Two C. elegans DM domain proteins, DMD-3 and MAB-3, function in late stages of male somatic gonad development.

To bring about sexual dimorphism in form, information from the sex determination pathway must trigger sex-specific modifications in developmental programs. DM-domain encoding genes have been found to be involved in sex determination in a multitude of animals, often at the level of male somatic gonad formation. Here we report our findings that the DM-domain transcription factors MAB-3 and DMD-3 function together in multiple steps during the late stages of C. elegans male somatic gonad development. Both mab-3 and dmd-3 are expressed in the linker cell and hindgut of L4 males and dmd-3 is also expressed in presumptive vas deferens cells. Furthermore, dmd-3, but not mab-3, expression in the linker cell is downstream of nhr-67, a nuclear hormone receptor that was previously shown to control late stages of linker cell migration. In mab-3; dmd-3 double mutant males, the last stage of linker cell migration is partially defective, resulting in aberrant linker cell shapes and often a failure of the linker cell to complete its migration to the hindgut. When mab-3; dmd-3 double mutant linker cells do complete their migration, they fail to be engulfed by the hindgut, indicating that dmd-3 and mab-3 activity are essential for this process. Furthermore, linker cell death and clearance are delayed in mab-3; dmd-3 double mutants, resulting in the linker cell persisting into adulthood. Finally, DMD-3 and MAB-3 function to activate expression of the bZIP transcription factor encoding gene zip-5 and downregulate the expression of the zinc metalloprotease ZMP-1 in the linker cell. Taken together, these results demonstrate a requirement for DM-domain transcription factors in controlling C. elegans male gonad formation, supporting the notion that the earliest DM-domain genes were involved in male somatic gonad development in the last common ancestor of the bilaterians.

Social and sexual behaviors in C. elegans: the first fifty years.

For the first 25 years after the landmark 1974 paper that launched the field, most C. elegans biologists were content to think of their subjects as solitary creatures. C. elegans presented no shortage of fascinating biological problems, but some of the features that led Brenner to settle on this species-in particular, its free-living, self-fertilizing lifestyle-also seemed to reduce its potential for interesting social behavior. That perspective soon changed, with the last two decades bringing remarkable progress in identifying and understanding the complex interactions between worms. The growing appreciation that C. elegans behavior can only be meaningfully understood in the context of its ecology and evolution ensures that the coming years will see similarly exciting progress.

Sexual modulation of sex-shared neurons and circuits in Caenorhabditis elegans.

Studies using the nematode C. elegans have provided unique insights into the development and function of sex differences in the nervous system. Enabled by the relative simplicity of this species, comprehensive studies have solved the complete cellular neuroanatomy of both sexes as well as the complete neural connectomes of the entire adult hermaphrodite and the adult male tail. This work, together with detailed behavioral studies, has revealed three aspects of sex differences in the nervous system: sex-specific neurons and circuits; circuits with sexually dimorphic synaptic connectivity; and sex differences in the physiology and functions of shared neurons and circuits. At all of these levels, biological sex influences neural development and function through the activity of a well-defined genetic hierarchy that acts throughout the body to translate chromosomal sex into the state of a master autosomal regulator of sexual differentiation, the transcription factor TRA-1A. This Review focuses on the role of genetic sex in implementing sex differences in shared neurons and circuits, with an emphasis on linking the sexual modulation of specific neural properties to the specification and optimization of sexually divergent and dimorphic behaviors. An important and unexpected finding from these studies is that chemosensory neurons are a primary focus of sexual modulation, with genetic sex adaptively shaping chemosensory repertoire to guide behavioral choice. Importantly, hormone-independent functions of genetic sex are the principal drivers of all of these sex differences, making nematodes an excellent model for understanding similar but poorly understood mechanisms that likely act throughout the animal kingdom. © 2016 Wiley Periodicals, Inc.

Sexual modulation of neural circuits and behavior in Caenorhabditis elegans.

Sex differences in behavior-both sex-specific and shared behaviors-are fundamental to nearly all animal species. One often overlooked mechanism by which these behavioral differences can be generated is through sex-specific modulation of shared circuitry (i.e., circuits present in both sexes). In vertebrates this modulation is likely regulated by hormone-dependent mechanisms as well as by somatic sex itself; invertebrate models have particular promise for understanding the latter of these. Here we review molecular and behavioral evidence of sexual modulation of shared circuitry in the nematode Caenorhabditis elegans. Multiple behaviors in this species, both copulatory and not, are modulated by the genetic sex of shared neurons and circuit. These studies are close to uncovering the molecular mechanisms by which somatic sex modulates neural function in the worm, mechanisms which may be well conserved in more complex organisms. Improving our understanding of the modulation of neural circuit development and function by somatic sex may lend important insight into sex differences in the mammalian nervous system which, in turn, may have important implications for sex biases in disease.

Distributed effects of biological sex define sex-typical motor behavior in Caenorhabditis elegans.

Sex differences in shared behaviors (for example, locomotion and feeding) are a nearly universal feature of animal biology. Though these behaviors may share underlying neural programs, their kinematics can exhibit robust differences between males and females. The neural underpinnings of these differences are poorly understood because of the often-untested assumption that they are determined by sex-specific body morphology. Here, we address this issue in the nematode Caenorhabditis elegans, which features two sexes with distinct body morphologies but similar locomotor circuitry and body muscle. Quantitative behavioral analysis shows that C. elegans and related nematodes exhibit significant sex differences in the dynamics and geometry of locomotor body waves, such that the male is generally faster. Using a recently proposed model of locomotor wave propagation, we show that sex differences in both body mechanics and the intrinsic dynamics of the motor system can contribute to kinematic differences in distinct mechanical contexts. By genetically sex-reversing the properties of specific tissues and cells, however, we find that sex-specific locomotor frequency in C. elegans is determined primarily by the functional modification of shared sensory neurons. Further, we find that sexual modification of body wall muscle together with the nervous system is required to alter body wave speed. Thus, rather than relying on a single focus of modification, sex differences in motor dynamics require independent modifications to multiple tissue types. Our results suggest shared motor behaviors may be sex-specifically optimized though distributed modifications to several aspects of morphology and physiology.

Behavioral evolution: No sex please, we're hermaphrodites.

Many 'hard-wired', innate animal behaviors are related to reproduction. So what happens when reproductive systems evolve? New research in nematodes has identified principles underlying the co-evolution of reproductive strategy and sexual behavior, revealing some surprises and raising intriguing new questions.

C. elegans males optimize mate-preference decisions via sex-specific responses to multimodal sensory cues.

For sexually reproducing animals, selecting optimal mates is important for maximizing reproductive fitness. In the nematode C. elegans, populations reproduce largely by hermaphrodite self-fertilization, but the cross-fertilization of hermaphrodites by males also occurs. Males' ability to recognize hermaphrodites involves several sensory cues, but an integrated view of the ways males use these cues in their native context to assess characteristics of potential mates has been elusive. Here, we examine the mate-preference behavior of C. elegans males evoked by natively produced cues. We find that males use a combination of volatile sex pheromones (VSPs), ascaroside sex pheromones, surface-associated cues, and other signals to assess multiple features of potential mates. Specific aspects of mate preference are communicated by distinct signals: developmental stage and sex are signaled by ascaroside pheromones and surface cues, whereas the presence of a self-sperm-depleted hermaphrodite is likely signaled by VSPs. Furthermore, males prefer to interact with virgin over mated, and well-fed over food-deprived, hermaphrodites; these preferences are likely adaptive and are also mediated by ascarosides and other cues. Sex-typical mate-preference behavior depends on the sexual state of the nervous system, such that pan-neuronal genetic masculinization in hermaphrodites generates male-typical social behavior. We also identify an unexpected role for the sex-shared ASH sensory neurons in male attraction to ascaroside sex pheromones. Our findings lead to an integrated view in which the distinct physical properties of various mate-preference cues guide a flexible, stepwise behavioral program by which males assess multiple features of potential mates to optimize mate preference.

C. elegans males optimize mate-choice decisions via sex-specific responses to multimodal sensory cues.

For sexually reproducing animals, selecting optimal mates is essential for maximizing reproductive fitness. Because the nematode C. elegans reproduces mostly by self-fertilization, little is known about its mate-choice behaviors. While several sensory cues have been implicated in males' ability to recognize hermaphrodites, achieving an integrated understanding of the ways males use these cues to assess relevant characteristics of potential mates has proven challenging. Here, we use a choice-based social-interaction assay to explore the ability of C. elegans males to make and optimize mate choices. We find that males use a combination of volatile sex pheromones (VSPs), ascaroside pheromones, surface-bound chemical cues, and other signals to robustly assess a variety of features of potential mates. Specific aspects of mate choice are communicated by distinct signals: the presence of a sperm-depleted, receptive hermaphrodite is likely signaled by VSPs, while developmental stage and sex are redundantly specified by ascaroside pheromones and surface-associated cues. Ascarosides also signal nutritional information, allowing males to choose well-fed over starved mates, while both ascarosides and surface-associated cues cause males to prefer virgin over previously mated hermaphrodites. The male-specificity of these behavioral responses is determined by both male-specific neurons and the male state of sex-shared circuits, and we reveal an unexpected role for the sex-shared ASH sensory neurons in male attraction to endogenously produced hermaphrodite ascarosides. Together, our findings lead to an integrated view of the signaling and behavioral mechanisms by which males use diverse sensory cues to assess multiple features of potential mates and optimize mate choice.

Developmental biology: A hole in the matrix.

Neurons must access the environment to gather information, but this exposure must be carefully managed. New work finds that glial cells, the non-neuronal component of the nervous system, control environmental access by stage- and sex-specific patterning of the extracellular matrix.

Sex-specificity of the C. elegans metabolome.

Recent studies of animal metabolism have revealed large numbers of novel metabolites that are involved in all aspects of organismal biology, but it is unclear to what extent metabolomes differ between sexes. Here, using untargeted comparative metabolomics for the analysis of wildtype animals and sex determination mutants, we show that C. elegans hermaphrodites and males exhibit pervasive metabolomic differences. Several hundred small molecules are produced exclusively or in much larger amounts in one sex, including a host of previously unreported metabolites that incorporate building blocks from nucleoside, carbohydrate, lipid, and amino acid metabolism. A subset of male-enriched metabolites is specifically associated with the presence of a male germline, whereas enrichment of other compounds requires a male soma. Further, we show that one of the male germline-dependent metabolites, an unusual dipeptide incorporating N,N-dimethyltryptophan, increases food consumption, reduces lifespan, and accelerates the last stage of larval development in hermaphrodites. Our results serve as a foundation for mechanistic studies of how the genetic sex of soma and germline shape the C. elegans metabolome and provide a blueprint for the discovery of sex-dependent metabolites in other animals.

The Wnt/beta-catenin asymmetry pathway patterns the atonal ortholog lin-32 to diversify cell fate in a Caenorhabditis elegans sensory lineage.

Each sensory ray of the Caenorhabditis elegans male tail comprises three distinct neuroglial cell types. These three cells descend from a single progenitor, the ray precursor cell, through several rounds of asymmetric division called the ray sublineage. Ray development requires the conserved atonal-family bHLH gene lin-32, which specifies the ray neuroblast and promotes the differentiation of its progeny. However, the mechanisms that allocate specific cell fates among these progeny are unknown. Here we show that the distribution of LIN-32 during the ray sublineage is markedly asymmetric, localizing to anterior daughter cells in two successive cell divisions. The anterior-posterior patterning of LIN-32 expression and of differentiated ray neuroglial fates is brought about by the Wnt/ß-catenin asymmetry pathway, including the Wnt ligand LIN-44, its receptor LIN-17, and downstream components LIT-1 (NLK), SYS-1 (ß-catenin), and POP-1 (TCF). LIN-32 asymmetry itself has an important role in patterning ray cell fates, because the failure to silence lin-32 expression in posterior cells disrupts development of this branch of the ray sublineage. Together, our results illustrate a mechanism whereby the regulated function of a proneural-class gene in a single neural lineage can both specify a neural precursor and actively pattern the fates of its progeny. Moreover, they reveal a central role for the Wnt/ß-catenin asymmetry pathway in patterning neural and glial fates in a simple sensory lineage.

Reprogramming the topology of the nociceptive circuit in C. elegans reshapes sexual behavior.

The effect of the detailed connectivity of a neural circuit on its function and the resulting behavior of the organism is a key question in many neural systems. Here, we study the circuit for nociception in C. elegans, which is composed of the same neurons in the two sexes that are wired differently. We show that the nociceptive sensory neurons respond similarly in the two sexes, yet the animals display sexually dimorphic behaviors to the same aversive stimuli. To uncover the role of the downstream network topology in shaping behavior, we learn and simulate network models that replicate the observed dimorphic behaviors and use them to predict simple network rewirings that would switch behavior between the sexes. We then show experimentally that these subtle synaptic rewirings indeed flip behavior. Interestingly, when presented with aversive cues, rewired males were compromised in finding mating partners, suggesting that network topologies that enable efficient avoidance of noxious cues have a reproductive "cost." Our results present a deconstruction of the design of a neural circuit that controls sexual behavior and how to reprogram it.

Sex-specific, pdfr-1-dependent modulation of pheromone avoidance by food abundance enables flexibility in C. elegans foraging behavior.

To make adaptive feeding and foraging decisions, animals must integrate diverse sensory streams with multiple dimensions of internal state. In C. elegans, foraging and dispersal behaviors are influenced by food abundance, population density, and biological sex, but the neural and genetic mechanisms that integrate these signals are poorly understood. Here, by systematically varying food abundance, we find that chronic avoidance of the population-density pheromone ascr#3 is modulated by food thickness, such that hermaphrodites avoid ascr#3 only when food is scarce. The integration of food and pheromone signals requires the conserved neuropeptide receptor PDFR-1, as pdfr-1 mutant hermaphrodites display strong ascr#3 avoidance, even when food is abundant. Conversely, increasing PDFR-1 signaling inhibits ascr#3 aversion when food is sparse, indicating that this signal encodes information about food abundance. In both wild-type and pdfr-1 hermaphrodites, chronic ascr#3 avoidance requires the ASI sensory neurons. In contrast, PDFR-1 acts in interneurons, suggesting that it modulates processing of the ascr#3 signal. Although a sex-shared mechanism mediates ascr#3 avoidance, food thickness modulates this behavior only in hermaphrodites, indicating that PDFR-1 signaling has distinct functions in the two sexes. Supporting the idea that this mechanism modulates foraging behavior, ascr#3 promotes ASI-dependent dispersal of hermaphrodites from food, an effect that is markedly enhanced when food is scarce. Together, these findings identify a neurogenetic mechanism that sex-specifically integrates population and food abundance, two important dimensions of environmental quality, to optimize foraging decisions. Further, they suggest that modulation of attention to sensory signals could be an ancient, conserved function of pdfr-1.

Neural sex modifies the function of a C. elegans sensory circuit.

Though sex differences in animal behavior are ubiquitous, their neural and genetic underpinnings remain poorly understood. In particular, the role of functional differences in the neural circuitry that is shared by both sexes has not been extensively investigated. We have addressed these issues with C. elegans olfaction, a simple innate behavior mediated by sexually isomorphic neurons. Though males respond to the same olfactory attractants as do hermaphrodites, we find that each sex has a characteristic repertoire of olfactory preferences. These are not secondary to other sex-specific behaviors and do not require signaling from the gonad. Sex-specific olfactory preferences are controlled by tra-1, the master regulator of C. elegans sexual differentiation. Moreover, the genetic masculinization of neurons in an otherwise wild-type hermaphrodite is sufficient to switch the sexual phenotype of olfactory preference behavior. These studies reveal novel and unexpected sex differences in a C. elegans sensory behavior that is exhibited by both sexes. Our results indicate that these differences are a function of the chromosomally determined sexual identity of shared neural circuitry.

C. elegans Males Integrate Food Signals and Biological Sex to Modulate State-Dependent Chemosensation and Behavioral Prioritization.

Dynamic integration of internal and external cues is essential for flexible, adaptive behavior. In C. elegans, biological sex and feeding state regulate expression of the food-associated chemoreceptor odr-10, contributing to plasticity in food detection and the decision between feeding and exploration. In adult hermaphrodites, odr-10 expression is high, but in well-fed adult males, odr-10 expression is low, promoting exploratory mate-searching behavior. Food-deprivation transiently activates male odr-10 expression, heightening food sensitivity and reducing food leaving. Here, we identify a neuroendocrine feedback loop that sex-specifically regulates odr-10 in response to food deprivation. In well-fed males, insulin-like (insulin/IGF-1 signaling [IIS]) and transforming growth factor ß (TGF-ß) signaling repress odr-10 expression. Upon food deprivation, odr-10 is directly activated by DAF-16/FoxO, the canonical C. elegans IIS effector. The TGF-ß ligand DAF-7 likely acts upstream of IIS and links feeding to odr-10 only in males, due in part to the male-specific expression of daf-7 in ASJ. Surprisingly, these responses to food deprivation are not triggered by internal metabolic cues but rather by the loss of sensory signals associated with food. When males are starved in the presence of inedible food, they become nutritionally stressed, but odr-10 expression remains low and exploratory behavior is suppressed less than in starved control males. Food signals are detected by a small number of sensory neurons whose activity non-autonomously regulates daf-7 expression, IIS, and odr-10. Thus, adult C. elegans males employ a neuroendocrine feedback loop that integrates food detection and genetic sex to dynamically modulate chemoreceptor expression and influence the feeding-versus-exploration decision.

Dynamic, Non-binary Specification of Sexual State in the C. elegans Nervous System.

Biological sex in animals is often considered a fixed, individual-level characteristic. However, not all sex-specific features are static: for example, C. elegans males (XO) can sometimes exhibit hermaphrodite (XX)-like feeding behavior [1, 2]. (C. elegans hermaphrodites are somatic females that transiently produce self-sperm.) Essentially all somatic sex differences in C. elegans are governed by the master regulator tra-1, whose activity is controlled by chromosomal sex and is necessary and sufficient to specify the hermaphrodite state [3]. One aspect of this state is high expression of the chemoreceptor odr-10. In hermaphrodites, high odr-10 expression promotes feeding, but in males, low odr-10 expression facilitates exploration [4]. However, males suppress this sex difference in two contexts: juvenile males exhibit high odr-10 expression and food deprivation activates odr-10 in adult males [4-6]. Remarkably, we find that both of these phenomena require tra-1. In juvenile (L3) males, tra-1 is expressed in numerous neurons; this expression diminishes as individuals mature into adulthood, a process that requires conserved regulators of sexual maturation. tra-1 remains expressed in a small number of neurons in adult males, where it likely has a permissive role in odr-10 activation. Thus, the neuronal functions of tra-1 are not limited to hermaphrodites; rather, tra-1 also acts in the male nervous system to transiently suppress a sexual dimorphism, developmentally and in response to nutritional stress. Our results show that the molecular and functional representation of sexual state in C. elegans is neither static nor homogeneous, challenging traditional notions about the nature of biological sex.

The Long Non-Coding RNA lep-5 Promotes the Juvenile-to-Adult Transition by Destabilizing LIN-28.

Biological roles for most long non-coding RNAs (lncRNAs) remain mysterious. Here, using forward genetics, we identify lep-5, a lncRNA acting in the C. elegans heterochronic (developmental timing) pathway. Loss of lep-5 delays hypodermal maturation and male tail tip morphogenesis (TTM), hallmarks of the juvenile-to-adult transition. We find that lep-5 is a ∼600 nt cytoplasmic RNA that is conserved across Caenorhabditis and possesses three essential secondary structure motifs but no essential open reading frames. lep-5 expression is temporally controlled, peaking prior to TTM onset. Like the Makorin LEP-2, lep-5 facilitates the degradation of LIN-28, a conserved miRNA regulator specifying the juvenile state. Both LIN-28 and LEP-2 associate with lep-5 in vivo, suggesting that lep-5 directly regulates LIN-28 stability and may function as an RNA scaffold. These studies identify a key biological role for a lncRNA: by regulating protein stability, it provides a temporal cue to facilitate the juvenile-to-adult transition.