Publications

The persistence of memory reflects not only how it is formed but also how forgetting is delayed. The mechanisms controlling forgetting remain obscure, and in particular it is unclear to what degree this process is actively regulated. We discovered that cold exposure delays forgetting of specific olfactory memories by more than eight-fold and that adaptation to cold abolishes this effect. To study the underlying mechanism, we performed RNA sequencing, mutant analyses and pharmacological assays. Here we show that regulation of membrane properties underlies the presence and absence of cold-induced delayed forgetting. Furthermore, lithium can also delay forgetting in cold-sensitive worms but not in cold-tolerant worms; this effect involves downregulation of the diacylglycerol pathway in AWC neurons and long-term suppression of activity in the downstream AIY interneurons. Thus, the genetic tractability of worms might be harnessed to study the mechanism of action of lithium and cold exposure and, more fundamentally, how memory is stored and lost.

The evolutionary paths taken by each sex within a given species sometimes diverge, resulting in behavioral differences. Given their distinct needs, the mechanism by which each sex learns from a shared experience is still an open question. Here, we reveal sexual dimorphism in learning: C. elegans males do not learn to avoid the pathogenic bacteria PA14 as efficiently and rapidly as hermaphrodites. Notably, neuronal activity following pathogen exposure was dimorphic: hermaphrodites generate robust representations, while males, in line with their behavior, exhibit contrasting representations. Transcriptomic and behavioral analysis revealed that the neuropeptide receptor npr-5, an ortholog of the mammalian NPY/NPF-like receptor, regulates male learning by modulating neuronal activity. Furthermore, we show the dependency of the males decision-making on their sexual status and demonstrate the role of npr-5 as a modulator of incoming sensory cues. Taken together, these findings illustrate how neuromodulators drive sex-specific behavioral plasticity in response to a shared experience.

Comparative inter-species single-cell atlases of Caenorhabditis whole embryos and larval nervous systems reveal deep conservation of cell type identities alongside extensive divergence in signaling pathways. These findings illustrate how stable cellular architectures support evolving molecular logic across development and behavior.

Sexually dimorphic instinctual behaviors that set females and males apart are found across animal clades. Recent studies in a variety of animal systems have provided deep insights into the neural circuits that guide sexually dimorphic behaviors, such as mating practices and social responses, and how sex differences in these circuits develop. Here, we discuss the neural circuits of several sexually dimorphic instinctual behaviors in rodents, flies, and worms - from mate attraction and aggression to pain perception and empathy. We highlight several salient similarities and differences between these circuits and reveal general principles that underlie the function and development of neural circuits for dimorphic behaviors.

Sex-specific behaviors are often attributed to differences in neuronal wiring and molecular composition, yet how genetic sex shapes the molecular architecture of the nervous system at the individual neuron level remains unclear. Here, we use single-cell RNA sequencing to profile the transcriptome of sex-shared neurons in adult Caenorhabditis elegans males and hermaphrodites. We uncover widespread molecular dimorphism across the nervous system, including in previously unrecognized neuron-types such as the touch receptors. Neuropeptides and signaling-related genes exhibit strong sex-biased expression, particularly in males, reinforcing the notion that neuropeptides are crucial for diversifying connectome outputs. Despite these differences, neurotransmitter identities remain largely conserved, indicating that functional dimorphism arises through modulatory, not identity-defining, changes. We show that sex-biased expression of neurotransmitter-related genes correlates with bias in outgoing synaptic connectivity and identify regulatory candidates for synaptic wiring, including both shared and sex-specific genes. This dataset provides a molecular framework for understanding how subtle regulatory differences tune conserved circuits to drive sex-specific behaviors.

Animal behavior varies across different timescales. This includes rapid shifts in behavior as animals transition between states and long-term changes that develop throughout an organism's life. This review presents the contributions of sex differences, individuality, and internal states to behavioral variability in the roundworm Caenorhabditis elegans. Sex is determined by chromosome composition, which directs neuronal development through gene regulation and experience to shape dimorphic behaviors. Genetically identical individuals within the same sex and reared in the same conditions still display distinctive, long-lasting behavioral traits that are controlled by neuromodulatory systems. At all life stages, internal states within the individual, shaped by external factors like food and stress, modulate behavior over minutes to hours. The interplay between these factors gives rise to rich behavioral diversity in C. elegans. These factors impact behavior in a sequential manner, as genetic sex, individuality, and internal states influence behavior over progressively finer timescales.

Dendrites are crucial for receiving information into neurons. Sensory experience affects the structure of these tree-like neurites, which, it is assumed, modifies neuronal function, yet the evidence is scarce, and the mechanisms are unknown. To study whether sensory experience affects dendritic morphology, we use the Caenorhabditis elegans' arborized nociceptor PVD neurons, under natural mechanical stimulation induced by physical contacts between individuals. We found that mechanosensory signals induced by conspecifics and by glass beads affect the dendritic structure of the PVD. Moreover, developmentally isolated animals show a decrease in their ability to respond to harsh touch. The structural and behavioral plasticity following sensory deprivation are functionally independent of each other and are mediated by an array of evolutionarily conserved mechanosensory amiloride-sensitive epithelial sodium channels (degenerins). Calcium imaging of the PVD neurons in a micromechanical device revealed that controlled mechanical stimulation of the body wall produces similar calcium dynamics in both isolated and crowded animals. Our genetic results, supported by optogenetic, behavioral, and pharmacological evidence, suggest an activity-dependent homeostatic mechanism for dendritic structural plasticity, that in parallel controls escape response to noxious mechanosensory stimuli.

Sex-specific traits and behaviors emerge during development by the acquisition of unique properties in the nervous system of each sex. However, the genetic events responsible for introducing these sex-specific features remain poorly understood. In this study, we create a comprehensive gene expression atlas of pure populations of hermaphrodites and males of the nematode Caenorhabditis elegans across development. We discover numerous differentially expressed genes, including neuronal gene families like transcription factors, neuropeptides, and G protein-coupled receptors. We identify INS-39, an insulin-like peptide, as a prominent male-biased gene expressed specifically in ciliated sensory neurons. We show that INS-39 serves as an early-stage male marker, facilitating the effective isolation of males in high-throughput experiments. Through complex and sex-specific regulation, ins-39 plays pleiotropic sexually dimorphic roles in various behaviors, while also playing a shared, dimorphic role in early life stress. This study offers a comparative sexual and developmental gene expression database for C. elegans. Furthermore, it highlights conserved genes that may underlie the sexually dimorphic manifestation of different human diseases.

Microorganisms use toxins to kill competing microorganisms or eukaryotic cells. Polymorphic toxins are proteins that encode carboxy-terminal toxin domains. Here we developed a computational approach to identify previously undiscovered, conserved toxin domains of polymorphic toxins within 105,438 microbial genomes. We validated nine short toxins, showing that they cause cell death upon heterologous expression in either Escherichia coli or Saccharomyces cerevisiae. Five cognate immunity genes that neutralize the toxins were also discovered. The toxins are encoded by 2.2% of sequenced bacteria. A subset of the toxins exhibited potent antifungal activity against various pathogenic fungi but not against two invertebrate model organisms or macrophages. Experimental validation suggested that these toxins probably target the cell membrane or DNA or inhibit cell division. Further characterization and structural analysis of two toxinimmunity protein complexes confirmed DNase activity. These findings expand our knowledge of microbial toxins involved in inter-microbial competition that may have the potential for clinical and biotechnological applications.

Information is transmitted between brain regions through the release of neurotransmitters from long-range projecting axons. Understanding how the activity of such long-range connections contributes to behavior requires efficient methods for reversibly manipulating their function. Chemogenetic and optogenetic tools, acting through endogenous G-protein-coupled receptor pathways, can be used to modulate synaptic transmission, but existing tools are limited in sensitivity, spatiotemporal precision or spectral multiplexing capabilities. Here we systematically evaluated multiple bistable opsins for optogenetic applications and found that the Platynereis dumerilii ciliary opsin (PdCO) is an efficient, versatile, light-activated bistable G-protein-coupled receptor that can suppress synaptic transmission in mammalian neurons with high temporal precision in vivo. PdCO has useful biophysical properties that enable spectral multiplexing with other optogenetic actuators and reporters. We demonstrate that PdCO can be used to conduct reversible loss-of-function experiments in long-range projections of behaving animals, thereby enabling detailed synapse-specific functional circuit mapping.

The correct interpretation of threat and reward is important for animal survival. Often, the decisions underlying these behavioral programs are mediated by volatile compounds in the animal's environment, which they detect and discriminate with specialized olfactory neurons along their body. Caenorhabditis (C.) elegans senses chemical stimuli with neurons located in the head and the tail of the animal, which mediate either attractive or aversive behaviors. How conflicting stimuli are processed in animals navigating different chemical gradients is poorly understood. Here, we conceived, created, and capitalized on a novel microfluidic device to enable automated and precise stimulation of head and tail neurons, either simultaneously or sequentially, while reading out neuronal activity in sensory and interneurons using genetically encoded calcium indicators. We achieve robust and programmable chemical pulses through the modulation of inlet pressures. To evaluate the device performance, we synchronized the flow control with microscopy data acquisition and characterized the flow properties in the fabricated devices. Together, our design has the potential to provide insight into the neural circuits and behavior of C. elegans simulating the experience of natural environments.

The poet W.B Yeats wrote that textquotedblleftAll that is personal soon rots, it must be packed in ice or salttextquotedblright. Here we show that in Caenorhabditis elegans nematodes, simple animals with just 302 neurons, memories are preserved on ice and in lithium salt. C. elegans nematodes can form associative memories, which are typically forgotten quickly. We discovered that when placed on ice, worms delay forgetting of specific olfactory memories by at least 8-fold. Delayed forgetting was canceled completely when the worms were gradually adapted to low temperatures, owing to a genetically-encoded program that turns acclimated worms cold-tolerant. RNA-seq, mutant analyses, and pharmacological assays revealed that regulation of membrane properties switches cold-induced delayed forgetting ON and OFF, and, remarkably, that lithium delays forgetting only in cold-sensitive but not cold-tolerant worms. We found that downregulation of the diacylglycerol pathway in the AWC sensory neurons is essential for lithium-mediated delayed forgetting, and using neuronal activity recordings located the memory trace to the downstream AIY interneurons. We suggest that the awesome genetic tractability of C. elegans might be harnessed to study the effects of lithium and cold temperatures on the brain, why it influences psychiatric disorders, and even more fundamentally how memory is stored and lost.

Dimorphic traits, shaped by both natural and sexual selection, ensure optimal fitness and survival of the organism. This includes neuronal circuits that are largely affected by different experiences and environmental conditions. Recent evidence suggests that sexual dimorphism of neuronal circuits extends to different levels such as neuronal activity, connectivity and molecular topography that manifest in response to various experiences, including chemical exposures, starvation and stress. In this review, we propose some common principles that govern experience-dependent sexually dimorphic circuits in both vertebrate and invertebrate organisms. While sexually dimorphic neuronal circuits are predetermined, they have to maintain a certain level of fluidity to be adaptive to different experiences. The first layer of dimorphism is at the level of the neuronal circuit, which appears to be dictated by sex-biased transcription factors. This could subsequently lead to differences in the second layer of regulation namely connectivity and synaptic properties. The third regulator of experience-dependent responses is the receptor level, where dimorphic expression patterns determine the primary sensory encoding. We also highlight missing pieces in this field and propose future directions that can shed light onto novel aspects of sexual dimorphism with potential benefits to sex-specific therapeutic approaches. Thus, sexual identity and experience simultaneously determine behaviours that ultimately result in the maximal survival success.

Molecular chaperones are essential cellular components that aid in protein folding and preventing the abnormal aggregation of disease-associated proteins. Mutations in one such chaperone, DNAJB6, were identified in patients with LGMDD1, a dominant autosomal disorder characterized by myofibrillar degeneration and accumulations of aggregated protein within myocytes. The molecular mechanisms through which such mutations cause this dysfunction, however, are not well understood. Here we employ a combination of solution NMR and biochemical assays to investigate the structural and functional changes in LGMDD1 mutants of DNAJB6. Surprisingly, we find that DNAJB6 disease mutants show no reduction in their aggregation-prevention activity in vitro, and instead differ structurally from the WT protein, affecting their interaction with Hsp70 chaperones. While WT DNAJB6 contains a helical element regulating its ability to bind and activate Hsp70, in LGMDD1 disease mutants this regulation is disrupted. These variants can thus recruit and hyperactivate Hsp70 chaperones in an unregulated manner, depleting Hsp70 levels in myocytes, and resulting in the disruption of proteostasis. Interfering with DNAJB6-Hsp70 binding, however, reverses the disease phenotype, suggesting future therapeutic avenues for LGMDD1.

The evolutionary paths taken by each sex within a given species sometimes diverge, resulting in behavioral differences that promote specific fitness outcomes for each sex. Given their distinct needs, the mechanism by which each sex learns from a shared experience is still an open question. We reveal a novel sexual dimorphism in learning: C. elegans males do not learn to avoid the pathogenic bacteria PA14 as efficiently and rapidly as hermaphrodites, even though their innate immunity recognizes the pathogen in a similar manner. Notably, we observe sexually dimorphic neuronal activity following pathogen exposure: hermaphrodites generate robust representations while males, in line with their behavior, exhibit contrasting representations, suggesting that a mechanism that modulates incoming sensory cues is at play. Transcriptomic and behavioral analysis revealed that the neuropeptide receptor npr-5, an ortholog of the mammalian NPY receptor, regulates male learning by modulating neuronal activity. Furthermore, we show the dependency of the males decision-making phenotype on their sexual status and demonstrate the pivotal role of npr-5 in this regulation as a sensory gatekeeper. Thus, collectively, we portray sex-specific plasticity in behavior toward a shared experience by modulating learning to fulfill the evolutionary needs.Competing Interest StatementThe authors have declared no competing interest.

Many animal behaviors are manifested differently in the two sexes of a given species, but how such sexual dimorphism is imprinted in the nervous system is not always clear. One mechanism involved is synaptic dimorphism, by which the same neurons exist in the two sexes, but form synapses that differ in features such as anatomy, molecular content or fate. While some evidence for synaptic dimorphism exists in humans and mammals, identifying these mechanisms in invertebrates has proven simpler, due to their smaller nervous systems and absence of external regulation by sex hormones. This review aims to present the current status of the field in invertebrates, the available toolkit for the study of synaptic dimorphism, and the standing questions that still remain incompletely answered.

The capacity of animals to respond to hazardous stimuli in their surroundings is crucial for their survival. In mammals, complex evaluations of the environment require large numbers and different subtypes of neurons. The nematode C. elegans avoids hazardous chemicals they encounter by reversing their direction of movement. How does the worms compact nervous system process the spatial information and direct motion change? We show here that a single interneuron, AVA, receives glutamatergic excitatory and inhibitory signals from head and tail sensory neurons, respectively. AVA integrates the spatially distinct and opposing cues, whose output instructs the animal's behavioral decision. We further find that the differential activation of AVA stems from distinct localization of inhibitory and excitatory glutamate-gated receptors along AVA's process and from different threshold sensitivities of the sensory neurons. Our results thus uncover a cellular mechanism that mediates spatial computation of nociceptive cues for efficient decision-making in C. elegans.

How sensory perception is processed by the two sexes of an organism is still only partially understood. Despite some evidence for sexual dimorphism in auditory and olfactory perception, whether touch is sensed in a dimorphic manner has not been addressed. Here we find that the neuronal circuit for tail mechanosensation in C. elegans is wired differently in the two sexes and employs a different combination of sex-shared sensory neurons and interneurons in each sex. Reverse genetic screens uncovered cell- and sex-specific functions of the alpha-tubulin mec-12 and the sodium channel tmc-1 in sensory neurons, and of the glutamate receptors nmr-1 and glr-1 in interneurons, revealing the underlying molecular mechanisms that mediate tail mechanosensation. Moreover, we show that only in males, the sex-shared interneuron AVG is strongly activated by tail mechanical stimulation, and accordingly is crucial for their behavioral response. Importantly, sex reversal experiments demonstrate that the sexual identity of AVG determines both the behavioral output of the mechanosensory response and the molecular pathways controlling it. Our results present extensive sexual dimorphism in a mechanosensory circuit at both the cellular and molecular levels.

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 \u201ccost.\u201d Our results present a deconstruction of the design of a neural circuit that controls sexual behavior and how to reprogram it.

Sex-specific behaviors are common in nature and are crucial for reproductive fitness and species survival. A key question in the field of sex/gender neurobiology is whether and to what degree the sex-shared nervous system differs between the sexes in the anatomy, connectivity and molecular identity of its components. An equally intriguing issue is how does the same sex-shared neuronal template diverge to mediate distinct behavioral outputs in females and males. This chapter aims to present the most up-to-date understanding of how this task is achieved in C. elegans. The vast majority of neurons in C. elegans are shared among the two sexes in terms of their lineage history, anatomical position and neuronal identity. Yet a substantial amount of evidence points to the hermaphrodite-male counterparts of some neurons expressing different genes and forming different synaptic connections. This, in turn, enables the same cells and circuits to transmit discrete signals in the two sexes and ultimately execute different functions. We review the various sex-shared behavioral paradigms that have been shown to be sexually dimorphic in recent years, discuss the mechanisms that underlie these examples, refer to the developmental regulation of neuronal dimorphism and suggest evolutionary concepts that emerge from the data.

Sexually dimorphic circuits underlie behavioral differences between the sexes, yet the molecular mechanisms involved in their formation are poorly understood. We show here that sexually dimorphic connectivity patterns arise in C. elegans through local ubiquitin-mediated protein degradation in selected synapses of one sex but not the other. Specifically, synaptic degradation occurs via binding of the evolutionary conserved E3 ligase SEL-10/FBW7 to a phosphodegron binding site of the netrin receptor UNC-40/DCC (Deleted in Colorectal Cancer), resulting in degradation of UNC-40. In animals carrying an undegradable unc-40 gain-of-function allele, synapses were retained in both sexes, compromising the activity of the circuit without affecting neurite guidance. Thus, by decoupling the synaptic and guidance functions of the netrin pathway, we reveal a critical role for dimorphic protein degradation in controlling neuronal connectivity and activity. Additionally, the interaction between SEL-10 and UNC-40 is necessary not only for sex-specific synapse pruning, but also for other synaptic functions. These findings provide insight into the mechanisms that generate sex-specific differences in neuronal connectivity, activity, and function.

Sex-specific synaptic connectivity is beginning to emerge as a remarkable, but little explored feature of animal brains. We describe here a novel mechanism that promotes sexually dimorphic neuronal function and synaptic connectivity in the nervous system of the nematode Caenorhabditis elegans. We demonstrate that a phylogenetically conserved, but previously uncharacterized Doublesex/Mab-3 related transcription factor (DMRT), dmd-4, is expressed in two classes of sex-shared phasmid neurons specifically in hermaphrodites but not in males. We find dmd-4 to promote hermaphrodite-specific synaptic connectivity and neuronal function of phasmid sensory neurons. Sex-specificity of DMD-4 function is conferred by a novel mode of posttranslational regulation that involves sex-specific protein stabilization through ubiquitin binding to a phylogenetically conserved but previously unstudied protein domain, the DMA domain. A human DMRT homolog of DMD-4 is controlled in a similar manner, indicating that our findings may have implications for the control of sexual differentiation in other animals as well.

The structural and functional properties of neurons have intrigued scientists since the pioneering work of Santiago Ramon y Cajal. Since then, emerging cutting- edge technologies, including light and electron microscopy, electrophysiology, biochemistry, optogenetics, and molecular biology, have dramatically increased our understanding of dendritic properties. This advancement was also facilitated by the establishment of different animal model organisms, from flies to mammals. Here we describe the emerging model system of a Caenorhabditis elegans polymodal neuron named PVD, whose dendritic tree follows a stereotypical structure characterized by repeating candelabra- like structural units. In the past decade, progress has been made in understanding PVD's functions, morphogenesis, regeneration, and aging, yet many questions still remain.

Injury triggers regeneration of axons and dendrites. Research has identified factors required for axonal regeneration outside the CNS, but little is known about regeneration triggered by dendrotomy. Here, we study neuronal plasticity triggered by dendrotomy and determine the fate of complex PVD arbors following laser surgery of dendrites. We find that severed primary dendrites grow toward each other and reconnect via branch fusion. Simultaneously, terminal branches lose self-avoidance and grow toward each other, meeting and fusing at the tips via an AFF-1-mediated process. Ectopic branch growth is identified as a step in the regeneration process required for bypassing the lesion site. Failure of reconnection to the severed dendrites results in degeneration of the distal end of the neuron. We discover pruning of excess branches via EFF-1 that acts to recover the original wild-type arborization pattern in a late stage of the process. In contrast, AFF-1 activity during dendritic auto-fusion is derived from the lateral seam cells and not autonomously from the PVD neuron. We propose a model in which AFF-1-vesicles derived from the epidermal seam cells fuse neuronal dendrites. Thus, EFF-1 and AFF-1 fusion proteins emerge as new players in neuronal arborization and maintenance of arbor connectivity following injury in Caenorhabditis elegans. Our results demonstrate that there is a genetically determined multi-step pathway to repair broken dendrites in which EFF-1 and AFF-1 act on different steps of the pathway. EFF-1 is essential for dendritic pruning after injury and extrinsic AFF-1 mediates dendrite fusion to bypass injuries.

Functional and anatomical sexual dimorphisms in the brain are either the result of cells that are generated only in one sex or a manifestation of sex-specific differentiation of neurons present in both sexes. The PHC neuron pair of the nematode C. elegans differentiates in a strikingly sex-specific manner. In hermaphrodites the PHC neurons display a canonical pattern of synaptic connectivity similar to that of other sensory neurons, but in males PHC differentiates into a densely connected hub sensory neuron/interneuron, integrating a large number of male-specific synaptic inputs and conveying them to both male-specific and sex-shared circuitry. We show that the differentiation into such a hub neuron involves the sex-specific scaling of several components of the synaptic vesicle machinery, including the vesicular glutamate transporter eat-4/VGLUT, induction of neuropeptide expression, changes in axonal projection morphology, and a switch in neuronal function. We demonstrate that these molecular and anatomical remodeling events are controlled cell autonomously by the phylogenetically conserved Doublesex homolog dmd-3, which is both required and sufficient for sex-specific PHC differentiation. Cellular specificity of dmd-3 action is ensured by its collaboration with non-sex-specific terminal selector-type transcription factors, whereas the sex specificity of dmd-3 action is ensured by the hermaphrodite-specific transcriptional master regulator of hermaphroditic cell identity tra-1, which represses the transcription of dmd-3 in hermaphrodite PHC. Taken together, our studies provide mechanistic insights into how neurons are specified in a sexually dimorphic manner.

Throughout evolution, sensory information is processed by the opposing sexes into distinct sexually dimorphic behaviors. At the level of the nervous system, the cause of these dimorphic behaviors remains largely unknown. Dimorphism may result from differential structural or signaling properties of the neuronal network. The nematode Caenorhabditis elegans is an ideal model to explore the genetic control of neuronal sex differences and the sexual regulation of the nervous system and behavior. The male and hermaphroditic nervous systems of C. elegans contain the same set of 294 neurons, but each sex also has its own set of sex-specific neurons. Comparisons of the C. elegans connectome between both sexes have revealed a striking dimension of sexual identity: specific neurons have sexually dimorphic synaptic connectivity. Here we review studies that try to elucidate how shared neuronal circuits are modulated to process sensory information in a sex-specific manner.

The aging brain undergoes structural changes that affect brain homeostasis, neuronal function and consequently cognition. The complex architecture of dendritic arbors poses a challenge to understanding age-dependent morphological alterations, behavioral plasticity and remodeling following brain injury. Here, we use the PVD polymodal neurons of C. elegans as a model to study how aging affects neuronal plasticity. Using confocal live imaging of C. elegans PVD neurons, we demonstrate age-related progressive morphological alterations of intricate dendritic arbors. We show that mutations in daf-2, which encodes an insulin-like growth factor receptor ortholog, fail to inhibit the progressive morphological aging of dendrites and do not prevent the minor decline in response to harsh touch during aging. We uncovered that PVD aging is characterized by a major decline in the regenerative potential of dendrites following experimental laser dendrotomy. Furthermore, the remodeling of transected dendritic trees by AFF-1-mediated self-fusion can be restored in old animals by daf-2 mutations, and can be differentially re-established by ectopic expression of the fusion protein AFF-1. Thus, ectopic expression of the fusogen AFF-1 in the PVD and mutations in daf-2 differentially rejuvenate some aspects of dendritic regeneration following injury.

Whether and how neurons that are present in both sexes of the same species can differentiate in a sexually dimorphic manner is not well understood. A comparison of the connectomes of the Caenorhabditis elegans hermaphrodite and male nervous systems reveals the existence of sexually dimorphic synaptic connections between neurons present in both sexes. Here we demonstrate sex-specific functions of these sex-shared neurons and show that many neurons initially form synapses in a hybrid manner in both the male and hermaphrodite pattern before sexual maturation. Sex-specific synapse pruning then results in the sex-specific maintenance of subsets of these connections. Reversal of the sexual identity of either the pre- or postsynaptic neuron alone transforms the patterns of synaptic connectivity to that of the opposite sex. A dimorphically expressed and phylogenetically conserved transcription factor is both necessary and sufficient to determine sex-specific connectivity patterns. Our studies reveal new insights into sex-specific circuit development.

Background: The molecular mechanisms that determine axonal growth potential are poorly understood. Intrinsic growth potential decreases with age, and thus one strategy to identify molecular pathways controlling intrinsic growth potential is by studying developing young neurons. The programmed and stereotypic remodeling of Drosophila mushroom body (MB) neurons during metamorphosis offers a unique opportunity to uncover such mechanisms. Despite emerging insights into MB γ-neuron axon pruning, nothing is known about the ensuing axon re-extension. Results: Using mosaic loss of function, we found that the nuclear receptor UNF (Nr2e3) is cell autonomously required for the re-extension of MB γ-axons following pruning, but not for the initial growth or guidance of any MB neuron type. We found that UNF promotes this process of developmental axon regrowth via the TOR pathway as well as a late axon guidance program via an unknown mechanism. We have thus uncovered a novel developmental program of axon regrowth that is cell autonomously regulated by the UNF nuclear receptor and the TOR pathway. Conclusions: Our results suggest that UNF activates neuronal re-extension during development. Taken together, we show that axon growth during developmental remodeling is mechanistically distinct from initial axon outgrowth. Due to the involvement of the TOR pathway in axon regeneration following injury, our results also suggests that developmental regrowth shares common molecular mechanisms with regeneration following injury.

The mechanisms controlling the formation and maintenance of neuronal trees are poorly understood. We examined the dynamic development of two arborized mechanoreceptor neurons (PVDs) required for reception of strong mechanical stimuli in Caenorhabditis elegans. The PVDs elaborated dendritic trees comprising structural units we call "menorahs." We studied how the number, structure, and function of menorahs were maintained. EFF-1, an essential protein mediating cell fusion, acted autonomously in the PVDs to trim developing menorahs. eff-1 mutants displayed hyperbranched, disorganized menorahs. Overexpression of EFF-1 in the PVD reduced branching. Neuronal pruning appeared to involve EFF-1dependent branch retraction and neurite-neurite autofusion. Thus, EFF-1 activities may act as a quality control mechanism during the sculpting of dendritic trees.

Eukaryotic cells have evolved diverged mechanisms to merge cells. Here, we discuss three types of cell fusion: (1) Non-self-fusion, cells with different genetic contents fuse to start a new organism and fusion between enveloped viruses and host cells; (2) Self-fusion, genetically identical cells fuse to form a multi-nucleated cell; and (3) Auto-fusion, a single cell fuses with itself by bringing specialized cell membrane domains into contact and transforming itself into a ring-shaped cell. This is a new type of selfish fusion discovered in C. elegans. We divide cell fusion into three stages: (1) Specification of the cell-fusion fate; (2) Cell attraction, attachment, and recognition; (3) Execution of plasma membrane fusion, cytoplasmic mixing and cytoskeletal rearrangements. We analyze cell fusion in diverse biological systems in development and disease emphasizing the mechanistic contributions of C. elegans to the understanding of programmed cell fusion, a genetically encoded pathway to merge specific cells.

Most readers of this review originated from a spermegg fusion event. Cell fusion is a process that is crucial at many intersections later during development. However, we do not know which molecules (fusogens) fuse the membranes of gametes to form zygotes, myoblasts to form myotubes in muscles, macrophages to form osteoclasts in bones, or cytotrophoblasts to form syncytiotrophoblasts in placentas. There are five gold standards that can be applied for the identification of genuine fusogens. Based on these criteria, a numerical score can be used to assess the likelihood of protein fusogenicity. We compare distinct families of candidate developmental, viral and intracellular fusogens and analyze current models of membrane fusion.

During cell-cell fusion, two cells' plasma membranes merge, allowing the cytoplasms to mix and form a syncytium. Little is known about the mechanisms of cell fusion. Here, we asked whether eff-1, shown previously to be essential for fusion in Caenorhabditis elegans, acts directly in the fusion machinery. We show that expression of EFF-1 transmembrane protein drives fusion of heterologous cells into multinucleate syncytia. We obtained evidence that EFF-1-mediated fusion involves a hemifusion intermediate characterized by membrane mixing without cytoplasm mixing. Furthermore, syncytiogenesis requires EFF-1 in both fusing cells. To test whether this mechanism also applies in vivo, we conducted genetic mosaic analysis of C. elegans and found that homotypic epidermal fusion requires EFF-1 in both cells. Thus, although EFF-1-mediated fusion shares characteristics with viral and intracellular fusion, including an apparent hemifusion step, it differs from these reactions in the homotypic organization of the fusion machinery.

Despite the identification of essential processes in which cell fusion plays spectacular roles such as in fertilization and development of muscle, bone, and placenta 1, 2, 3, 4, 5, there are no identified proteins that directly mediate developmental cell fusion reactions. C. elegans has recently become among the best-characterized models to use for studying developmental cell fusion 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15. The eff-1 (epithelial fusion failure) gene encodes novel type I membrane proteins required for epithelial cell fusion. Analysis of eff-1 mutants showed that cell fusion normally restricts routes for cell migration and establishes body and organ shape and size [ 5, 8, 9, 11]. Here, we explored cell fusion by using time-lapse confocal and electron microscopy of different organs. We found that ectopic expression of eff-1 is sufficient to fuse epithelial cells that do not normally fuse. This ectopic fusion results in cytoplasmic content mixing and disappearance of apical junctions, starting less than 50 min after the start of eff-1 transcription. We found that eff-1 is necessary to initiate and expand multiple microfusion events between pharyngeal muscle cells. Surprisingly, eff-1 is not required to fuse the gonadal anchor cell to uterine cells. Thus, eff-1 is sufficient and essential for most but not all cell fusion events during C. elegans development.