Maureen M Barr (2001) International C. elegans Meeting "Lov neurotransmission"

Maureen M Barr

[International C. elegans Meeting, 2001]

The nervous system of the adult C. elegans male possesses 381 neurons to the hermaphrodite's 302. These additional sex-specific neurons are required for male mating behaviors, namely, response to hermaphrodite contact, backing, turning, location of vulva (Lov), spicule insertion, and sperm transfer. We are specifically studying Lov behavior at the cellular, genetic, and molecular levels. The HOA and HOB hook neurons are required for Lov behavior (1) and the putative sensory receptor lov-1 is expressed in HOB (2). How does signaling initiated by the LOV-1 receptor culminate in Lov behavior? We are interested in identifying the neurotransmitters and neuropeptides used by the HOA and HOB mechano- and chemosensory neurons, respectively (3). Anne Hart and Arif Nathoo have identified 21 putative n europeptide like protein ( nlp ) genes in C. elegans and made transcriptional GFP fusions (4) which we have screened for expression in the adult male tail. An nlp-8::gfp transcriptional fusion is expressed uniformly throughout the HOB neuron. Expression is also observed in other non-sex, non-stage specific cells. We have constructed a full length nlp-8::gfp translational fusion and observed expression only in HOB, suggesting nlp-8 may be a Lov neuropeptide. We are exploring the role of nlp-8 in Lov behavior using genetic and molecular approaches. Liu, K. S. and P. W. Sternberg. 1995. Neuron 14:79-89. Barr, M. M. and P. W. Sternberg. 1999. Nature 401: 386-389. Sulston, J. E. et al. 1980. Developmental Biology 78:542-576. Nathoo, A., R. Moeller, and A. Hart. 1999. International Worm Meeting abstract 621.

Barr MM et al. (1997) International C. elegans Meeting "HERMAPHRODITE SIGNALS FOR MALE MATING"

Sternberg PW, Liu KK, Barr MM

[International C. elegans Meeting, 1997]

Although the hermaphrodite is a behaviorally passive mating partner, her vulva and uterus provide cues to the male. The vulva may signal the male hook sensillum, causing the male to stop backing and to attempt spicule insertion. The uterus may signal male sperm release. We are interested in the cellular basis of both hermaphrodite cues and male responses. To elucidate the nature of vulva and uterine cues, intact or neuron-ablated males were paired with ablated or mutant hermaphrodites. We found that the secondary vulva cells are both necessary and sufficient for vulva location. The male hook and post-cloacal sensilla may be involved in sensing and discriminating secondary cell characteristics. Males can transfer sperm to hermaphrodites with a defective vulval-uterine connection, suggesting the uterine signal may be diffusible. SPV-ablated males can transfer sperm to uterine-ablated hermaphrodites, supporting a role for SPV as a negative regulator of sperm transfer and sensor of an inhibitory uterine signal. We are now carrying out more refined ablation studies and behavioral analysis to limit these hermaphrodite cues to a single cell or minimal group of cells. We hope to determine whether chemosensation, mechanosensation, or both are involved in male behavioral responses.

Barr MM et al. (1996) West Coast Worm Meeting "VULVA LOCATION: HOW A MALE GETS TO WHERE HE'S GOING"

Hajdu-Cronin YM, Sternberg PW, Barr MM, Liu KK

[West Coast Worm Meeting, 1996]

C. elegans male mating behavior involves multiple steps, including response to the hermaphrodite, backing along her body, turning via a deep ventral bend at the end of her body, vulva location, spicule insertion, and sperm transfer. The male must integrate multiple signals and behave according to the movements of the disinterested, otherwise self-fertilizing hermaphrodite. Vulva location requires input from three sensilla: the hook, the post-cloacal sensilla (p.c.s.), and the spicules (1). The step of vulva location is complex: neuronal circuitry is partially redundant and this functional overlap allows for behavioral adaptation. Vulva location behavior is an excellent model system to dissect neuronal redundancy and behavioral plasticity. By screening for copulation defective (Cod) mutants, several vulva location mutants have been isolated. However, none exhibit severe behavioral or mating efficiency defects. As different sensory pathways appear to be affected in cod-13(sy420)III and cod-12(sy419)II, we are analyzing these mutants at genetic and cellular (via laser ablations) levels. sy420 most closely resembles hook-ablation (passes the vulva frequently) while sy419 mimics p.c.s.-ablation (locates but easily loses the vulva). sy420 maps to the interval defined by dpy-17 lon-1. Hook-ablated sy419 males behave distinctly from hook-ablated wild-type males. Hook-minus wild-type males pass the vulva and circle the hermaphrodite for several minutes before initiating a slow search that requires the p.c.s. and spicules (1). Hookless sy419 males exhibit a constitutive vulva location behavior involving spicule protraction at random positions along the hermaphrodite's body often coupled with premature sperm release. These behaviors in sy419 are vulva-dependent but uterus-independent, suggesting a possible defect in sensory feedback regulation. Combination ablations are being performed to determine which neurons are required for this aberrant behavior. Vulva location Cod mutants may affect partially redundant genes and a synergistic Cod phenotype may be produced by two mutations. Since hook ablation augments the behavioral defects of a sy419 male, sy419 may provide a sensitive background to obtain mutants with hook-like defects. (1) Neuron (1995) 4: 79-89.

MM Barr et al. (1999) International C. elegans Meeting "Genetic analysis of male mating behavior: What's lov got to do with it?"

PW Sternberg, MM Barr

[International C. elegans Meeting, 1999]

Vulva location behavior is an attractive model system for studying sensory perception in that information processing may be followed from stimulus (the hermaphrodite vulva) to sensory receptor (the male hook and post cloacal sensilla [pcs]) and ultimately to behavioral response (stop). We have delimited the vulva location signal to the cellular level. Hook neurons sense the 2deg lineage-derived vulva cells while the pcs respond to the 1deg lineage-derived cells. Ablation studies, SEM analysis, and interspecific behavioral observations suggest mechanosensation and chemosensation may be involved in vulva location behavior. We have initiated a genetic analysis of vulva location behavior, first examining the mating behavior of existing sensory mutants. Only cilium structure mutants were vulva location defective. The observation that only mutants defective in many sensory behaviors exhibit vulva location defects while males with specific sensory defects (such as Mec, Odr, and non-cilia defective Osm and Che mutants) behave essentially as wild type suggests a different set of gene products/molecules mediate vulva location behavior. We next screened for mutants defective specifically in vulva location behavior. lov-1 (for location of vulva defective) is required for two male sensory behaviors, response and vulva location. lov-1 encodes a putative membrane protein with a mucin-like, serine-threonine rich amino terminus followed by two blocks of homology to human polycystins encoded by the autosomal dominant polycystic kidney disease (ADPKD) genes. lov-1 is specifically expressed in adult male sensory neurons of the rays, hook, and head, mediating response, vulva location, and potentially chemotaxis to hermaphrodites, respectively. Polarized localization of LOV-1::GFP to ciliated sensory endings is consistent with a role in sensory reception and signaling.

Barr MM et al. (2000) Midwest Worm Meeting "Polycystic Kidney Disease (PKD) gene homologs and male mating behavior"

Barr MM, Sternberg PW

[Midwest Worm Meeting, 2000]

We have identified two genes, lov-1 and pkd-2, that are required for the male sensory behaviors of response and vulva location (1). lov-1 and pkd-2 encode PKD1 and PKD2 homologues, respectively. Autosomal Dominant Polycystic Kidney Disease affects 1 in 1,000 individuals and mutation in either the PKD1 or PKD2 gene accounts for 95% of all cases. Hence, we are using the lov-1 and pkd-2 pathway(s) to study the cell and molecular biology of PKD. lov-1::gfp and pkd-2::gfp are coexpressed and colocalized in the male sensory neurons of the rays, hook, and head. The male sensory neurons of lov-1 and pkd-2 mutants appear wild type. Combined, these observations suggest several possible functions for the polycystins in male mating behavior. lov-1 and pkd-2 may potentially function in sensory signal transduction, as molecular scaffolds, in the establishment and/or maintenance of neuronal cell polarity, or developmentally in the ultrastructural assembly of male-specific cilia. To test the hypothesis that lov-1 and pkd-2 act in concert, genetic interactions between lov-1 and pkd-2 are being examined. Additionally, other components that may be involved in the LOV-1/PKD-2 polycystin pathway are being characterized. For example, Tg737, the mouse Autosomal Recessive Polycystic Kidney Disease gene, encodes a protein that interacts with polycystin 1. Interestingly, the domain of interaction is conserved between LOV-1 and PKD1 (1). We have identified a Tg737 homolog in the C. elegans genome database and are analyzing its role in sensory behaviors. (1) Barr MM, Sternberg PW. Nature. 1999 Sep 23;401(6751):386-9.

Maureen M Barr et al. (2001) International C. elegans Meeting "The C. elegans Autosomal Dominant Polycystic Kidney Disease Genes lov-1 and pkd-2 act in the same pathway"

Paul W Sternberg, Maureen M Barr

[International C. elegans Meeting, 2001]

Autosomal Dominant Polycystic Kidney Disease (ADPKD) strikes 1 in 1000 individuals, often resulting in end-stage renal failure. Mutations in either PKD1 or PKD2 account for 95% of all cases. Recently, polycystin-1 and polycystin-2 (encoded by PKD1 and PKD2, respectively) have been shown to form a cation channel in vitro (1). Here we show that the C. elegans homologues of PKD1 and PKD2, lov-1 and pkd-2 (2), act in the same pathway in vivo . Mutations in either lov-1 or pkd-2 result in identical male sensory behavioral defects. Furthermore, pkd-2; lov-1 double mutants are phenotypically indistinguishable from lov-1 and pkd-2 single mutants, indicating that lov-1 and pkd-2 act together. LOV-1 and PKD-2 protein colocalize to male-specific chemosensory neurons and concentrate in cilia. Consistent with a role in sensory signal transduction, basodendritic localization of PKD-2 is essential for function. In contrast to defects in the C. elegans Autosomal Recessive PKD gene osm-5 (3), the cilia of C. elegans ADPKD mutants are wild type as judged by electron microscopy. This system provides a genetically tractable model for determining molecular mechanisms underlying PKD. 1. Hanaoka, K. et al. Nature 408, 990-994 (2000). 2. Barr, M.M. & Sternberg, P.W. Nature 401, 386-9 (1999). 3. Qin, H., Rosenbaum, J.L. & Barr, M.M. Curr Biol 11. 457-461 (2001).

Barr MM et al. (1998) West Coast Worm Meeting "Signals for C. elegans male mating behavior: A cellular and genetic approach to Lov (location of vulva)"

Barr MM, Sternberg PW

[West Coast Worm Meeting, 1998]

We are analyzing the genetic control of stereotyped behavior in the nematode Caenorhabditis elegans. Although the hermaphrodite is behaviorally passive, her vulva is thought to provide cues to the male (Liu and Sternberg, Neuron (1995) 14, 79-89). In this study, we focused on the cellular basis of hermaphrodite vulva location cues and neuronal basis of the male response. Vulva location behavior is executed by a minimum of eight sensory neurons with overlapping and redundant function. The vulva was considered as two parts: the 1º cell slit and the 2º cell lips. The 2º vulva cells appear to stimulate the male hook neurons HOA and HOB while the 1º vulva cells act as a cue to the post cloacal sensilla. The simplest interpretation of these data is that the male recognizes a pattern generated by the vulva. Disrupting this pattern interferes with normal behavioral response. It is not clear if the pattern encodes mechanosensory (a characteristic shape) or chemosensory (a combination of cell-specific chemicals) information, or both. SEM analysis of the adult vulva supports a model in which the vulva acts as a mechanosensory signal. Four structural characteristics of the vulva distinguish it from the rest of the hermaphrodite body. First, the anterior-posterior lips of the vulva protrude to form a bilobed bump. Second, the vulva slit divides the lips so that a crevice results. Third, a pair of left-right flaps surround the edges of the vulva. Finally, the annuli ridges that encircle the worm (in both sexes at all stages) are missing from the vulva cuticle. The obvious differences in morphology between the vulva and cuticle suggest mechanosensation may be involved in vulva location behavior. We hypothesize that the vulva acts not as a single cue but rather as a compilation of mechanostimuli to the male. Genetic analysis of behavior may provide insight into sensory reception and nervous system function. We examined several classes of behavioral mutants that are defective in chemotaxis, odorant response, osmotic avoidance, or mechanosensation. Only mutants defective in all sensory neurons exhibited defects in vulva location, suggesting that a distinct set of molecules may mediate vulva location behavior. To identify components necessary for a proper male behavioral response, we performed genetic screens to identify lov (for location of vulva defective) mutants. lov-1 males are response and vulva location defective but are wild-type for other behaviors (Mec+, Not+, Osm+, FITC+). We have rescued lov-1 mating defects by germline transformation with cosmid ZK945 and are in the process of delimiting rescue to a single gene.

Wang, Juan et al. (2017) International Worm Meeting "Biogenesis and Function of Social Extracellular Vesicles (EVs)."

Barr, Maureen, Wang, Juan, Silva, Malan

[International Worm Meeting, 2017]

Extracellular vesicles are emerging as an important aspect of intercellular communication by delivering a parcel of proteins, lipids even nucleic acids to specific target cells over short or long distances (Maas 2017). A subset of C. elegans ciliated neurons release EVs to the environment and elicit changes in male behaviors in a cargo-dependent manner (Wang 2014, Silva 2017). Our studies raise many questions regarding these social communicating EV devices. Why is the cilium the donor site? What mechanisms control ciliary EV biogenesis? How are bioactive functions encoded within EVs? EV detection is a challenge and obstacle because of their small size (100nm). However, we possess the first and only system to visualize and monitor GFP-tagged EVs in living animals in real time. We are using several approaches to define the properties of an EV-releasing neuron (EVN) and to decipher the biology of ciliary-released EVs. To identify mechanisms regulating biogenesis, release, and function of ciliary EVs we took an unbiased transcriptome approach by isolating EVNs from adult worms and performing RNA-seq. We identified 335 significantly upregulated genes, of which 61 were validated by GFP reporters as expressed in EVNs (Wang 2015). By characterizing components of this EVN parts list, we discovered new components and pathways controlling EV biogenesis, EV shedding and retention in the cephalic lumen, and EV environmental release. We also identified cell-specific regulators of EVN ciliogenesis and are currently exploring mechanisms regulating EV cargo sorting. Our genetically tractable model can make inroads where other systems have not, and advance frontiers of EV knowledge where little is known. Maas, S. L. N., Breakefield, X. O., & Weaver, A. M. (2017). Trends in Cell Biology. Silva, M., Morsci, N., Nguyen, K. C. Q., Rizvi, A., Rongo, C., Hall, D. H., & Barr, M. M. (2017). Current Biology. Wang, J., Kaletsky, R., Silva, M., Williams, A., Haas, L. A., Androwski, R. J., Landis JN, Patrick C, Rashid A, Santiago-Martinez D, Gravato-Nobre M, Hodgkin J, Hall DH, Murphy CT, Barr, M. M. (2015).Current Biology. Wang, J., Silva, M., Haas, L. A., Morsci, N. S., Nguyen, K. C. Q., Hall, D. H., & Barr, M. M. (2014). Current Biology.

Haas, Leonard A. et al. (2011) International Worm Meeting "The Effect of Hermaphrodite Sperm Status on C. elegans Sexual Attraction."

Haas, Leonard A., Barr, Maureen, Morsci, Natalia

[International Worm Meeting, 2011]

Sexual behaviors are modulated by numerous sensory signals. We use C. elegans to study how hermaphrodite-derived signals regulate male mating behavior. Male-specific genes lov-1 and pkd-2 are homologous to human autosomal dominant polycystic disease (ADPKD) genes. lov-1 and pkd-2 mutants are defective in multiple male sensory behaviors, including contact-based response (Barr and Sternberg 1999; Barr et al. 2001). Strikingly, lov-1 and pkd-2 males exhibited a threefold increase in response to sperm-depleted N2 or sperm-less fog-2 hermaphrodites. However, this improved response elicitation was decreased when fog-2 hermaphrodites were supplemented with male sperm. Wild-type males exposed to a mixture of sperm-depleted and self-sperm containing hermaphrodites also prefer sperm-depleted hermaphrodites. We conclude that hermaphrodite sperm status affects male mating behaviors via an unidentified ADPKD-independent pathway. We wanted to determine the nature of the signal that causes the increase in attractiveness to sperm-depleted hermaphrodites, using pkd-2 males as a sensitized background. We considered three possible sources that caused the increased attractiveness of sperm-depleted hermaphrodites: spermatogenesis, sperm activation, or the presence of fertilized oocytes. Through experiments with spe-19 and spe-38, we concluded that inactivated spermatids failed to inhibit hermaphrodite attractiveness; however, fertilization defective spermatozoa were capable of inhibiting hermaphrodite attractiveness. Although we do not know the molecular mechanism by which sperm signaling inhibits hermaphrodite mating cue, we show that it is triggered during or after sperm activation. We conclude that the reproductive state of the hermaphrodite impacts male behavior and mate choice. We are currently testing the different aspects of sperm activation for its effect on hermaphrodite attractiveness.

Renee L Engle et al. (2003) International Worm Meeting "Genetic analysis of IFT and ARPKD: Isolating osm-5 suppressors"

Renee L Engle, Joel Rosenbaum, Hongmin Qin, Maureen M Barr

[International Worm Meeting, 2003]

Intraflagellar transport (IFT) is required for the formation and maintenance of C. elegans sensory cilia. Mutations in IFT motors, Complex A, or Complex B polypeptides result in defective cilia. OSM-5 has been characterized as a Complex B IFT polypeptide (1). osm-5::gfp is expressed in ciliated sensory neurons in both hermaphrodites and males and indicates a role in the formation and maintenance of cilia (1). Mutations in osm-5 result in stunted cilia and can be characterized by dye-filling defects, disruption of sensory behaviors (2), and abnormal male mating behavior (3). osm-5 is homologous to the Chlamydomonas gene IFT88 and the mouse autosomal recessive polycystic kidney disease (ARPKD) gene Tg737; mutations in these genes lead to defective ciliogenesis (2, 4, 5). Emerging evidence suggests that cilia have a role in polycystic kidney disease (PKD) (6, review).To identify genes that interact directly or indirectly with the IFT complex, we are implementing an osm-5 suppression screen. osm-5(mn397) is a missense mutation in a conserved residue in the second tetratricopeptide repeat (TPR) domain (1) and is characterized by dye-filling defects (Dyf-) (2). A suppressor of osm-5(mn397) will have a normal dye uptake (Dyf+) phenotype, while the missense allele, osm-5(mn397), will remain Dyf-. We will use a Copas Biosort to categorize and separate the Dyf+ suppressor phenotype from the Dyf- phenotype. This will enable us to quickly and efficiently screen millions of mutagenized animals for suppressors. 1. Qin, H., Rosenbaum, J.L., and Barr, M.M. Curr Biol 11, 457-461 (2001); 2. Perkins, L.A. et al. Dev Biol 117, 456-487 (1986); 3. Barr, M.M., Sternberg, P.W. Nature 401, 386-389 (1999); 4. Pazour, G.L. et al. J Cell Biol 151, 709-718 (2000); 5. Murcia, N.S. et al. Development 127, 2347-2355 (2000); 6. Pazour, G.J. and Rosenbaum, J.L. Trends Cell Biol 12, 551-555 (2002).