Knowledge of connectivity in the nervous system is essential to understanding its function. Here we describe connectomes for both adult sexes of the nematode Caenorhabditis elegans, an important model organism for neuroscience research. We present quantitative connectivity matrices that encompass all connections from sensory input to end-organ output across the entire animal, information that is necessary to model behaviour. Serial electron microscopy reconstructions that are based on the analysis of both new and previously published electron micrographs update previous results and include data on the male head. The nervous system differs between sexes at multiple levels. Several sex-shared neurons that function in circuits for sexual behaviour are sexually dimorphic in structure and connectivity. Inputs from sex-specific circuitry to central circuitry reveal points at which sexual and non-sexual pathways converge. In sex-shared central pathways, a substantial number of connections differ in strength between the sexes. Quantitative connectomes that include all connections serve as the basis for understanding how complex, adaptive behavior is generated.

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 the 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.

SUMMARY 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 neurons of the nematode C. elegans differentiate in a strikingly sex-specific manner. While in hermaphrodites the PHC neurons display a canonical pattern of synaptic connectivity similar to that of other sensory neurons, PHC differentiates into a densely connected hub sensory/interneuron in males, 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 sex-specificity of dmd-3 action is ensured by the hermaphrodite-specific, transcriptional master regulator of hermaphroditic cell identity, tra-1, which represses transcription of dmd-3 in hermaphrodite PHC. Taken together, our studies provide mechanistic insights into how neurons are specified in a sexually dimorphic manner.

One goal of modern day neuroscience is the establishment of molecular maps that assign unique features to individual neuron types. Such maps provide important starting points for neuron classification, for functional analysis, and for developmental studies aimed at defining the molecular mechanisms of neuron identity acquisition and neuron identity diversification. In this resource paper, we describe a nervous system-wide map of the potential expression sites of 244 members of the largest gene family in the C. elegans genome, rhodopsin-like (class A) G-protein-coupled receptor (GPCR) chemoreceptors, using classic gfp reporter gene technology. We cover representatives of all sequence families of chemoreceptor GPCRs, some of which were previously entirely uncharacterized. Most reporters are expressed in a very restricted number of cells, often just in single cells. We assign GPCR reporter expression to all but two of the 37 sensory neuron classes of the sex-shared, core nervous system. Some sensory neurons express a very small number of receptors, while others, particularly nociceptive neurons, coexpress several dozen GPCR reporter genes. GPCR reporters are also expressed in a wide range of inter- and motorneurons, as well as non-neuronal cells, suggesting that GPCRs may constitute receptors not just for environmental signals, but also for internal cues. We observe only one notable, frequent association of coexpression patterns, namely in one nociceptive amphid (ASH) and two nociceptive phasmid sensory neurons (PHA, PHB). We identified GPCRs with sexually dimorphic expression and several GPCR reporters that are expressed in a left/right asymmetric manner. We identified a substantial degree of GPCR expression plasticity; particularly in the context of the environmentally-induced dauer diapause stage when one third of all tested GPCRs alter the cellular specificity of their expression within and outside the nervous system. Intriguingly, in a number of cases, the dauer-specific alterations of GPCR reporter expression in specific neuron classes are maintained during postdauer life and in some case new patterns are induced post-dauer, demonstrating that GPCR gene expression may serve as traits of life history. Taken together, our resource provides an entry point for functional studies and also offers a host of molecular markers for studying molecular patterning and plasticity of the nervous system.

A core principle of nervous system organization is the diversification of neuron classes into subclasses that share large sets of features but differ in select traits. We describe here a molecular mechanism necessary for motor neurons to acquire subclass-specific traits in the nematode Caenorhabditis elegans. Cholinergic motor neuron classes of the ventral nerve cord can be subdivided into subclasses along the anterior-posterior (A-P) axis based on synaptic connectivity patterns and molecular features. The conserved COE-type terminal selector UNC-3 not only controls the expression of traits shared by all members of a neuron class, but is also required for subclass-specific traits expressed along the A-P axis. UNC-3, which is not regionally restricted, requires region-specific cofactors in the form of Hox proteins to co-activate subclass-specific effector genes in post-mitotic motor neurons. This intersectional gene regulatory principle for neuronal subclass diversification may be conserved from nematodes to mice.DOI: http://dx.doi.org/10.7554/eLife.25751.001

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Bone tissue engineering requires the upregulation of several regenerative stages, including a critical early phase of angiogenesis. Previous studies have suggested that a sequential delivery of platelet-derived growth factor (PDGF) to bone morphogenetic protein-2 (BMP-2) could promote angiogenic tubule formation when delivered to in vitro cocultures of human umbilical vein endothelial cells (HUVECs) and human mesenchymal stem cells (hMSCs). However, it was previously unclear that this PDGF to BMP-2 delivery schedule will result in cell migration into the scaffolding system and affect the later expression of bone markers. Additionally, a controlled delivery system had not yet been engineered for programmed sequential presentation of this particular growth factor. By combining alginate matrices with calcium phosphate scaffolding, a programmed growth factor delivery schedule was achieved. Specifically, a combination of alginate microspheres, alginate hydrogels, and a novel blend of resorbable calcium phosphate-based cement (ReCaPP) was used. PDGF and BMP-2 were sequentially released from this hybrid calcium phosphate/alginate scaffold with the desired 3-day overlap in PDGF to BMP-2 delivery. Using a three-dimensional coculture model, we observed that this sequence of PDGF to BMP-2 delivery influenced both cellular infiltration and alkaline phosphatase (ALP) expression. It was found that the presence of early PDGF delivery increased the distance of cell infiltration into the calcium phosphate/alginate scaffolding in comparison to early BMP-2 delivery and simultaneous PDGF+BMP-2 delivery. It was also observed that hMSCs expressed a greater amount of ALP+ staining in response to scaffolds delivering the sequential PDGF to BMP-2 schedule, when compared with scaffolds delivering no growth factor, or PDGF alone. Importantly, hMSCs cultured with scaffolds releasing the PDGF to BMP-2 schedule showed similar amounts of ALP staining to hMSCs cultured with BMP-2 alone, suggesting that the sequential schedule of PDGF to BMP-2 presentation promotes differentiation of hMSCs toward an osteoblast phenotype while also increasing cellular infiltration of the scaffold.

Differences between female and male brains exist across the animal kingdom and extend from molecular to anatomical features. Here we show that sexually dimorphic anatomy, gene expression and function in the nervous system can be modulated by past experiences. In the nematode Caenorhabditis elegans, sexual differentiation entails the sex-specific pruning of synaptic connections between neurons that are shared by both sexes, giving rise to sexually dimorphic circuits in adult animals. We discovered that starvation during juvenile stages is memorized in males to suppress the emergence of sexually dimorphic synaptic connectivity. These circuit changes result in increased chemosensory responsiveness in adult males following juvenile starvation. We find that an octopamine-mediated starvation signal dampens the production of serotonin (5-HT) to convey the memory of starvation. Serotonin production is monitored by a 5-HT1A serotonin receptor homologue that acts cell-autonomously to promote the pruning of sexually dimorphic synaptic connectivity under well-fed conditions. Our studies demonstrate how life history shapes neurotransmitter production, synaptic connectivity and behavioural output in a sexually dimorphic circuit.