Ghosh S et al. (2017) Worm "Non-neuronal cell outgrowth in C. elegans."

Ghosh S, Vetrone SA, Sternberg PW

[Worm, 2017]

Cell outgrowth is a hallmark of some non-migratory developing cells during morphogenesis. Understanding the mechanisms that control cell outgrowth not only increases our knowledge of tissue and organ development, but can also shed light on disease pathologies that exhibit outgrowth-like behavior. C. elegans is a highly useful model for the analysis of genes and the function of their respective proteins. In addition, C. elegans also has several cells and tissues that undergo outgrowth during development. Here we discuss the outgrowth mechanisms of nine different C. elegans cells and tissues. We specifically focus on how these cells and tissues grow outward and the interactions they make with their environment. Through our own identification, and a meta-analysis, we also identify gene families involved in multiple cell outgrowth processes, which defined potential C. elegans core components of cell outgrowth, as well as identify a potential stepwise cell behavioral cascade used by cells undergoing outgrowth.

Ghosh DD et al. (2017) Curr Opin Neurobiol "Multisensory integration in C. elegans."

Zhang Y, Nitabach MN, Ghosh DD, Harris G

[Curr Opin Neurobiol, 2017]

Multisensory integration is a neural process by which signals from two or more distinct sensory channels are simultaneously processed to form a more coherent representation of the environment. Multisensory integration, especially when combined with a survey of internal states, provides selective advantages for animals navigating complex environments. Despite appreciation of the importance of multisensory integration in behavior, the underlying molecular and cellular mechanisms remain poorly understood. Recent work looking at how Caenorhabditis elegans makes multisensory decisions has yielded mechanistic insights into how a relatively simple and well-defined nervous system employs circuit motifs of defined features, synaptic signals and extrasynaptic neurotransmission, as well as neuromodulators in processing and integrating multiple sensory inputs to generate flexible and adaptive behavioral outputs.

Ghosh, Rajarshi et al. (2013) International Worm Meeting "Genetics of praziquantel resistance in C. elegans."

Tenenbaum, Conrad, Levinson, Anya, Kruglyak, Leonid, Ghosh, Rajarshi

[International Worm Meeting, 2013]

The anthelmintic praziquantel (PRZ) is widely used for treating parasitic flatworm infections in human. It is the most-used drug for treating schistosomasis, a debilitating fluke-borne disease. Despite this prevalence, the target of PRZ is still unknown. Dependence on a single drug with unknown target, for a disease affecting 240 million people worldwide, makes the problem of emergence of drug resistance a special concern. The target and mechanism of action of PRZ has been elusive perhaps because it is thought that nematodes are unaffected by this drug. Indeed we found that the laboratory strain N2 is resistant to PRZ relative to other wild isolates of C. elegans. However, we observed that, as in flatworms, PRZ induces spastic paralysis in several nematode species and wild isolates of C. elegans. To identify the genetic targets of PRZ, we mutagenized N2 and Hawaiian (CB4856) strains and selected mutants resistant to high dose of PRZ. We isolated nine and two mutants in the N2 and CB4856 background respectively. Through whole genome sequencing, complementation tests, RNAi and transgenic rescue we identified the first genes contributing to PRZ resistance in C.elegans. Interestingly different genes in the N2 and CB4856 background conferred PRZ resistance. Tissue specific knockdown experiments and epistatsis analysis suggested that PRZ disrupts osmoregulation in C. elegans. We also found that CB4856 animals were significantly more sensitive to PRZ than N2 animals. To identify genetic basis of individual differences in responses to PRZ, we took a quantitative trait loci mapping approach and identified a significant locus on chromosome IV. Currently we are using transgenic rescue to fine map the QTL and testing the interactions between the QTL and the genes identified through mutagenesis. Using classical and quantitative genetic approaches and by studying wild isolates beyond the laboratory strain, we uncovered novel insights into mechanism of action of PRZ .

Ghosh, Rajarshi et al. (2013) International Worm Meeting "Evolution of avermectin resistance in C. briggsae."

Kruglyak, Leonid, Cutter, Asher, Jovelin, Richard, Ghosh, Rajarshi, Wang, Wei, Thomas, Cristel

[International Worm Meeting, 2013]

Several nematode species have evolved resistance to the widely used anthelmintic avermectins (AVM). AVM is produced naturally by S.avermitilis, a ubiquitous soil bacterium. As many nematodes spend part of their life cycle in contact with soil, they are likely to encounter S.avermitilis. Widespread AVM resistance may be a result of different nematode species' ability to counter a common selective pressure, namely the toxins produced by S. avermitilis. To test this hypothesis we surveyed AVM resistance in diverse nematode species. We found that resistance to AVM and to S. avermitilis was prevalent in this phylum. To identify the genetic basis of natural AVM resistance we focused on C. briggsae. We found that two divergent isolates of C. briggsae differed significantly in their responses to AVM. Using QTL mapping approach with these two strains , we identified a significant locus on Chromosome II underlying responses to AVM. We also surveyed 50 isolates of C. briggsae for responses to AVM and found that they exhibit wide variation. The pattern of variation in responses to AVM correlated significantly with the observed phylogeographic pattern in C. briggsae, with temperate isolates being more likely to be resistant than tropical ones. To gain insights into the evolution AVM resistance in C. briggsae, we obtained whole genome sequences of these isolates. Using this data we confirmed that glc-1, the causative gene for natural AVM resistance in C. elegans, is the result of a duplication of another GluCl subunit in the elegans lineage. Thus glc-1 is absent in C. briggsae suggesting that the genetic mechanisms of natural resistance to AVM in C. briggsae are likely different from C. elegans. The sequence data will help us determine if variation in candidate targets for AVM correlate with the pattern of resistance in C. briggsae and map the genetic basis of differences in responses to AVM in C. briggsae .

Ghosh, D. D. et al. (2015) International Worm Meeting "Neuroendocrine reinforcement of a dynamic multisensory decision."

Sanders, T., Chase, D.L., Hong, S., Koelle, M.R., Ghosh, D. D., Cohen, N., Nitabach, M.N.

[International Worm Meeting, 2015]

To navigate complex natural environments containing both dangerous and valuable items, animals must make economic decisions on the basis of information transduced by multiple senses. However, detailed underlying neural mechanisms of multisensory decision making remain poorly understood. Here we confronted worms with a multisensory decision in which the reward of food must be balanced with the threat of desiccation imposed by a hyperosmotic barrier intervening between the worm and a source of food odor. We find that this decision is modulated by food deprivation. To identify neural substrates underlying this decision, we focused on the RIM interneuron, which is advantageously positioned to transduce integrated multisensory information into locomotor outputs. Consistent with this hypothesis, we find that the activation of a neuropeptide receptor in RIM sets the balance of threat and reward in this decision, with greater receptor activation biasing the worm against crossing the dangerous barrier. Unexpectedly, however, RIM controls the decision not by synaptic signaling to the downstream premotor command circuit, but rather by extrasynaptic aminergic signaling directly onto the primary osmosensory neuron to tune its sensitivity. Additionally, our results suggest that this neuromodulator relay is suppressed in food deprived states, thereby providing a link between internal state, neural network activity, and decision making. Finally, to characterize the complex and dynamic interplay between neuromodulator activity, neuron state, and behavior in the decision making arena, we reproduced the paradigm in silico [1]. Computational modeling revealed how non-linear sensory integration in RIM modulates neuromodulator circuit activity to implement the decision. Taken together, these studies reveal a cellular and molecular mechanism for a dynamic multisensory decision. Intriguingly, our results identified organizational circuit principles conserved between mammalian and C. elegans multisensory decision making. Therefore our studies have broad implications for understanding principles underlying multisensory decision making in Metazoans.1Sanders, T., Ghosh, D.D., Nitabach, M.N., and Cohen, N. "Nonlinear sensory integration in C. elegans: a computational model." International C. elegans meeting.

Ghosh I et al. (1998) Mol Biochem Parasitol "Thioredoxin peroxidases from Brugia malayi."

Scott AL, Eisinger SW, Raghavan N, Ghosh I

[Mol Biochem Parasitol, 1998]

Parasite-derived antioxidant proteins have been implicated in playing an important role in protection against the oxygen radicals that are generated during aerobic metabolism and in defense against host immune cell attack. Here we report that filarial nematodes include the thioredoxin peroxidase/thiol-specific antioxidant (TPx/TSA) family of antioxidant proteins as part of their complex defense against radical-mediated damage. At the protein level, the TPx/TSA from Brugia malayi (Bm-TPx-1) was approximately 50% identical and approximately 60% similar to TPx/TSAs from mammals, amphibians and yeast. Bm-TPx-1 was also approximately 60% identical to putative TPx proteins from a related filarial nematode, Onchocerca volvulus, and from the free-living nematode Caenorhabditis elegans. That B. malayi may express multiple forms of molecules with TPx/TSA activity was indicated by the identification of a B. malayi gene encoding a second, distinct member of the TPx/TSA family (Bm-tpx-2). Bm-tpx-1 was found to be transcribed in all stages of the parasite present in the mammalian host and the 25 kDa translation product was present in all of the developmental stages studied. The results of immunohistochemical, immunofluorescent and immunoprecipitation studies showed Bm-TPx-1 to be localized in the cells of the hypodermis/lateral chord in adult parasites and not to be present at the surface or in excretory/secretory products. The distribution in the parasite suggests that Bm-TPx-1 may play its major role in countering radicals produced within cells. A recombinant form of Bm-TPx-1 was biologically active and capable of protecting DNA from oxygen radical-mediated damage. Thioredoxin peroxidases may prove to be a critical component in the parasite's defense against injury caused by oxygen radicals derived from endogenous and exogenous sources.

Ghosh DD et al. (2016) Neuron "Neural Architecture of Hunger-Dependent Multisensory Decision Making in C.elegans."

Ghosh DD, Sanders T, Koelle MR, Chase DL, McCurdy LY, Nitabach MN, Cohen N, Hong S

[Neuron, 2016]

Little is known about how animals integrate multiple sensory inputs in natural environments to balance avoidance of danger with approach to things of value. Furthermore, the mechanistic link between internal physiological state and threat-reward decision making remains poorly understood. Here we confronted C.elegans worms with the decision whether to cross a hyperosmotic barrier presenting the threat of desiccation to reach a source of food odor. We identified a specific interneuron that controls this decision via top-down extrasynaptic aminergic potentiation of the primary osmosensory neurons to increase their sensitivity to the barrier. We also establish that food deprivation increases the worm's willingness to cross the dangerous barrier by suppressing this pathway. These studies reveal a potentially general neural circuit architecture for internal state control of threat-reward decision making.

Ghosh Roy, A. et al. (2009) International Worm Meeting "Dissecting Calcium/cAMP mediated axon regeneration pathways."

Jin, Y., Goncharov, A., Chisholm, A.D., Ghosh Roy, A., Wu, Z.

[International Worm Meeting, 2009]

We are interested in the molecular pathways that promote the regenerative response of axons after injury. Injury of axons of cultured neurons causes a transient elevation of intracellular calcium. Pharmacological elevation of calcium and cAMP are long known to enhance the ability of axons to regenerate in vitro and in vivo. However the molecular mechanisms that govern the Ca/cAMP mediated axonal regeneration are not clearly understood. We previously reported that the lateral touch neurons ALM and PLM show robust regenerative responses after injury (Wu et al., PNAS, 2007). We report here that calcium and cAMP play critical roles in the regenerative responses of these neurons. Using genetically encoded Ca sensors we have found that laser axotomy triggers a rapid local elevation of intracellular calcium that propagates bidirectionally away from the axotomy site. The amplitude of this calcium transient correlates with the extent of total axonal regrowth. Further, gain of function in the voltage-gated calcium channel, egl-19 increases the amplitude of the calcium transient and accelerates regenerative growth, suggesting calcium levels are a critical determinant of regrowth kinetics. We find that genetic elevation of calcium enhances regeneration in several ways. The injured axon enters the regeneration phase earlier compared to controls, extends faster, and more often fuses with the severed distal fragment. Our ultrastructural analysis shows that in these cases the regrowing axon has physically fused membranes with the distal fragment. This fusion process appears not to require known fusogens such as EFF-1 or AFF-1. Mutations that chronically elevate neuronal cAMP promote regeneration in a similar manner with the exception that the regenerating process also has an increased tendency to extend ventral synaptic branches. Reduction of neuronal cAMP by overexpression of PDE-4 blocked this ventral branch regrowth, indicating that cAMP functions cell autonomously to promote growth. Activation of protein kinase A has similar effects on regeneration, suggesting PKA is the major effector of cAMP. We further find that different bZip transcription factors are required for distinct aspects of the regenerative response, suggesting calcium/cAMP acts via multiple transcription factors. Our studies indicate that calcium and cAMP have conserved signaling roles and are rate limiting for regeneration. We are currenty exploring the relationship between calcium/cAMP signaling and other regeneration pathways.

Shipa R Ghosh et al. (2005) International Worm Meeting "Identifying novel components of muscle in C. elegans"

Ian A Hope, Shipa R Ghosh

[International Worm Meeting, 2005]

The smooth sinusoidal movements of C. elegans are effected by 95 obliquely striated bodywall muscle cells. There are also non-striated single-sarcomere muscles; the pharyngeal, intestinal, anal and sex-specific muscles. Force generated by the myofilament lattice of bodywall muscle is transmitted to the external cuticle through the attachment structures of the dense bodies, M-lines and fibrous organelles. Attachment structures are also present in the single-sarcomere muscle cells. Additional components of the lattice and associated attachment structures may be identified by investigating the subcellular localisation of endogenous proteins fused to GFP. The twin resources of the Promoterome and ORFeome are being used to construct Promoter::ORF::GFP fusions in a high-throughput manner using Multisite Gateway recombinational cloning. Transgenic lines are generated using these constructs to study the expression patterns of ORF::GFP fusion proteins in vivo. The genes selected for inclusion in this project were chosen because their respective promoters had been shown previously to drive expression of reporter genes in muscle cells. Including ORFs within Promoter::ORF::GFP constructs may reveal the subcellular localisation of the native protein. The genes W05F2.4, F07C3.4 and F02A9.3 were used to establish procedures and expression patterns will be presented. Relative localisation of the fusion proteins with well-known components of muscle structures will be investigated using reporter genes of differing colours. This would also facilitate visualisation of the various components of the contractile apparatus and associated attachment structures assembling throughout development. After initially identifying novel components, techniques such as RNAi and yeast two-hybrid experiments may be employed to clarify the roles and interactions of these proteins.

Ghosh A et al. (2024) mBio "Translation initiation or elongation inhibition triggers contrasting effects on Caenorhabditis elegans survival during pathogen infection."

Singh J, Ghosh A

[mBio, 2024]

Diverse microbial pathogens are known to attenuate host protein synthesis. Consequently, the host mounts a defense response against protein translation inhibition, leading to increased transcript levels of immune genes. The seemingly paradoxical upregulation of immune gene transcripts in response to blocked protein synthesis suggests that the defense mechanism against translation inhibition may not universally benefit host survival. However, a comprehensive assessment of host survival on pathogens upon blockage of different stages of protein synthesis is currently lacking. Here, we investigate the impact of knockdown of various translation initiation and elongation factors on the survival of Caenorhabditis elegans exposed to Pseudomonas aeruginosa. Intriguingly, we observe opposing effects on C. elegans survival depending on whether translation initiation or elongation is inhibited. While translation initiation inhibition enhances survival, elongation inhibition decreases it. Transcriptomic studies reveal that translation initiation inhibition activates a bZIP transcription factor ZIP-2-dependent innate immune response that protects C. elegans from P. aeruginosa infection. In contrast, inhibiting translation elongation triggers both ZIP-2-dependent and ZIP-2-independent immune responses that, while effective in clearing the infection, are detrimental to the host. Thus, our findings reveal the opposing roles of translation initiation and elongation inhibition in C. elegans survival during P. aeruginosa infection, highlighting distinct transcriptional reprogramming that may underlie these differences.Importance: Several microbial pathogens target host protein synthesis machinery, potentially limiting the innate immune responses of the host. In response, hosts trigger a defensive response, elevating immune gene transcripts. This counterintuitive response can have either beneficial or harmful effects on host survival. In this study, we conduct a comprehensive analysis of the impact of knocking down various translation initiation and elongation factors on the survival of Caenorhabditis elegans exposed to Pseudomonas aeruginosa. Intriguingly, inhibiting initiation and elongation factors has contrasting effects on C. elegans survival. Inhibiting translation initiation activates immune responses that protect the host from bacterial infection, while inhibiting translation elongation induces aberrant immune responses that, although clear the infection, are detrimental to the host. Our study reveals divergent roles of translation initiation and elongation inhibition in C. elegans survival during P. aeruginosa infection and identifies differential transcriptional reprogramming that could underlie these differences.