Singh, A. et al. (2019) International Worm Meeting "Role of neuronal-peptides in intestinal and organismal aging."

Singh, A., Hansen , M., Chalasani , S.

[International Worm Meeting, 2019]

Inter-tissue communications are vital for organism development and survival. This becomes particularly relevant in the context of aging, as various tissues have been shown to age at different rates. One particular challenge is to identify age-altered, cross-tissue signals that can modulate organ health and organismal survival. To identify such signals, we engineered C. elegans for tissue-specific RNAi to probe neuron-to-intestine peptide signals in modulating intestinal health. We focused on the readily quantifiable age-associated increase in intestinal leakiness, which is conserved from worms to mammals. Interestingly, we found that abrogating the secretion of neuronal peptides delayed age-related intestinal leakiness, and extended survival. These findings imply that neuronal peptides might play a role in timing intestinal aging. Next, we posited that neuronal peptides likely exert their effect on intestinal health by signaling through, either G-coupled protein receptor- (GPCR-) or non-GPCR class of peptide-receptors. Interestingly, the intestine-specific knockdown of both G-protein gamma subunit encoding genes, as well as the insulin/IGF-1-like receptor daf-2,cell-autonomously delayed age-associated intestinal leakiness. These results indicate that aged neurons may release peptide signals that are sensed by DAF-2/InR and/or possibly GPCR-like receptors on the intestine, modifying the integrity of intestinal cells in aged animals. To identify the specific neuronal peptide(s) that exert a pro-aging effect on intestine, we knocked-down individual insulin-like peptides specifically in the neurons. Our preliminary data indicate that knockdown of at least one insulin gene delayed the age-related intestinal leakiness, similar to the effect of blocking peptide secretion in neurons. These studies implicate a novel role for neuronal peptides in modifying intestinal health. In future experiments, we aim to establish the precise neuronal circuitry of intestine-modifying peptide signals, and the key intestine-specific genes and pathways that modulate intestinal aging downstream of neuronal peptides. As a long-term goal, this study seeks to establish the functional conservation of neuronal peptides in the etiology of intestinal and organismal aging.

Singh RN et al. (1977) International C. elegans Meeting "Some observations on moulting"

Sulston JE, Singh RN

[International C. elegans Meeting, 1977]

Nirupama Singh et al. (2008) Development & Evolution Meeting "Identification of Novel Target Genes of CeTwist Homodimers"

Ann Corsi, Peng Wang, Nirupama Singh

[Development & Evolution Meeting, 2008]

Our laboratory is interested in the protein, Twist, a transcription factor that functions as a heterodimer and homodimer in the developing mesoderm in many organisms. Mutations, in the human gene encoding the Twist protein and in genes that encode its downstream target genes, lead to craniosynostotic diseases characterized by premature fusion of skull sutures. Twist functions as a homodimer and heterodimer in humans however a detailed understanding of the effect on target genes for each dimer is not known. We are interested in investing how CeTwist functions in mesoderm development of C. elegans. We have already identified a number of target genes of CeTwist heterodimers but not of homodimers. In C. elegans having a group of target genes that respond to each dimer will help to elucidate this problem. Previous work in our laboratory provides some evidence for CeTwist functions as both the homodimer and heterodimer. Our aim is to find novel target genes of CeTwist homodimers. Finding novel target genes of CeTwist homodimers may also help us to identify human homologues which are mutated in related craniosynostotic syndromes but have not been identified yet. To find homodimer targets, the approach we are taking is to overexpress CeTwist by using an inducible heat shock promoter and finding the potential target genes that are overexpressed using Affymetrix oligonucleotide microarrays. Using this approach, we found a total of 106 genes which were upregulated. From this list, 15 genes were given high priority for further study on the basis of their expression level, description, protein domains, and homologues in other organisms. Using transcriptional GFP reporters, we are testing the expression pattern of these genes. We are interested in a pattern that may partially overlap the CeTwist expression pattern. In this project, 13 of 16 reporter genes have been constructed using standard molecular techniques. Transgenic animals harboring the reporter genes are made by microinjection. So far, transgenic lines have been isolated for 10 of the reporter genes. While examining the expression pattern of these genes, one of them seems to be interesting and will be examined further. The next step is to carefully examine the expression pattern of the remaining constructs to determine which genes are the best candidates to be CeTwist homodimer targets and also to reexamine the microarray data to determine second priority genes for further investigation.

Singh, M. et al. (2019) International Worm Meeting "CSR-1 catalytic-dependent and -independent activity on germline transcriptome."

Cecere, G., Loew, D., Dingli, F., Cornes, E., Quarato, P., Didier, C., Li, B., Singh, M.

[International Worm Meeting, 2019]

Caenorhabditis elegans has the presence of a large family of worm-specific Argonaute proteins (WAGOs). Among these WAGOs, only one Argonaute protein - CSR-1, is known to be essential and possess a catalytic activity in vitro. CSR-1 loads small RNAs antisense to most of the germline-expressed mRNAs. Recent studies indicate that the interaction between CSR-1 and its germline mRNA targets can result in 1) direct cleavage of the CSR-1 targets, and/or 2) protection from piRNA-mediated silencing. However, there is still a lack of understanding of the mechanism of CSR-1-mediated gene regulation and how these two activities of CSR-1 are compatible. Here, we combined a worm-sorting strategy with proteomic, biochemical, and high-throughput genomic approaches to dissect the role of CSR-1 catalytic activity-dependent and independent regulation of target genes at precise developmental stages. We observed that CSR-1 cleavage activity occurs on cytoplasmic mRNAs and results in the upregulation of a small subset of its targets characterized by a high density of small RNAs loaded onto CSR-1. These targets include CSR-1-interacting RNAi and p-granule factors identified by mass-spectrometry, indicating that this post-transcriptional slicing is a mode of feedback regulation. Mutation in the catalytic site does not affect the ability of CSR-1 to load small RNAs but impacts the biogenesis of small RNAs derived from the coding regions of its target mRNAs. Further, we show that upon complete removal of CSR-1 protein, piRNA-dependent downstream Argonaute proteins load small RNAs from a subset of CSR-1 targets and downregulate them at the transcriptional level. Overall, our study demonstrates mechanistic details for the two functions (protection and cleavage) attributed to CSR-1 which can, in fact, co-exist but regulate different sets of germline transcripts.

Singh H et al. (1996) East Coast Worm Meeting "mec-4(d) as a reverse genetic ablation tool"

Singh H, Driscoll MA, Tavernarakis NN

[East Coast Worm Meeting, 1996]

Singh H et al. (1996) West Coast Worm Meeting "mec-4(d) AS A REVERSE GENETIC ABLATION TOOL"

Tavernarakis NN, Singh H, Driscoll MA

[West Coast Worm Meeting, 1996]

Sequencing of gene-rich regions of Caenorhabditis elegans' 100 Mb genome is expected to be completed by the end of 1996 (Consortium report, 1996). Many genes have been predicted, some of which have been implicated by homology in development and disease. Clearly, reverse genetic studies will become increasingly important in C. elegans molecular biology. When a predicted gene of interest is identified, it is first essential to ask whether the gene corresponds to a gene previously identified by mutation, a question that is commonly addressed by DNA microinjection complementation experiments. If a mutation in the gene does not exist, it can be constructed by transposon-generated excision. Unfortunately, these experiments require a fair time investment and a bit of guessing about candidate loci before an answer is reached. We present here that genetic ablation with mec-4(d) is an efficient means of indicating likely mutant phenotypes of a gene of interest, along with obtaining an idea about its expression pattern, facilitating reverse genetic studies. Dominant mutations in mec-4 cause degeneration of the six touch neurons. It has been postulated that MEC-4 forms (part of) a multisubunit ion channel, which in aberrant form, causes osmotic imbalance and consequent cell necrosis. Expression of hsp-16p/mec-4(d) in transgenics induced degeneration of many different cell types all over the worm body, indicating a broad necrotic potency for mec-4(d). We used regulatory sequences from well characterized worm genes (including mec-7, unc-54, myo-2, unc-4) to express mec-4(d) and observed specific ablation or disruption of function of cells in which the toxic allele is expressed. Such a strategy was also employed with glr-1 by Maricq et al., (Nature 378:78-81). One example of an application of this technique for reverse genetics is our analysis of T28D9_7, a predicted ORF from the genome sequencing project which is also ~34% similar to mec-4. The putative regulatory sequences of T28D9_7 were fused upstream of mec-4(d). In integrated transgenics, the ablation fusion killed the same cells in which the T28D9_7/lac-Z fusion was expressed (T28D9_7 shows embryonic and L1 expression). The integrated ablation transgenics were uncoordinated and showed touch abnormalities. In this instance, there were no candidate unc or touch abnormal mutations in LGII to which T28D9_7 maps. In the case of another predicted gene R13A1_4, the ablation phenotype immediately led us to identify unc-8 as a candidate gene, which was confirmed shortly thereafter (see abstract by Tavernarakis et al.). In conclusion, we find that ablation of cells using mec-4(d) can greatly aid in determining the potential phenotype of a mutation, in cases where the function of the cell is essential for the expression of the mutant phenotype. This strategy also helps in indentifying the cells expressing the gene since ablation of specific cell types results in pertinent behaviorial abnormalities. This works particularly well for genes expressed in the nervous system. The mec-4(d) ablation vector, which houses a promoterless cassette and a polylinker to facilitate cloning is available for use.

Singh, Akanksha (2021) International Worm Meeting "CRISPR- Nanobodies from C. elegans as an therapeutic approach for Erythroblastosis Fetalis"

Singh, Akanksha

[International Worm Meeting, 2021]

Erythroblastosis fetalis is a consequence of incompatibility amongst Rh-negative antigen from mother and Rh-positive antigen of foetus thus resulting in hemolysis. The hemolysis usually results in respiratory problems, neurological disorders or even heart failure. C.elegans is known to be homologous to some extent with human genome and produces monoclonal antibodies. The nanobodies from C. elegans are engineered with CRISPR Cas 9 technology where Cas 9 protein has been incorporated into nanobodies that will accelerate its efficiency. The engineered nanobodies will target genes like RHD and RhCE that are prominent in RBC membranous environment. The nanobodies with Cas-9 might block the interaction of Rh-negative antibodies with Rh-positive antibodies of foetus as by refusing the recruitment of Rh-negative antibodies in placenta. Antibodies from the C. elegans are extracted and engineered to produce nanobodies with encoded Cas 9 protein. The haemoglobin obtained from foetus are mixed with haemoglobin from mother with Rh-antigens in vitro. After a period of 24-48 hours, when engineered nanobodies are treated with blood by ELISA technique indicated reduced count of Rh-positive antibodies thus blocking RHD gene not allowing the interaction with Rh-negative antibodies. The aim of the study was to study molecular interaction occurring after exposure of nanobodies to Rh- antigens present in foetal blood. Keywords: Erythroblastosis fetalis, Rh-negative, hemolysis, C. elegans, CRISPR Cas 9, RHD , RHCE, haemoglobin, nanobodies.

Singh, Komudi et al. (2011) International Worm Meeting "Notch signaling regulates chemosensation and behavioral adaptation to stress."

Singh, Komudi, Hart, Anne C.

[International Worm Meeting, 2011]

A role for Notch signaling in development is well established, but several studies now support a role for Notch signaling in the adult nervous system. The molecular mechanisms and targets critical for Notch function in neuronal signaling and behavior are unclear. OSM-11 is one of the DOS (Delta and OSM) motif proteins that act with DSL domain ligands to activate LIN-12 Notch receptors in vulval cell fate specification (Komatsu et al., 2008). Loss of osm-11 causes defective osmotic avoidance (Wheeler & Thomas, 2006) and, as we report here, impairs avoidance of 1-octanol and increases dispersal due to increased locomotion speed and decreased spontaneous reversal rates. The role of osm-11 and Notch pathway genes in octanol avoidance response was addressed in depth and suggested a redundant role for lin-12 and glp-1 Notch receptors in this chemosensory behavior. Furthermore, dissection of cellular site of action for Notch receptors suggested that the two receptors functioned in different/non-overlapping population of neurons. Studies in the past have shown that increased external osmolarity (hyperosmotic stress) leads to increased synthesis of the osmolyte glycerol in adult C. elegans (Lamitina, et al 2006). Under these unfavorable conditions, animals might also be expected to alter their behavior. We find that adaptation to hyperosmolarity impairs octanol response and increases dispersal, which are defects observed in osm-11 animals. Additionally, we find that the physiological and behavioral consequences of hyperosmotic stress adaptation are genetically separable. The role of Notch pathway genes in these potentially adaptive changes is under investigation.

Singh, J. et al. (2019) International Worm Meeting "A fight-and-flight response against bacterial pathogens in Caenorhabditis elegans."

Aballay, A., Singh, J.

[International Worm Meeting, 2019]

The ability to distinguish harmful and beneficial microbes is critical for the survival of an organism. To effectively counteract microbial pathogens, metazoans have evolved mechanisms to recognize them and deploy defenses at molecular and behavioral levels. Upon exposure to the bacterial pathogen Pseudomonas aeruginosa, C. elegans elicits an innate immune response that results in the activation of microbial-killing pathways and a behavioral pathogen-avoidance response. Here, we show that bloating of the intestinal lumen caused by pathogen infection elicits the microbial aversion behavior. Bloating of the intestinal lumen also activates defense responses including an innate immune response, even in the absence of bacterial pathogens or live bacteria. Neuroendocrine pathway genes are upregulated by intestinal bloating and are required for the microbial aversion behavior. We propose that bloating of the intestine may be perceived as a danger signal that activates an immune fight-and-flight response. These results suggest that inputs from the intestine can affect host behavior by modulating neuroendocrine signaling.

Singh, Varsha et al. (2011) International Worm Meeting "Neuronal control of stress response and innate immunity in C. elegans."

Singh, Varsha, Aballay, Alejandro, Sun, Jingru

[International Worm Meeting, 2011]

Heat Shock Protein (HSP) and Unfolded Protein Response (UPR) genes are upregulated in response to various stresses including heat shock, oxidative stress and ER stress. In C. elegans, HSP and UPR genes are also required for an adequate immune response to pathogens. Recent studies have shown that thermosensory neurons can regulate HSP gene induction at the organismal level. Increasing evidence also indicates that the nervous system of C. elegans controls avoidance to certain pathogens and activation of innate immune pathways. To identify neurons capable of controlling the activation of HSP or UPR genes as well as innate immune responses to pathogens, we screened mutants, in genes involved in the function of sensory neurons, for expression of stress proteins in response to bacterial pathogens and their pathogen susceptibility. Mutation in thermosensory neurons affected not only the expression of stress proteins but also pathogen susceptibility. Additional sensory neurons suppressed innate immunity while others enhanced innate immunity.