurons, whereas a synthetic Vc1.1 analogue that is inactive at GABABR did not. Human DRG neurons expressed GABABR and its downstream effector channels CaV2.2 and CaV2.3. Mouse colonic DRG neurons exhibited high GABABR, CaV2.2 and CaV2.3 expression, with upregulation of the CaV2.2 exon-37a variant during CVH. Vc1.1 inhibited mouse colonic afferents ex vivo and nociceptive signalling of noxious CRD into the spinal cord in vivo, with greatest efficacy observed during CVH. A selective GABABR antagonist prevented Vc1.1-induced inhibition, whereas blocking both CaV2.2 and CaV2.3 caused inhibition comparable with Vc1.1 alone. CONCLUSIONS: Vc1.1-mediated activation of GABABR is a novel mechanism for reducing the excitability of human DRG neurons. Vc1.1-induced activation of GABABR on the peripheral endings of colonic afferents reduces nociceptive signalling. The enhanced antinociceptive actions of Vc1.1 during CVH suggest it is a novel candidate for the treatment for CVP.

Distinct afferent innervation patterns within the human proximal and distal esophageal mucosa.

Little is known about the mucosal phenotype of the proximal human esophagus. There is evidence to suggest that the proximal esophagus is more sensitive to chemical and mechanical stimulation compared with the distal. This may have physiological relevance (e.g., in prevention of aspiration of gastroesophageal refluxate), but also pathological relevance (e.g., in reflux perception or dysphagia). Reasons for this increased sensitivity are unclear but may include impairment in mucosal barrier integrity or changes in sensory innervation. We assessed mucosal barrier integrity and afferent nerve distribution in the proximal and distal esophagus of healthy human volunteers. In 10 healthy volunteers baseline proximal and distal esophageal impedance was measured in vivo. Esophageal mucosal biopsies from the distal and proximal esophagus were taken, and baseline transepithelial electrical resistance (TER) was measured in Ussing chambers. Biopsies were examined immunohistochemically for presence and location of calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers. In a further four healthy volunteers we investigated for colocalization of CGRP and protein gene product (PGP) 9.5 immunoreactivity in nerve fibers. Baseline impedance was higher in the proximal than in the distal esophagus [2,936 Ω (SD578) vs. 2,229 Ω (SD821); P = 0.03], however, baseline TER was not significantly different between them. Mucosal CGRP-immunoreactive nerves were found in the epithelium of both proximal and distal esophagus, but were located more superficially in the proximal mucosa compared with the distal [11.5 (SD7) vs. 21.7 (SD5) cell layers from lumen, P = 0.002] 19% of proximal, and 10% of distal mucosal PGP-immunoreactive fibers colocalized with CGRP. PGP-immunoreactive fibers were also significantly closer to the luminal surface in the proximal compared with the distal esophagus (P < 0.001). We conclude that mucosal barrier integrity is similar in proximal and distal esophagus, but proximal mucosal afferent nerves are in a more superficial location. The enhanced sensitivity to reflux-evoked symptoms of the proximal esophagus most likely has an anatomical basis.

Escape behavior elicited by single, channelrhodopsin-2-evoked spikes in zebrafish somatosensory neurons.

Somatosensory neurons in teleosts and amphibians are sensitive to thermal, mechanical, or nociceptive stimuli [1, 2]. The two main types of such cells in zebrafish--Rohon-Beard and trigeminal neurons--have served as models for neural development [3-6], but little is known about how they encode tactile stimuli. The hindbrain networks that transduce somatosensory stimuli into a motor output encode information by using very few spikes in a small number of cells [7], but it is unclear whether activity in the primary receptor neurons is similarly efficient. To address this question, we manipulated the activity of zebrafish neurons with the light-activated cation channel, Channelrhodopsin-2 (ChR2) [8, 9]. We found that photoactivation of ChR2 in genetically defined populations of somatosensory neurons triggered escape behaviors in 24-hr-old zebrafish. Electrophysiological recordings from ChR2-positive trigeminal neurons in intact fish revealed that these cells have extremely low rates of spontaneous activity and can be induced to fire by brief pulses of blue light. Using this technique, we find that even a single action potential in a single sensory neuron was at times sufficient to evoke an escape behavior. These results establish ChR2 as a powerful tool for the manipulation of neural activity in zebrafish and reveal a degree of efficiency in coding that has not been found in primary sensory neurons.

Longitudinal intravital imaging nerve degeneration and sprouting in the toes of spared nerve injured mice.

Neuropathic pain is pain caused by damage to the somatosensory nervous system. Both degenerating injured nerves and neighboring sprouting nerves can contribute to neuropathic pain. However, the mesoscale changes in cutaneous nerve fibers over time after the loss of the parent nerve has not been investigated in detail. In this study, we followed the changes in nerve fibers longitudinally in the toe tips of mice that had undergone spared nerve injury (SNI). Nav1.8-tdTomato, Thy1-GFP and MrgD-GFP mice were used to observe the small and large cutaneous nerve fibers. We found that peripheral nerve plexuses degenerated within 3 days of nerve injury, and free nerve endings in the epidermis degenerated within 2 days. The timing of degeneration paralleled the initiation of mechanical hypersensitivity. We also found that some of the Nav1.8-positive nerve plexuses and free nerve endings in the fifth toe survived, and sprouting occurred mostly from 7 to 28 days. The timing of the sprouting of nerve fibers in the fifth toe paralleled the maintenance phase of mechanical hypersensitivity. Our results support the hypotheses that both injured and intact nerve fibers participate in neuropathic pain, and that, specifically, nerve degeneration is related to the initiation of evoked pain and nerve sprouting is related to the maintenance of evoked pain.

Whole-brain efferent and afferent connectivity of mouse ventral tegmental area melanocortin-3 receptor neurons.

The mesolimbic dopamine (DA) system is involved in the regulation of multiple behaviors, including feeding, and evidence demonstrates that the melanocortin system can act on the mesolimbic DA system to control feeding and other behaviors. The melanocortin-3 receptor (MC3R) is an important component of the melanocortin system, but its overall role is poorly understood. Because MC3Rs are highly expressed in the ventral tegmental area (VTA) and are likely to be the key interaction point between the melanocortin and mesolimbic DA systems, we set out to identify both the efferent projection patterns of VTA MC3R neurons and the location of the neurons providing afferent input to them. VTA MC3R neurons were broadly connected to neurons across the brain but were strongly connected to a discrete set of brain regions involved in the regulation of feeding, reward, and aversion. Surprisingly, experiments using monosynaptic rabies virus showed that proopiomelanocortin (POMC) and agouti-related protein (AgRP) neurons in the arcuate nucleus made few direct synapses onto VTA MC3R neurons or any of the other major neuronal subtypes in the VTA, despite being extensively labeled by general retrograde tracers injected into the VTA. These results greatly contribute to our understanding of the anatomical interactions between the melanocortin and mesolimbic systems and provide a foundation for future studies of VTA MC3R neurons and the circuits containing them in the control of feeding and other behaviors.

Identification of E2/E3 ubiquitinating enzymes and caspase activity regulating Drosophila sensory neuron dendrite pruning.

Ubiquitin-proteasome system (UPS) is a multistep protein degradation machinery implicated in many diseases. In the nervous system, UPS regulates remodeling and degradation of neuronal processes and is linked to Wallerian axonal degeneration, though the ubiquitin ligases that confer substrate specificity remain unknown. Having shown previously that class IV dendritic arborization (C4da) sensory neurons in Drosophila undergo UPS-mediated dendritic pruning during metamorphosis, we conducted an E2/E3 ubiquitinating enzyme mutant screen, revealing that mutation in ubcD1, an E2 ubiquitin-conjugating enzyme, resulted in retention of C4da neuron dendrites during metamorphosis. Further, we found that UPS activation likely leads to UbcD1-mediated degradation of DIAP1, a caspase-antagonizing E3 ligase. This allows for local activation of the Dronc caspase, thereby preserving C4da neurons while severing their dendrites. Thus, in addition to uncovering E2/E3 ubiquitinating enzymes for dendrite pruning, this study provides a mechanistic link between UPS and the apoptotic machinery in regulating neuronal process remodeling.

Interferon gamma gene expression in sensory neurons: evidence for autocrine gene regulation.

We explored expression and possible function of interferon-gamma (IFN-gamma) in cultured fetal (E15) rat dorsal root ganglion neurons combining whole cell patch-clamp electrophysiology with single cell reverse transcriptase polymerase chain reaction and confocal laser immunocytochemistry. Morphologically, we located IFN-gamma protein in the cytoplasm of the neurons in culture as well as in situ during peri- and postnatal development. Transcripts for classic IFN-gamma and for its receptor were determined in probes of cytoplasm sampled from individual cultured neurons, which had been identified by patch clamp electrophysiology. In addition, the cultured neurons expressed both chains of the IFN-gamma receptor. Locally produced IFN-gamma acts back on its cellular source. Phosphorylation and nuclear translocation of the IFN-inducible transcriptional factor STAT1 as well as IFN-gamma-dependent expression of major histocompatibility complex class I molecules on the neuronal membrane were noted in untreated cultures. However, both processes were substantially blocked in the presence of antibodies neutralizing IFN-gamma. Our findings indicate a role of IFN-gamma in autocrine regulation of sensory neurons.

Identification of intrinsic primary afferent neurons in mouse jejunum.

BACKGROUND: The gut is the only organ system with intrinsic neural reflexes. Intrinsic primary afferent neurons (IPANs) of the enteric nervous system initiate intrinsic reflexes, form gut-brain connections, and undergo considerable neuroplasticity to cause digestive diseases. They remain inaccessible to study in mice in the absence of a selective marker. Advillin is used as a marker for primary afferent neurons in dorsal root ganglia. The aim of this study was to test the hypothesis that advillin is expressed in IPANs of the mouse jejunum. METHODS: Advillin expression was assessed with immunohistochemistry and using transgenic mice expressing an inducible Cre recombinase under the advillin promoter were used to drive tdTomato and the genetically encoded calcium indicator GCaMP5. These mice were used to characterize the morphology and physiology of advillin-expressing enteric neurons using confocal microscopy, calcium imaging, and whole-cell patch-clamp electrophysiology. KEY RESULTS: Advillin is expressed in about 25% of myenteric neurons of the mouse jejunum, and these neurons demonstrate the requisite properties of IPANs. Functionally, they demonstrate calcium responses following mechanical stimuli of the mucosa and during antidromic action potentials. They have Dogiel type II morphology with neural processes that mostly remain within the myenteric plexus, but also project to the mucosa and express NeuN and calcitonin gene-related peptide (CGRP), but not nNOS. CONCLUSIONS AND INFERENCES: Advillin marks jejunal IPANs providing accessibility to this important neuronal population to study and model digestive disease.

Altered expression of P2X3 in vagal and spinal afferents following esophagitis in rats.

Purinergic P2X(3) receptors are predominantly expressed in small diameter primary afferent neurons and activation of these receptors by adenosine triphosphate is reported to play an important role in nociceptive signaling. The objective of this study was to investigate the expression of P2X(3) receptors in spinal and vagal sensory neurons and esophageal tissues following esophagitis in rats. Two groups of rats were used including 7 days fundus-ligated (7D-ligated) esophagitis and sham-operated controls. Esophagitis was produced by ligating the fundus and partial obstruction of pylorus that initiated reflux of gastric contents. The sham-operated rats underwent midline incision without surgical manipulation of the stomach. Expressions of P2X(3) receptors in thoracic dorsal root ganglia (DRGs), nodose ganglia (NGs), and esophageal tissues were evaluated by RT-PCR, western blot and immunohistochemistry. Esophageal neurons were identified by retrograde transport of Fast Blue from the esophagus. There were no significant differences in P2X(3) mRNA expressions in DRGs (T1-T3) and NGs between 7D-ligated and sham-operated rats. However, there was an upregulation of P2X(3) mRNA in DRGs (T6-T12) and in the esophageal muscle. At protein level, P2X(3) exhibited significant upregulation both in DRGs and in NGs of rats having chronic esophagitis. Immunohistochemical analysis exhibited a significant increase in P2X(3) and TRPV1 co-expression in DRGs and NGs in 7D-ligated rats compared to sham-operated rats. The present findings suggest that chronic esophagitis results in upregulation of P2X(3) and its co-localization with TRPV1 receptor in vagal and spinal afferents. Changes in P2X(3) expression in vagal and spinal sensory neurons may contribute to esophageal hypersensitivity following acid reflux-induced esophagitis.

Depletion of 43-kD growth-associated protein in primary sensory neurons leads to diminished formation and spreading of growth cones.

The 43-kD growth-associated protein (GAP-43) is a major protein kinase C (PKC) substrate of growing axons, and of developing nerve terminals and glial cells. It is a highly hydrophilic protein associated with the cortical cytoskeleton and membranes. In neurons it is rapidly transported from the cell body to growth cones and nerve terminals, where it accumulates. To define the role of GAP-43 in neurite outgrowth, we analyzed neurite regeneration in cultured dorsal root ganglia (DRG) neurons that had been depleted of GAP-43 with any of three nonoverlapping antisense oligonucleotides. The GAP-43 depletion procedure was specific for this protein and an antisense oligonucleotide to the related PKC substrate MARCKS did not detectably affect GAP-43 immunoreactivity. We report that neurite outgrowth and morphology depended on the levels of GAP-43 in the neurons in a substrate-specific manner. When grown on a laminin substratum, GAP-43-depleted neurons extended longer, thinner and less branched neurites with strikingly smaller growth cones than their GAP-43-expressing counterparts. In contrast, suppression of GAP-43 expression prevented growth cone and neurite formation when DRG neurons were plated on poly-L-ornithine. These findings indicate that GAP-43 plays an important role in growth cone formation and neurite outgrowth. It may be involved in the potentiation of growth cone responses to external signals affecting process formation and guidance.

Localization of Drosophila retinal degeneration B, a membrane-associated phosphatidylinositol transfer protein.

The Drosophila retinal degeneration B (rdgB) mutation causes abnormal photoreceptor response and light-enhanced retinal degeneration. Immunoblots using polyclonal anti-rdgB serum showed that rdgB is a 160-kD membrane protein. The antiserum localized the rdgB protein in photoreceptors, antennae, and regions of the Drosophila brain, indicating that the rdgB protein functions in many sensory and neuronal cells. In photoreceptors, the protein localized adjacent to the rhabdomeres, in the vicinity of the subrhabdomeric cisternae. The rdgB protein's amino-terminal 281 residues are > 40% identical to the rat brain phosphatidylinositol transfer protein (PI-TP). A truncated rdgB protein, which contains only this amino-terminal domain, possesses a phosphatidylinositol transfer activity in vitro. The remaining 773 carboxyl terminal amino acids have additional functional domains. Nitrocellulose overlay experiments reveal that an acidic amino acid domain, adjacent to the PI transfer domain, binds 45Ca+2. Six hydrophobic segments are found in the middle of the putative translation product and likely function as membrane spanning domains. These results suggest that the rdgB protein, unlike the small soluble PI-TPs, is a membrane-associated PI-TP, which may be directly regulated by light-induced changes in intracellular calcium.

Axonal neuregulin signals cells of the oligodendrocyte lineage through activation of HER4 and Schwann cells through HER2 and HER3.

We are interested in the signaling between axons and glia that leads to myelination and maintenance of the myelin internode, and we have focused on the role of neuregulins and their receptors. Neuregulins are a family of ligands that includes heregulin, neu differentiation factor, glial growth factor, and the acetylcholine receptor-inducing activity. Three signal transducing transmembrane receptors for neuregulins, which bear significant homology to the EGF receptor, are currently known: HER2 (erbB2), HER3 (erbB3), and HER4 (erbB4). We have found that oligodendrocite-type II astrocyte (O2A) progenitor cells and mature oligodendrocytes express HER2 and HER4 but no HER3. Schwann cells express HER2 and HER3 but little HER4. In O2A progenitor cells and oligodendrocytes, recombinant neuregulin induces the rapid tyrosine phosphorylation of only HER4. HER2 is not phosphorylated in cells of the oligodendrocyte lineage, but a physical interaction between HER2 and HER4 was detected in coimmunoprecipitation experiments. In Schwann cells, neuregulin induces the phosphorylation of both HER2 and HER3. Coimmunoprecipitation experiments indicate that receptor activation in Schwann cells results in the formation of HER2:HER3 heterodimers. Neuregulin localized immunocytochemically was present on neurites of cultured dorsal root ganglion neurons, and it was released into the medium in a form that promoted receptor tyrosine phosphorylation. Neuregulins therefore meet important criteria expected of molecules involved in axonal-glial signaling. The use of unique neuregulin receptor combinations in oligodendrocytes and Schwann cells likely results in recruitment of different signaling pathways and thus provides a basis for different biological responses.

Identification of a peptide specific for Aplysia sensory neurons by PCR-based differential screening.

In order to identify genes specific for the sensory neurons of Aplysia, a miniaturized differential screening method based on the polymerase chain reaction and applicable to small amounts of tissue was used. One messenger RNA was isolated that is expressed in every mechanoreceptor sensory cluster of the Aplysia central nervous system. This messenger RNA encodes a peptide that seems to function as an inhibitory cotransmitter. The peptide selectively inhibits certain postsynaptic cells but not others and thereby allows the sensory neurons to achieve target-specific synaptic actions.

The LIM homeobox gene ceh-14 confers thermosensory function to the AFD neurons in Caenorhabditis elegans.

In Caenorhabditis elegans three pairs of neurons, AFD, AIY, and AIZ, play a key role in thermosensation. The LIM homeobox gene ceh-14 is expressed in the AFD thermosensory neurons. ceh-14 mutant animals display athermotactic behaviors, although the neurons are still present and differentiated. Two other LIM homeobox genes, ttx-3 and lin-11, function in the two interneurons AIY and AIZ, respectively. Thus, the three key thermosensory neurons are specified by three different LIM homeobox genes. ceh-14 ttx-3 lin-11 triple mutant animals have a basic cryophilic thermotaxis behavior indicative of a second thermotaxis pathway. Misexpression of ceh-14 in chemosensory neurons can restore thermotactic behavior without impairing the chemosensory function. Thus, ceh-14 confers thermosensory function to neurons.

A regulatory subunit of the cAMP-dependent protein kinase down-regulated in aplysia sensory neurons during long-term sensitization.

Binding of cAMP by the five neuronal isoforms (N1-5) of the regulatory (R) subunit of the Aplysia cAMP-dependent protein kinase is diminished in sensory neurons stimulated to produce long-term presynaptic facilitation. To determine how the cAMP-binding activity of the R subunits is lost, we isolated cDNAs encoding N4, which is a homolog of mammalian RI. Immunoblots with antisera raised against the R protein overexpressed in E. coli show that the diminished binding activity, which occurs in long-term facilitation, results from coordinate loss of R protein isoforms. No change was detected in the amount of transcripts for R subunits, suggesting that the down-regulation results from enhanced proteolytic turnover.

Chemosuppression of retinal axon growth by the mouse optic chiasm.

To determine whether diffusible guidance cues direct retinal axon growth and divergence at the optic chiasm, we cocultured mouse retinal and chiasm explants in collagen gels. The chiasm reduced retinal neurite lengths and numbers, but did not affect commissural or pontine neurite growth. This reduction in growth was equal for all retinal quadrants and occurred without reorienting the direction of neurite extension. The floor plate, another midline guidance locus, also suppressed retinal neurite outgrowth, whereas cortex or cerebellum explants did not. Growth suppression was not mediated by netrin-1, which instead enhanced retinal neurite extension. We propose that chemosuppression may be a general guidance mechanism that acts in intermediate targets to prime growth cones to perceive other, more specific cues.

neurogenin1 is essential for the determination of neuronal precursors for proximal cranial sensory ganglia.

The NEUROGENINS (NGNs) are neural-specific basic helix-loop-helix (bHLH) transcription factors. Mouse embryos lacking ngn1 fail to generate the proximal subset of cranial sensory neurons. ngn1 is required for the activation of a cascade of downstream bHLH factors, including NeuroD, MATH3, and NSCL1. ngn1 is expressed by placodal ectodermal cells and acts prior to neuroblast delamination. Moreover, NGN1 positively regulates the Delta homolog DLL1 and can be negatively regulated by Notch signaling. Thus, ngn1 functions similarly to the proneural genes in Drosophila. However, the initial pattern of ngn1 expression appears to be Notch independent. Taken together with the fact that ectopic ngn1 expression can convert ectodermal cells to neurons in Xenopus (Ma et al., 1996), these data and those of Fode et al. (1998 [this issue of Neuron]) identify ngns as vertebrate neuronal determination genes, analogous to myoD and myf5 in myogenesis.

Extracellular proteins needed for C. elegans mechanosensation.

The mec-5 and mec-9 genes encode putative extracellular proteins that allow a set of six touch receptor neurons in C. elegans to respond to gentle touch. MEC-5 is a collagen made by the epidermal cells that surround the touch cells. Mutations causing touch insensitivity affect the Gly-X-Y repeats of this collagen. mec-9 produces two transcripts, the larger of which is expressed in the touch cells and two PVD neurons. This transcript encodes a protein with 5 Kunitz-type protease inhibitor domains, 6 EGF-like repeats (2 of the Ca(2+)-binding type), and a glutamic acid-rich region. Missense mutations causing touch insensitivity affect both the EGF-like and Kunitz domains. Since mec-9 loss of function mutations dominantly enhance the touch insensitive phenotype of several mec-5 mutations, MEC-5 and MEC-9 may interact. We propose that these proteins provide an extracellular attachment point for the mechanosensory channels of the touch cells.