Abstract
Sex differences in behaviour extend to cognitive-like processes such as learning, but the underlying dimorphisms in neural circuit development and organization that generate these behavioural differences are largely unknown. Here we define at the single-cell level—from development, through neural circuit connectivity, to function—the neural basis of a sex-specific learning in the nematode Caenorhabditis elegans. We show that sexual conditioning, a form of associative learning, requires a pair of male-specific interneurons whose progenitors are fully differentiated glia. These neurons are generated during sexual maturation and incorporated into pre-exisiting sex-shared circuits to couple chemotactic responses to reproductive priorities. Our findings reveal a general role for glia as neural progenitors across metazoan taxa and demonstrate that the addition of sex-specific neuron types to brain circuits during sexual maturation is an important mechanism for the generation of sexually dimorphic plasticity in learning.
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References
Kimura, K.-I., Ote, M., Tazawa, T. & Yamamoto, D. Fruitless specifies sexually dimorphic neural circuitry in the Drosophila brain. Nature 438, 229–233 (2005)
Ruta, V. et al. A dimorphic pheromone circuit in Drosophila from sensory input to descending output. Nature 468, 686–690 (2010)
Yang, C. F. et al. Sexually dimorphic neurons in the ventromedial hypothalamus govern mating in both sexes and aggression in males. Cell 153, 896–909 (2013)
Rideout, E. J., Dornan, A. J., Neville, M. C., Eadie, S. & Goodwin, S. F. Control of sexual differentiation and behavior by the doublesex gene in Drosophila melanogaster. Nature Neurosci. 13, 458–466 (2010)
Stowers, L. & Logan, D. W. Sexual dimorphism in olfactory signaling. Curr. Opin. Neurobiol. 20, 770–775 (2010)
Nottebohm, F. & Arnold, A. P. Sexual dimorphism in vocal control areas of the songbird brain. Science 194, 211–213 (1976)
Keleman, K. et al. Dopamine neurons modulate pheromone responses in Drosophila courtship learning. Nature 489, 145–149 (2012)
Sulston, J. E., Albertson, D. G. & Thomson, J. N. The Caenorhabditis elegans male: postembryonic development of nongonadal structures. Dev. Biol. 78, 542–576 (1980)
Jarrell, T. A. et al. The connectome of a decision-making neural network. Science 337, 437–444 (2012)
Sakai, N. et al. A sexually conditioned switch of chemosensory behavior in C. elegans. PLoS ONE 8, e68676 (2013)
Sulston, J. E., Schierenberg, E., White, J. G. & Thomson, J. N. The embryonic cell lineage of the nematode Caenorhabditis elegans. Dev. Biol. 100, 64–119 (1983)
Sulston, J. E. & Horvitz, H. R. Post-embryonic cell lineages of the nematode, Caenorhabditis elegans. Dev. Biol. 56, 110–156 (1977)
White, J. G., Southgate, E., Thomson, J. N. & Brenner, S. The structure of the nervous system of the nematode Caenorhabditis elegans. Phil. Trans. R. Soc. Lond. B 314, 1–340 (1986)
Hall, D. H. & Russell, R. L. The posterior nervous system of the nematode Caenorhabditis elegans: serial reconstruction of identified neurons and complete pattern of synaptic interactions. J. Neurosci. 11, 1–22 (1991)
Ward, S., Thomson, N., White, J. G. & Brenner, S. Electron microscopical reconstruction of the anterior sensory anatomy of the nematode Caenorhabditis elegans.?2UU. J. Comp. Neurol. 160, 313–337 (1975)
Varshney, L. R., Chen, B. L., Paniagua, E., Hall, D. H. & Chklovskii, D. B. Structural properties of the Caenorhabditis elegans neuronal network. PLOS Comput. Biol. 7, e1001066 (2011)
Barrios, A. Exploratory decisions of the Caenorhabditis elegans male: a conflict of two drives. Semin. Cell Dev. Biol. 33, 10–17 (2014)
Ryan, D. A. et al. Sex, age, and hunger regulate behavioral prioritization through dynamic modulation of chemoreceptor expression. Curr. Biol. 24, 2509–2517 (2014)
Saeki, S., Yamamoto, M. & Iino, Y. Plasticity of chemotaxis revealed by paired presentation of a chemoattractant and starvation in the nematode Caenorhabditis elegans. J. Exp. Biol. 204, 1757–1764 (2001)
Vellai, T., McCulloch, D., Gems, D. & Kovács, A. L. Effects of sex and insulin/insulin-like growth factor-1 signaling on performance in an associative learning paradigm in Caenorhabditis elegans. Genetics 174, 309–316 (2006)
Srinivasan, J. et al. A blend of small molecules regulates both mating and development in Caenorhabditis elegans. Nature 454, 1115–1118 (2008)
Barrios, A., Nurrish, S. & Emmons, S. W. Sensory regulation of C. elegans male mate-searching behavior. Curr. Biol. 18, 1865–1871 (2008)
Barr, M. M. & Sternberg, P. W. A polycystic kidney-disease gene homologue required for male mating behaviour in C. elegans. Nature 401, 386–389 (1999)
Barr, M. M. & García, L. R. Male mating behavior. WormBook. http://dx.doi.org/10.1895/wormbook.1.78.1 (2006)
Lipton, J., Kleemann, G., Ghosh, R., Lints, R. & Emmons, S. W. Mate searching in Caenorhabditis elegans: a genetic model for sex drive in a simple invertebrate. J. Neurosci. 24, 7427–7434 (2004)
Janssen, T. et al. Discovery and characterization of a conserved pigment dispersing factor-like neuropeptide pathway in Caenorhabditis elegans. J. Neurochem. 111, 228–241 (2009)
Barrios, A., Ghosh, R., Fang, C., Emmons, S. W. & Barr, M. M. PDF-1 neuropeptide signaling modulates a neural circuit for mate-searching behavior in C. elegans. Nature Neurosci. 15, 1675–1682 (2012)
Egger, B., Chell, J. M. & Brand, A. H. Insights into neural stem cell biology from flies. Phil. Trans. R. Soc. Lond. B 363, 39–56 (2008)
Oikonomou, G. & Shaham, S. The glia of Caenorhabditis elegans. Glia 59, 1253–1263 (2011)
Hong, Y., Roy, R. & Ambros, V. Developmental regulation of a cyclin-dependent kinase inhibitor controls postembryonic cell cycle progression in Caenorhabditis elegans. Development 125, 3585–3597 (1998)
Zarkower, D. Somatic sex determination. WormBook. http://dx.doi.org/10.1895/wormbook.1.84.1 (2006)
Hodgkin, J. A genetic analysis of the sex-determining gene, tra-1, in the nematode Caenorhabditis elegans. Genes Dev. 1, 731–745 (1987)
Procko, C., Lu, Y. & Shaham, S. Sensory organ remodeling in Caenorhabditis elegans requires the zinc-finger protein ZTF-16. Genetics 190, 1405–1415 (2012)
Hao, L., Johnsen, R., Lauter, G., Baillie, D. & Bürglin, T. R. Comprehensive analysis of gene expression patterns of hedgehog-related genes. BMC Genomics 7, 280 (2006)
White, J. Q. et al. The sensory circuitry for sexual attraction in C. elegans males. Curr. Biol. 17, 1847–1857 (2007)
Lee, K. & Portman, D. S. Neural sex modifies the function of a C. elegans sensory circuit. Curr. Biol. 17, 1858–1863 (2007)
Mowrey, W. R., Bennett, J. R. & Portman, D. S. Distributed effects of biological sex define sex-typical motor behavior in Caenorhabditis elegans. J. Neurosci. 34, 1579–1591 (2014)
WormBase. AMsoR http://www.wormbase.org/species/all/anatomy_term/WBbt:0003929#01-10
Jarriault, S., Schwab, Y. & Greenwald, I. A Caenorhabditis elegans model for epithelial–neuronal transdifferentiation. Proc. Natl Acad. Sci. USA 105, 3790–3795 (2008)
Okada, T. S. Transdifferentiation: Flexibility in Cell Differentiation (Clarendon Press, 1991)
Eguchi, G. & Kodama, R. Transdifferentiation. Curr. Opin. Cell Biol. 5, 1023–1028 (1993)
Noctor, S. C., Martínez-Cerdeño, V., Ivic, L. & Kriegstein, A. R. Cortical neurons arise in symmetric and asymmetric division zones and migrate through specific phases. Nature Neurosci. 7, 136–144 (2004)
Doetsch, F. The glial identity of neural stem cells. Nature Neurosci. 6, 1127–1134 (2003)
Ninkovic, J. & Gotz, M. Fate specification in the adult brain—lessons for eliciting neurogenesis from glial cells. Bioessays 35, 242–252 (2013)
Zuryn, S. et al. Sequential histone-modifying activities determine the robustness of transdifferentiation. Science 345, 826–829 (2014)
Arnold, A. P. Developmental plasticity in neural circuits controlling birdsong: sexual differentiation and the neural basis of learning. J. Neurobiol. 23, 1506–1528 (1992)
WormAtlas (2002–2015) (eds Altun, Z. F. et al.) http://www.wormatlas.org/colorcode.htm
Bargmann, C. I. & Avery, L. Laser killing of cells in Caenorhabditis elegans. Methods Cell Biol. 48, 225–250 (1995)
Hall, D. H., Hartwieg, E. & Nguyen, K. C. Q. Modern electron microscopy methods for C. elegans. Methods Cell Biol. 107, 93–149 (2012)
Saalfeld, S., Fetter, R., Cardona, A. & Tomancak, P. Elastic volume reconstruction from series of ultra-thin microscopy sections. Nature Methods 9, 717–720 (2012)
Cardona, A. et al. TrakEM2 software for neural circuit reconstruction. PLoS ONE 7, e38011 (2012)
Xu, M. et al. Computer assisted assembly of connectomes from electron micrographs: application to Caenorhabditis elegans. PLoS ONE 8, e54050 (2013)
Smoot, M. E., Ono, K., Ruscheinski, J., Wang, P.-L. & Ideker, T. Cytoscape 2.8: new features for data integration and network visualization. Bioinformatics 27, 431–432 (2011)
Tursun, B., Patel, T., Kratsios, P. & Hobert, O. Direct conversion of C. elegans germ cells into specific neuron types. Science 331, 304–308 (2011)
Altun-Gultekin, Z. et al. A regulatory cascade of three homeobox genes, ceh-10, ttx-3 and ceh-23, controls cell fate specification of a defined interneuron class in C. elegans. Development 128, 1951–1969 (2001)
Barrios, A., Ghosh, R., Fang, C., Emmons, S. W. & Barr, M. M. PDF-1 neuropeptide signaling modulates a neural circuit for mate-searching behavior in C. elegans. Nature Neuroscience 15, 1675–7682 (2012)
Zahn, T. R., Macmorris, M. A., Dong, W., Day, R. & Hutton, J. C. IDA-1, a Caenorhabditis elegans homolog of the diabetic autoantigens IA-2 and phogrin, is expressed in peptidergic neurons in the worm. J. Comp. Neurol. 429, 127–143 (2000)
Stefanakis, N., Carrera, I. & Hobert, O. Regulatory logic of pan-neuronal gene expression in C. elegans. Neuron 87, 733–750 (2015)
Mckay, S. J. et al. Gene expression profiling of cells, tissues, and developmental stages of the nematode C. elegans. Cold Spring Harb Quant. Biol. 68, 159–169 (2015)
Altun, Z. F., Chen B., Wang Z. W. & Hall, D. H. High resolution map of Caenorhabditis elegans gap junction proteins. Developmental Dynamics 238, 1936–1950 (2009)
Serrano-Saiz, E. et al. Modular control of glutamatergic neuronal identity in C. elegans by distinct homeodomain proteins. Cell 155, 659–673 (2013)
Kratsios, P., Stolfi, A., Levine, M. & Hobert, O. Coordinated regulation of cholinergic motor neuron traits through a conserved terminal selector gene. Nature Neuroscience 15, 205–214 (2011)
Bell, L. R., Stone, S., Yochem, J., Shaw, J. E. & Herman, R. K. The molecular identities of the Caenorhabditis elegans intraflagellar transport genes dyf-6, daf-10 and osm-1. Genetics 173, 1275–1286 (2006)
Gerisch, B., Weitzel, C., Kober-Eisermann, C., Rottiers, V. & Antebi, A. A hormonal signaling pathway influencing C. elegans metabolism, reproductive development, and life span. Dev. Cell 1, 841–851 (2001)
Alkema, M. J., Hunter-Ensor, M., Ringstad, N. & Horvitz, H. R. Tyramine functions independently of octopamine in the Caenorhabditis elegans nervous system. Neuron 46, 247–260 (2005)
Kim, K. & Li, C. Expression and regulation of an FMRFamide-related neuropeptide gene family in Caenorhabditis elegans. J. Comp. Neurol. 475, 540–550 (2004)
Beets, I. et al. Vasopressin/oxytocin-related signaling regulates gustatory associative learning in C. elegans. Science 338, 543–545 (2012)
Gruninger, T. R., Gualberto, D. G., LeBoeuf, B. & García, L. R. Integration of male mating and feeding behaviors in Caenorhabditis elegans. J. Neurosci. 26, 169–179 (2006)
Yoshimura, S., Murray, J. I., Lu, Y., Waterston, R. H. & Shaham, S. mls-2 and vab-3 control glia development, hlh-17/Olig expression and glia-dependent neurite extension in C. elegans. Development 135, 2263–2275 (2008)
Haklai-Topper, L. et al. The neurexin superfamily of Caenorhabditis elegans. Gene Expr. Patterns 11, 144–150 (2011)
Gower, N. J. D. et al. Dissection of the promoter region of the inositol 1,4,5-trisphosphate receptor gene, itr-1, in C. elegans: a molecular basis for cell-specific expression of IP3R isoforms. J. Mol. Biol. 306, 145–157 (2011)
Heiman, M. G. & Shaham, S. DEX-1 and DYF-7 establish sensory dendrite length by anchoring dendritic tips during cell migration. Cell 137, 344–355 (2009)
Acknowledgements
We would like to acknowledge M. Barr, in whose laboratory A.B. discovered the MCMs; WormAtlas for illustrations (reproduced with permission); T. Jarrell for contributions to the EM reconstruction; and W. Letton for the generation of strains and preliminary ablation studies. We thank M. Boxem, D. Portman, H. Baylis, L. Bianchi and R. Garcia for strains and reagents; M. Zhen, O. Hobert, I. Carrera, N. Stefanakis and S. Shaham, for unpublished reagents. Purified ascarosides were a gift from F. Schroeder to the Barr laboratory. Additional strains were obtained from the CGC, which is funded by NIH grant P40 OD010440. We thank L. Cochella, I. Carrera, S. Jarriault, and several of our close colleagues in CDB and NPP at University College London for discussions and comments on the manuscript; C. Barnes for advice on statistical analysis. This work was supported by a Master it! Scholarship Scheme (Malta and EU) to M.S., by NIH grant OD010943 to D.H.H., by Marie Curie CIG grant 618779 to R.J.P. and by a grant from The G. Harold and Leila Y. Mathers Charitable Foundation to S.W.E.; S.J.C. is supported by NIH grant 5T32GM007491; R.J.P. is a Wellcome Trust Research Career Development Fellow 095722/Z/11/Z; A.B. is supported by the Wellcome Trust Institutional Strategic Support Fund 097815/Z/11/A.
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M.S., T.F., R.J.P. and A.B. conceived and performed the development and behaviour experiments. S.J.C., K.C.Q.N., S.W.E. and D.H.H. performed the ultrastructural analysis of the MCMs. S.J.C. and S.W.E. reconstructed the connectivity of the MCMs from serial EM sections. R.J.P. and A.B. co-wrote the manuscript and discussed it with all the authors.
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Extended data figures and tables
Extended Data Figure 1 The MCMs are newly identified male-specific neurons.
a, WormAtlas-style diagram depicting the morphology and position of one of the bilateral pair of MCM neurons in the head of a male worm and its projection within the nerve ring and along the ventral cord. b, Volumetric reconstruction of the MCML cell body and projection based on tracing of serial EM sections. c, Co-expression of transgenes for neuronal markers in the rab-3-positive cells identified as MCMs (indicated with dashed red circles). All photographs are lateral views of animals oriented anterior to the left and dorsal to the top except for ric-19, which are dorsal views. Transgenes are listed in Extended Data Table 1. pdf-1 (neuropeptide pigment dispersing factor); snb-1 (synaptobrevin); ida-1 (tyrosine phosphatse-like receptor, orthologue of mammalian phogrin); ric-19 (rab-2 effector); nca-1 (NALCN Na+ channel subunit); ccb-1 (voltage-gated Ca2+ channel subunit); unc-36 (voltage-gated Ca2+ channel subunit); inx-3 (gap junction innexin). D, dorsal; L, left; R, right; V, ventral. d, Diagram of the neurons that directly connect to and from the MCMs. Triangles, sensory neurons; octagons, interneurons and unidentified neurons. The thickness of the arrows is proportional to the anatomical strength of the connections (Extended Data Table 2).
Extended Data Figure 2 The MCMs are not required for other male-specific behaviours.
a, Response of intact and MCM-ablated males (inIs179(ida-1::gfp);him-8(e1489) and otIs356(rab-3::rfp)him-5(e1490)) to dilutions of ascaroside pheromones (Ascr). Graphs represent Tukey box plots of logarithmic transformations of the data; n, number of independent events (that is, entry in scoring region). t-test with Bonferroni correction was used for statistical analysis. ***P < 0.001; **P < 0.01; *P < 0.05; n.s., no statistically significant difference (P ≥ 0.05). b, Response efficiency to mate contact of intact, MCM-ablated and pdf-1(tm1996) mutant males measured as the proportion of responses out of total contacts with an hermaphrodite. Intact and MCM-ablated animals were inIs179(ida-1::gfp);him-8(e1489). Wild-type animals were him-5(e1490). A response indicates that the male placed its tail ventral down on the mate’s body and backed along it to make a turn. c, d, Proportion of good turns (c) and location of vulva efficiency (d) of intact and MCM-ablated males (inIs179(ida-1::gfp);him-8(e1489) and otIs356(rab-3::rfp)him-5(e1490)). e, Fertility (measured as proportion of cross-progeny) of intact and MCM-ablated males (otIs356(rab-3::rfp)him-5(e1490)). For b–e, n, number of individual animals tested. Error bars indicate s.e.m. Mann–Whitney U-test was used for statistical analysis. *P < 0.05; n.s., no statistically significant difference (P ≥ 0.05). f, Mate-searching behaviour, measured as PL values (probability of leaving food per hour) in the absence or presence of mates, of intact and MCM-ablated males (otIs356(rab-3::rfp)him-5(e1490)). n, number of individual animals tested. Two independent population assays were performed on different days. Maximum likelihood statistical analysis was used to compare PL values. Error bars indicate s.e.m. ***P <0.001; n.s., no statistically significant difference (P ≥ 0.05).
Extended Data Figure 3 The MCMs arise from a division of the AMso glial cell.
All photographs are lateral views of animals oriented anterior to the left and dorsal to the top. a, Fluorescent photographs showing the two cells expressing rnr-1::gfp co-labelled with the glial marker ptr-10::rfp and the neuronal marker rab-3::rfp in the head of males at the early and late L4 stages. The AMso and MCM cell bodies are indicated with dashed lines. b, Fluorescent images of the AMso cell body and its projection at two time points during cell division. Photos are overexposed for visualization of the projection, indicated by arrows. The chromosomes are labelled with a histone::rfp transgene, and the AMso cell body is indicated by dashed lines.
Extended Data Figure 4 AMso plasticity is regulated by AMso genetic sex.
a, Diagram of the AMso and MCM lineage. b, c, Proportion of individuals with MCMs in control animals and animals expressing sex-reversing transgenes in AMso. b, AMso masculinization with grl-2::fem-3::SL2::mCherry transgenes (oleEx18 and oleEx24). c, AMso feminization with grl-2::tra-2IC::SL2::mCherry transgenes (oleEx19 and oleEx23) and ztf-16::tra-2IC::SL2::mCherry transgene oleEx22. MCM cell fate was identified with ida-1::gfp or rab-3::yfp reporter transgenes. In the head, the grl-2 promoter drives expression in AMso and the excretory duct and pore cells, and the ztf-16 glial enhancer drives expression in the AMso and amphid sheath glia. # indicates an independent transgenic array line for each manipulation. χ2 test was used for statistical analysis; ***P < 0.001; n.s., no statistical significant difference (P ≥ 0.05); n = number of animals scored.
Extended Data Figure 5 The MCMs lose molecular and structural characteristics of glia after birth.
a, Proportion of MCMs with presence of the glial marker ptr-10::myrRfp or the neuronal marker ida-1::gfp at different stages after MCM birth. b, Electron micrograph of a cross-section of an adult male head showing the MCM and AMso cell body ultrastructure. Neighbouring tissues are colour coded following WormAtlas (http://www.wormatlas.org/colorcode.htm). Purple (pharynx), muscle (green), hypodermis (light cream), AMso (amphid socket, pink). The dendrites of the amphid neurons (amphid bundle) are not colored.
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Sammut, M., Cook, S., Nguyen, K. et al. Glia-derived neurons are required for sex-specific learning in C. elegans. Nature 526, 385–390 (2015). https://doi.org/10.1038/nature15700
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DOI: https://doi.org/10.1038/nature15700
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