We use the nematode Caenorhabditis elegans to investigate aspects of nerve cell development and function. The wealth of developmental, anatomical, genetic, and molecular information available for C. elegans provides a powerful and multifaceted approach to these studies. Our work has focused on the study of a set of six neurons that are the sensory receptors for gentle touch (the touch receptor neurons, or TRNs), to address two questions: 1) How is neuronal cell fate determined? and 2) What is the molecular basis of mechanosensation, a sensory modality that underlies a variety of senses (e.g., touch, hearing, and balance)? We also work on neuronal degeneration, microtubule structure and function, and channel structure and function, and we develop methodologies to further scientific discovery.
We initially approached TRN development and function by mutational analysis, obtaining more than 450 mutations (in 18 genes) that produce a touch-insensitive phenotype. These touch genes are needed for the generation, specification, and function of the TRNs. The first two groups contain genes that regulate touch cell development, and the last group (function) contains genes that are developmental targets of this regulation.
Many of the genes that regulate TRN differentiation are transcription factors, and we have identified transcription factors that specify, maintain, and restrict cell fate. Some of these proteins act as classical selector factors, which direct the production of cell-characteristic proteins. Other transcription factors restrict and allow the selectors to act. Among these latter transcription factors are repressors of TRN differentiation that allow other cells to differentiate using the same selectors and inhibitors of these repressors that ensure that the TRNs can still differentiate as TRNs. We have also identified another class of transcription factors that do not themselves direct differentiation, but instead reduce stochastic variability and ensure robust TRN differentiation. We call such transcription factors (which include Hox proteins) “guarantors.” In addition to acting as guarantors, Hox transcription factors also specify regionally different subtypes of TRNs, e.g., by causing posterior TRNs to develop as bipolar neurons in distinction to the anterior cells (the apparent TRN ground state), which are monopolar neurons.
Twelve touch genes from our original collection are needed for TRN function. Using genetics, molecular biology, and electrophysiology, we have identified a transduction channel, the MEC-42MEC-10 heterotrimer, that underlies the touch response in the TRNs. Other genes encode proteins that are needed for optimum channel activity. These include the cholesterol-binding membrane protein MEC-2, the channel-specific chaperone MEC-6, and the extracellular matrix proteins MEC-1, MEC-5, and MEC-9. We are currently studying how these and other proteins expressed in these cells transduce touch. Our current model is that the channel complex is associated with the extracellular matrix. This tethering can lead to movement of the complex in the membrane leading to its opening.
Genetic Analysis of Touch Sensitivity (Terese Lawry, Matt Walker, Ben Wesley, Zhenhao Guo)
The Million Mutation Project (MMP) is a set of 2006 strains derived after chemical mutagenesis whose genomes have been sequenced (Thompson et al. Genome Res. 23, 1749-1762, 2013). Several groups have used this collection to examine the phenotypes caused the mutation of candidate genes (e.g., Mathew et al. PLOS Neg. Trop. Dis. 10: e0005058; Bulger et al. G3 7: 257-268, 2017; Chen et al. J. Cell Sci. 132: jcs227660, 2019), and a subset of the MMP strains has been screened for strains giving particular phenotypes (Wang et al. Develop. Biol. 399: 306-314, 2015; Timbers et al. PLoS Genet. 12: e1006235, 2016).
We are exploring the usefulness of the entire set of MMP strains as a genetic screening tool. The advantages of this approach over conventional screens include the ability 1) to identify both phenotype-causing and non-phenotype mutations in any gene of interest, and 2) to screen for more subtle phenotypes (e.g., partial or less penetrant phenotypes) because the strains are homozygous for most of the mutations. We are using these strains for several projects and are developing methods to most easily go from the strains to the causative mutations.
We initially screened all 2006 strains for defects in touch sensitivity and identified 105 strains with defects in touch sensitivity. Sixty-five of the strains had mutations in previously identified genes needed for touch. We are currently characterizing the remaining strains. Many of these strains have weak touch insensitivity phenotypes, so they would have been missed in our previous screens.
Identification of Gene Products that Normally Reduce Touch Sensitivity.
Virtually every biological system is balanced with processes that increase activity and those that decrease it. Because of the extreme sensitivity of the C. elegans TRNs, touch insensitive animals are much easier to find than touch supersensitive animals. Nonetheless, we have found sensitized systems that allowed us to identify genes whose loss increases touch sensitivity: mfb-1, uba-1, and cav-1 mutants increase touch sensitivity reduced by the loss of insulin signaling and mec-10 and mec-19 overcome poml-1’s suppression of mec-4d deaths. Recently, we discovered two new sensitized systems that we are exploiting to identify additional genes whose products normally inhibit touch sensitivity. The study of such mutants will address a major hole in our understanding of mechanosensation.
Structural Analysis of the TRN Touch Transduction Complex (Zhenhao Guo and Swastik De)
TRN mechanosensation requires the MEC-42MEC-10 transduction complex and several other TRN proteins. To gain a greater understanding of how this complex works, we have begun to purify and isolate the channel proteins for cryo-electron microscopy in collaboration with Joachim Frank’s group. We hope is that the structural analysis will lead us to a better understanding of the interactions of the channel subunits and how the channel may be gated.
Touch Sensitivity in other Nematodes (Hieu Hoang)
We have begun to look at TRN development and function in other nematodes and compare them to the information we already have for C. elegans. Our goal is not only to identify key core components needed in the development and function of these cells, but also to see how cell differentiation and function are modulated evolutionarily. We also wish to identify key amino acids in the TRN proteins by their conservation. In addition to studying TRN morphology and arrangement in other nematodes and comparing sequences and expression patterns of conserved touch sensitivity genes, we are also using CRISPR/Cas 9 mutagenesis to systematically generate knock-out and knock-in mutations of mec gene homologs in these species. We are starting this study by examining the touch genes in several other Caenorhabditis species and Pristionchus pacificus and will expand it to additional species.
Neuronal ensheathment (Brian Coblitz, eMalick Njie, and Matt Walker)
Neuronal ensheathment occurs in many multicellular organisms, but the underlying molecular mechanisms are poorly understood. We are studying this process using the TRNs, which become ensheathed by the surrounding epidermis (called the hypodermis) immediately after the third of the four larval molts. Using forward and reverse genetics (including screen Million Mutation strains, we identified 22 ensheathment genes. These genes are important for the mechanosensory ECM, adhesion complexes, axon guidance, and axonal transport. We recently discovered a synthetic ensheathment phenotype that should enable us to identified other components needed for ensheathment.
Sensitivity and Resistance to Benomyl (Zhenhao Guo, Dimitri Cadet, Kevin Chaikelson, Daniela Plaza, Liliana Seoror, Bryan Uceda-Alvarez, and Sophie López)
Over thirty years ago we discovered that loss of the b-tubulin gene ben-1 resulted in the complete loss of sensitivity to several benzimidazoles, drugs that are usually powerful anthelmintics. Because of the greater sensitivity afforded by examining the Million Mutation Project strains, we are screening these strains for mutants with either increased resistance or increased sensitivity to the benzimidazole benomyl. Several strains with partial resistance or increased sensitivity have been identified. Since none of these partially defective strains are mutated in the ben-1 gene, they should provide new insights into how C. elegans reacts to this class of drugs.