Research Summary

Survival requires strategies to identify and attract mates. How sensory neurons receive sex- specific signals and how multiple sensory stimuli are integrated to produce innate, stereotyped behaviors is poorly understood. To attack this problem,  the Barr Laboratory studies the molecular basis of sex-specific and sensory behaviors in the nematode Caenorhabditis elegans, which is tractable to molecular genetic, cell biological, and physiological approaches. There are three main areas of research in our laboratory, all using C. elegans as a simple animal model. First, we are interested in sensory biology and behaviors. Second, we study receptor trafficking and signaling in cilia. Finally, we have developed a C. elegans model for Nephronophthisis, a human genetic disease of cilia. The unifying theme of all three areas is the understanding of how a ciliated sensory neuron is specified in form and function.

Sensory Biology
Sexual behaviors are evoked by a wide variety of sensory cues and generated by specialized sensory neurons that sense mate-derived signals. In C. elegans, male sexual behaviors are driven by long- and short-range chemical and contact-based signals from a potential mate. We are interested in identifying the genes, molecules, and neural circuits that control sexually dimorphic sensory behaviors. Recently, we identified a Caenorhabditis elegans hermaphrodite-derived cue that stimulates male behavioral response and is dynamically regulated by her reproductive status (Morsi, Haas, and Barr, submitted). Wild-type males preferentially responded to older hermaphrodites. Increased sex appeal of older hermaphrodites was potent enough to stimulate robust response from mating-deficient pkd-2 and lov-1 polycystin mutant males. High response of pkd-2 males towards older hermaphrodites was independent of chemoattractant short- chain ascarosides, but was contingent on the absence of active sperm in the hermaphrodites. The enhanced pkd-2 male response towards spermless hermaphrodites was blocked by prior insemination or by genetic ablation of the ceh-18-dependent sperm-sensing pathway of the hermaphrodite somatic gonad. Our work suggests an interaction between sperm and the soma of the hermaphrodite that has a negative but reversible effect on mating response of males, the phenomenon to date attributed to gonochoristic species only. Future efforts will be aimed at identifying the attraction molecule(s) and defining the polycystin-independent sensory pathway.

Dendrite morphology and plasticity profoundly affect neuronal signaling and behavioral outputs. Little is known regarding the molecular signals governing morphogenesis of dendrite structure. During non-dauer stages of C. elegans development, the inner labial (IL2) neurons, a set of six ciliated putative chemosensory neurons, display a bipolar morphology with an unbranched axon and dendrite. We found that during the dauer stage, the IL2 neurons exhibit hierarchal dendritic branching and a switch from a bipolar to a multipolar morphology (Figure 1. Schroeder, Androwski, and Barr, in preparation).

  Figure 1: IL2 neurons in dauer (left) and non-dauer (right) hermaphrodites,
                 labeled with Pklp-6::GFP (top). Bottom cartoon illustration
                 modified from Wormatlas.org 

 

Following recovery from the dauer stage, the branches are incompletely resorbed. Using GFP
reporters, we analyzed the timing of both branch formation and resorption in relation to dauer formation and recovery, respectively. To identify molecules and pathways regulating branch formation, we are using a candidate gene approach as well as a forward genetic screen. To determine the function of the branches, we will use electrophysiology and calcium imaging of IL2 neurons. As various developmental disorders are associated with defects in dendrite structure, dauer IL2 branching may serve as a new and rapid model to understand the molecular basis of arborization and dendritic pruning that underlie these disorders.

Polycystins and Cilia in C. elegans
Cilia are cellular organelles that are essential for human development, organ function, fertility, and sensation of touch, sight, smell, taste, and hearing.    In the last decade, much has been learned about intraflagellar transport (IFT), the core machinery controlling ciliogenesis, and the connection between cilia and human disease. Despite the profound medical importance of cilia in human health, how cilia are specialized in form and function remains poorly understood. Cilia adopt diverse morphologies and express unique repertoires of receptors and signaling molecules necessary for different roles in various tissues. A better understanding of the mechanisms controlling trafficking to, within, and from cilia will be important in identifying therapeutic targets to combat diseases associated with cilia.

We were the first to discover that the polycystins localized to cilia, providing an important link between the cilium and human disease. Autosomal Dominant Polycystic Kidney Disease (ADPKD) affects 1:400-1000, with 95% of all cases being caused by mutations in the polycystin- encoding genes PKD1 and PKD2.    Our group was also the first to connect vesicular trafficking, IFT, and ubiquitin-mediated downregulation to polycystin ciliary receptor localization. The evolutionarily conserved polycystin genetic pathway, ciliary localization, and sensory function make the nematode an attractive model to study ciliary formation, morphogenesis, specialization, and signaling in the context of human genetic diseases of cilia. We have continued to exploit C. elegans for new gene discovery and are poised to provide new insights into polycystin ciliary localization and function. By studying the mechanism of action of our newly cloned cil (PKD- 2 ciliary localization) genes, we will determine how molecular motors (Barr and Morsci, in press), phosphoinositides, tubulin post-translational modifications (O’Hagan et al, in revision), and ubiquitination impact ciliary receptor trafficking, issues relevant to cilia biology and many human genetic diseases.

To identify new genes and pathways regulating PKD-2 ciliary trafficking and function, we performed a cell-type specific RNA interference screen, knocking down 100 PC interactors identified from the literature and scoring PKD-2::GFP localization and male mating behaviors. We are currently validating these candidates. Once the logistics of screening and validating are worked out, we will complete a genome-wide RNAi screen. Neuron-specific RNAi is a revolutionary technique for the lab, where we have been limited to time consuming, yet very fruitful, forward genetics screens. A particular advantage of this RNAi methodology is the ability to study adult male-specific function of normally essential genes. We expect that our results will significantly advance our understanding of TRP polycystin function and trafficking, as well as ciliary membrane receptor trafficking in general.