Time-lapse imaging and cell-specific expression profiling reveal dynamic branching and molecular determinants of a multi-dendritic nociceptor in C. elegans

AbstractNociceptive neurons innervate the skin with complex dendritic arbors that respond to pain-evoking stimuli such as harsh mechanical force or extreme temperatures. Here we describe the structure and development of a model nociceptor, the PVD neuron of C. elegans, and identify transcription factors that control morphogenesis of the PVD dendritic arbor. The two PVD neuron cell bodies occupy positions on either the right (PVDR) or left (PVDL) sides of the animal in posterior–lateral locations. Imaging with a GFP reporter revealed a single axon projecting from the PVD soma to the ventral cord and an elaborate, highly branched arbor of dendritic processes that envelop the animal with a web-like array directly beneath the skin. Dendritic branches emerge in a step-wise fashion during larval development and may use an existing network of peripheral nerve cords as guideposts for key branching decisions. Time-lapse imaging revealed that branching is highly dynamic with active extension and withdrawal and that PVD branch overlap is prevented by a contact-dependent self-avoidance, a mechanism that is also employed by sensory neurons in other organisms. With the goal of identifying genes that regulate dendritic morphogenesis, we used the mRNA-tagging method to produce a gene expression profile of PVD during late larval development. This microarray experiment identified>2,000 genes that are 1.5X elevated relative to all larval cells. The enriched transcripts encode a wide range of proteins with potential roles in PVD function (e.g., DEG/ENaC and Trp channels) or development (e.g., UNC-5 and LIN-17/frizzled receptors). We used RNAi and genetic tests to screen 86 transcription factors from this list and identified eleven genes that specify PVD dendritic structure. These transcription factors appear to control discrete steps in PVD morphogenesis and may either promote or limit PVD branching at specific developmental stages. For example, time-lapse imaging revealed that MEC-3 (LIM homeodomain) is required for branch initiation in early larval development whereas EGL-44 (TEAD domain) prevents ectopic PVD branching in the adult. A comparison of PVD-enriched transcripts to a microarray profile of mammalian nociceptors revealed homologous genes with potentially shared nociceptive functions. We conclude that PVD neurons display striking structural, functional and molecular similarities to nociceptive neurons from more complex organisms and can thus provide a useful model system in which to identify evolutionarily conserved determinants of nociceptor fate

Photochemistry Of Monochloro Complexes Of Copper(ii) In Methanol Probed By Ultrafast Transient Absorption Spectroscopy

Ultrafast transient absorption spectra in the deep to near UV range (212-384 nm) were measured for the [Cu-II(MeOH)(5)Cl](+) complexes in methanol following 255-nm excitation of the complex into the ligand-to-metal charge-transfer excited state. The electronically excited complex undergoes sub-200 fs radiationless decay, predominantly via back electron transfer, to the hot electronic ground state followed by fast vibrational relaxation on a 0.4-4 Ps time scale. A minor photochemical channel is Cu-Cl bond dissociation, leading to the reduction of copper(H) to copper(I) and the formation of MeOH center dot Cl charge-transfer complexes. The depletion of ground-state [Cu-II(MeOH)(5)Cl](+) perturbs the equilibrium between several forms of copper(II) complexes present in solution. Complete re-equilibration between [Cu-II(MeOH)(5)Cl](+) and [Cu-II(MeOH)(4)Cl-2] is established on a 10-500 ps time scale, slower than methanol diffusion, suggesting that the involved ligand exchange mechanism is dissociative

Ozone observations and a model of marine boundary layer photochemistry during SAGA 3

A major purpose of the third joint Soviet‐American Gases and Aerosols (SAGA 3) oceanographic cruise was to examine remote tropical marine O3 and photochemical cycles in detail. On leg 1, which took place between Hilo, Hawaii, and Pago‐Pago, American Samoa, in February and March 1990, shipboard measurements were made of O3, CO, CH4, nonmethane hydrocarbons (NMHC), NO, dimethyl sulfide (DMS), H2S, H2O2, organic peroxides, and total column O3. Postcruise analysis was performed for alkyl nitrates and a second set of nonmethane hydrocarbons. A latitudinal gradient in O3 was observed on SAGA 3, with O3 north of the intertropical convergence zone (ITCZ) at 15–20 parts per billion by volume (ppbv) and less than 12 ppbv south of the ITCZ but never ≤3 ppbv as observed on some previous equatorial Pacific cruises (Piotrowicz et al., 1986; Johnson et al., 1990). Total column O3 (230–250 Dobson units (DU)) measured from the Akademik Korolev was within 8% of the corresponding total ozone mapping spectrometer (TOMS) satellite observations and confirmed the equatorial Pacific as a low O3 region. In terms of number of constituents measured, SAGA 3 may be the most photochemically complete at‐sea experiment to date. A one‐dimensional photochemical model gives a self‐consistent picture of O3‐NO‐CO‐hydrocarbon interactions taking place during SAGA 3. At typical equatorial conditions, mean O3 is 10 ppbv with a 10–15% diurnal variation and maximum near sunrise. Measurements of O3, CO, CH4, NMHC, and H2O constrain model‐calculated OH to 9 × 105 cm−3 for 10 ppbv O3 at the equator. For DMS (300–400 parts per trillion by volume (pptv)) this OH abundance requires a sea‐to‐air flux of 6–8 × 109 cm−2 s−1, which is within the uncertainty range of the flux deduced from SAGA 3 measurements of DMS in seawater (Bates et al., this issue). The concentrations of alkyl nitrates on SAGA 3 (5–15 pptv total alkyl nitrates) were up to 6 times higher than expected from currently accepted kinetics, suggesting a largely continental source for these species. However, maxima in isopropyl nitrate and bromoform near the equator (Atlas et al., this issue) as well as for nitric oxide (Torres and Thompson, this issue) may signify photochemical and biological sources of these species

The brain is a DJ using neuropeptides as sensory crossfaders

Sensory loss induces cross-modal plasticity, often resulting in altered performance in remaining sensory modalities. Whereas much is known about the macroscopic mechanisms underlying cross-modal plasticity, only scant information exists about its cellular and molecular underpinnings. We found that Caenorhabditis elegans nematodes deprived of a sense of body touch exhibit various changes in behavior, associated with other unimpaired senses. We focused on one such behavioral alteration, enhanced odor sensation, and sought to reveal the neuronal and molecular mechanisms that translate mechanosensory loss into improved olfactory acuity. To this end, we analyzed in mechanosensory mutants food-dependent locomotion patterns that are associated with olfactory responses and found changes that are consistent with enhanced olfaction. The altered locomotion could be reversed in adults by optogenetic stimulation of the touch receptor (mechanosensory) neurons. Furthermore, we revealed that the enhanced odor response is related to a strengthening of inhibitory AWC→AIY synaptic transmission in the olfactory circuit. Consistently, inserting in this circuit an engineered electrical synapse that diminishes AWC inhibition of AIY counteracted the locomotion changes in touch-deficient mutants. We found that this cross-modal signaling between the mechanosensory and olfactory circuits is mediated by neuropeptides, one of which we identified as FLP-20. Our results indicate that under normal function, ongoing touch receptor neuron activation evokes FLP-20 release, suppressing synaptic communication and thus dampening odor sensation. In contrast, in the absence of mechanosensory input, FLP-20 signaling is reduced, synaptic suppression is released, and this enables enhanced olfactory acuity; these changes are long lasting and do not represent ongoing modulation, as revealed by optogenetic experiments. Our work adds to a growing literature on the roles of neuropeptides in cross-modal signaling, by showing how activity-dependent neuropeptide signaling leads to specific cross-modal plastic changes in neural circuit connectivity, enhancing sensory performance.status: publishe

C. elegans SWAN-1 Binds to EGL-9 and Regulates HIF-1-Mediated Resistance to the Bacterial Pathogen Pseudomonas aeruginosa PAO1

Pseudomonas aeruginosa is a nearly ubiquitous human pathogen, and infections can be lethal to patients with impaired respiratory and immune systems. Prior studies have established that strong loss-of-function mutations in the egl-9 gene protect the nematode C. elegans from P. aeruginosa PAO1 fast killing. EGL-9 inhibits the HIF-1 transcription factor via two pathways. First, EGL-9 is the enzyme that targets HIF-1 for oxygen-dependent degradation via the VHL-1 E3 ligase. Second, EGL-9 inhibits HIF-1-mediated gene expression through a VHL-1-independent mechanism. Here, we show that a loss-of-function mutation in hif-1 suppresses P. aeruginosa PAO1 resistance in egl-9 mutants. Importantly, we find stabilization of HIF-1 protein is not sufficient to protect C. elegans from P. aeruginosa PAO1 fast killing. However, mutations that inhibit both EGL-9 pathways result in higher levels of HIF-1 activity and confer resistance to the pathogen. Using forward genetic screens, we identify additional mutations that confer resistance to P. aeruginosa. In genetic backgrounds that stabilize C. elegans HIF-1 protein, loss-of-function mutations in swan-1 increase the expression of hypoxia response genes and protect C. elegans from P. aeruginosa fast killing. SWAN-1 is an evolutionarily conserved WD-repeat protein belonging to the AN11 family. Yeast two-hybrid and co-immunoprecipitation assays show that EGL-9 forms a complex with SWAN-1. Additionally, we present genetic evidence that the DYRK kinase MBK-1 acts downstream of SWAN-1 to promote HIF-1-mediated transcription and to increase resistance to P. aeruginosa. These data support a model in which SWAN-1, MBK-1 and EGL-9 regulate HIF-1 transcriptional activity and modulate resistance to P. aeruginosa PAO1 fast killing

HIF-1 Modulates Dietary Restriction-Mediated Lifespan Extension via IRE-1 in Caenorhabditis elegans

Dietary restriction (DR) extends lifespan in various species and also slows the onset of age-related diseases. Previous studies from flies and yeast have demonstrated that the target of rapamycin (TOR) pathway is essential for longevity phenotypes resulting from DR. TOR is a conserved protein kinase that regulates growth and metabolism in response to nutrients and growth factors. While some of the downstream targets of TOR have been implicated in regulating lifespan, it is still unclear whether additional targets of this pathway also modulate lifespan. It has been shown that the hypoxia inducible factor-1 (HIF-1) is one of the targets of the TOR pathway in mammalian cells. HIF-1 is a transcription factor complex that plays key roles in oxygen homeostasis, tumor formation, glucose metabolism, cell survival, and inflammatory response. Here, we describe a novel role for HIF-1 in modulating lifespan extension by DR in Caenorhabditis elegans. We find that HIF-1 deficiency results in extended lifespan, which overlaps with that by inhibition of the RSKS-1/S6 kinase, a key component of the TOR pathway. Using a modified DR method based on variation of bacterial food concentrations on solid agar plates, we find that HIF-1 modulates longevity in a nutrient-dependent manner. The hif-1 loss-of-function mutant extends lifespan under rich nutrient conditions but fails to show lifespan extension under DR. Conversely, a mutation in egl-9, which increases HIF-1 activity, diminishes the lifespan extension under DR. This deficiency is rescued by tissue-specific expression of egl-9 in specific neurons and muscles. Increased lifespan by hif-1 or DR is dependent on the endoplasmic reticulum (ER) stress regulator inositol-requiring protein-1 (IRE-1) and is associated with lower levels of ER stress. Therefore, our results demonstrate a tissue-specific role for HIF-1 in the lifespan extension by DR involving the IRE-1 ER stress pathway

The varitint-waddler (Va) deafness mutation in TRPML3 generates constitutive, inward rectifying currents and causes cell degeneration

Varitint-waddler (Va and VaJ) mice are deaf and have vestibular impairment, with inner ear defects that include the degeneration and loss of sensory hair cells. The semidominant Va mutation results in an alanine-to-proline substitution at residue 419 (A419P) of the presumed ion channel TRPML3. Another allele, VaJ, has the A419P mutation in addition to an I362T mutation. We found that hair cells, marginal cells of stria vascularis, and other cells lining the cochlear and vestibular endolymphatic compartments express TRPML3. When heterologously expressed in LLC-PK1-CL4 epithelial cells, a culture model for hair cells, TRPML3 accumulated in lysosomes and in espin-enlarged microvilli that resemble stereocilia. We also demonstrated that wild-type TRPML3 forms channels that are blocked by Gd3+, have a conductance of 50–70 pS and, like many other TRP channels, open at very positive potentials and thus rectify outwardly. In addition to this outward current, TRPML3(419P) and (I362T+A419P) generated a constitutive inwardly rectifying current that suggests a sensitivity to hyperpolarizing negative potentials and that depolarized the cells. Cells expressing TRPML3(A419P) or (I362T+A419P), but not wild-type TRPML3, died and were extruded from the epithelium in a manner reminiscent of degenerating hair cells in Va mice. The increased open probability of TRPML3(A419P) and (I362T+A419P) at physiological potentials likely underlies hair cell degeneration and deafness in Va and VaJ mice