Visualizing the organization and differentiation of the male-specific nervous system of C. elegans

Sex differences in the brain are prevalent throughout the animal kingdom and particularly well appreciated in the nematode C. elegans, where male animals contain a little studied set of 93 male-specific neurons. To make these neurons amenable for future study, we describe here how a multicolor reporter transgene, NeuroPAL, is capable of visualizing the distinct identities of all male specific neurons. We used NeuroPAL to visualize and characterize a number of features of the male-specific nervous system. We provide several proofs of concept for using NeuroPAL to identify the sites of expression of gfp-tagged reporter genes and for cellular fate analysis by analyzing the effect of removal of several developmental patterning genes on neuronal identity acquisition. We use NeuroPAL and its intrinsic cohort of more than 40 distinct differentiation markers to show that, even though male-specific neurons are generated throughout all four larval stages, they execute their terminal differentiation program in a coordinated manner in the fourth larval stage. This coordinated wave of differentiation, which we call “just-in-time" differentiation, couples neuronal maturation programs with the appearance of sexual organs.

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Visualizing the organization and differentiation of the male-specific nervous system of C. elegans

10.1101/2021.04.06.438718 ◽

2021 ◽

Author(s):

Tessa Tekieli ◽

Eviatar Yemini ◽

Amin Nejatbakhsh ◽

Erdem Varol ◽

Robert W Fernandez ◽

...

Keyword(s):

Nervous System ◽

Just In Time ◽

Differentiation Markers ◽

C Elegans ◽

Larval Stages ◽

Fourth Larval Stage ◽

Developmental Patterning ◽

Male Specific ◽

Cluster Gene ◽

The Brain

Sex differences in the brain are prevalent throughout the animal kingdom and particularly well appreciated in the nematode C. elegans. While 294 neurons are shared between the two sexes, the nervous system of the male contains an additional 93 male-specific neurons, most of which have received very little attention so far. To make these neurons amenable for future study, we describe here how a multicolor, multipromoter reporter transgene, NeuroPAL, is capable of visualizing the distinct identities of all male specific neurons. We used this tool to visualize and characterize a number of features of the male-specific nervous system. We provide several proofs of concept for using NeuroPAL to identify the sites of expression of gfp-tagged reporter genes. We demonstrate the usage of NeuroPAL for cellular fate analysis by analyzing the effect of removal of developmental patterning genes, including a HOX cluster gene (egl-5), a miRNA (lin-4) and a proneural gene (lin-32/Ato), on neuronal identity acquisition within the male-specific nervous system. We use NeuroPAL and its intrinsic cohort of more than 40 distinct differentiation markers to show that, even though male-specific neurons are generated throughout all four larval stages, they execute their terminal differentiation program in a coordinated manner in the fourth larval stage that is concomitant with male tale retraction. This wave of differentiation couples neuronal maturation programs with the appearance of sexual organs. We call this wave 'just-in-time' differentiation by its analogy to the mechanism of 'just-in-time' transcription of metabolic pathway genes.

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Natural sensory context drives diverse brain-wide activity during C. elegans mating

10.1101/2020.09.09.289454 ◽

2020 ◽

Cited By ~ 1

Author(s):

Vladislav Susoy ◽

Wesley Hung ◽

Daniel Witvliet ◽

Joshua E. Whitener ◽

Min Wu ◽

...

Keyword(s):

Nervous System ◽

Behavioral Context ◽

C Elegans ◽

Behavioral Dynamics ◽

Brain Circuits ◽

Natural Context ◽

Neuronal Imaging ◽

Complex Sequences ◽

Motor Actions ◽

The Brain

AbstractNatural goal-directed behaviors often involve complex sequences of many stimulus-triggered components. Understanding how brain circuits organize such behaviors requires mapping the interactions between an animal, its environment, and its nervous system. Here, we use continuous brain-wide neuronal imaging to study the full performance of mating by the C. elegans male. We show that as each mating unfolds in its own sequence of component behaviors, the brain operates similarly between instances of each component, but distinctly between different components. When the full sensory and behavioral context is taken into account, unique roles emerge for each neuron. Functional correlations between neurons are not fixed, but change with behavioral dynamics. From the contribution of individual neurons to circuits, our study shows how diverse brain-wide dynamics emerge from the integration of sensory perception and motor actions within their natural context.

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In silico analysis of the transcriptional regulatory logic of neuronal identity specification throughout the C. elegans nervous system

eLife ◽

10.7554/elife.64906 ◽

2021 ◽

Vol 10 ◽

Author(s):

Lori Glenwinkel ◽

Seth R Taylor ◽

Kasper Langebeck-Jensen ◽

Laura Pereira ◽

Molly B Reilly ◽

...

Keyword(s):

Nervous System ◽

Transcription Factors ◽

Single Cell ◽

Expression Profiles ◽

Neuronal Cell ◽

In Silico Analysis ◽

Specific Gene ◽

Neuron Type ◽

C Elegans ◽

Neuronal Identity

The generation of the enormous diversity of neuronal cell types in a differentiating nervous system entails the activation of neuron type-specific gene batteries. To examine the regulatory logic that controls the expression of neuron type-specific gene batteries, we interrogate single cell expression profiles of all 118 neuron classes of the Caenorhabditis elegans nervous system for the presence of DNA binding motifs of 136 neuronally expressed C. elegans transcription factors. Using a phylogenetic footprinting pipeline, we identify cis-regulatory motif enrichments among neuron class-specific gene batteries and we identify cognate transcription factors for 117 of the 118 neuron classes. In addition to predicting novel regulators of neuronal identities, our nervous system-wide analysis at single cell resolution supports the hypothesis that many transcription factors directly co-regulate the cohort of effector genes that define a neuron type, thereby corroborating the concept of so-called terminal selectors of neuronal identity. Our analysis provides a blueprint for how individual components of an entire nervous system are genetically specified.

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The Prop1-like homeobox gene unc-42 specifies the identity of synaptically connected neurons

eLife ◽

10.7554/elife.64903 ◽

2021 ◽

Vol 10 ◽

Cited By ~ 1

Author(s):

Emily G Berghoff ◽

Lori Glenwinkel ◽

Abhishek Bhattacharya ◽

HaoSheng Sun ◽

Erdem Varol ◽

...

Keyword(s):

Nervous System ◽

Transcription Factors ◽

Homeobox Gene ◽

Synaptic Connectivity ◽

Axon Pathfinding ◽

C Elegans ◽

Neuronal Identity ◽

Anatomical Analysis ◽

Neuropeptide Receptors ◽

Functional Circuitry

Many neuronal identity regulators are expressed in distinct populations of cells in the nervous system, but their function is often analyzed only in specific isolated cellular contexts, thereby potentially leaving overarching themes in gene function undiscovered. We show here that the Caenorhabditis elegans Prop1-like homeobox gene unc-42 is expressed in 15 distinct sensory, inter- and motor neuron classes throughout the entire C. elegans nervous system. Strikingly, all 15 neuron classes expressing unc-42 are synaptically interconnected, prompting us to investigate whether unc-42 controls the functional properties of this circuit and perhaps also the assembly of these neurons into functional circuitry. We found that unc-42 defines the routes of communication between these interconnected neurons by controlling the expression of neurotransmitter pathway genes, neurotransmitter receptors, neuropeptides, and neuropeptide receptors. Anatomical analysis of unc-42 mutant animals reveals defects in axon pathfinding and synaptic connectivity, paralleled by expression defects of molecules involved in axon pathfinding, cell-cell recognition, and synaptic connectivity. We conclude that unc-42 establishes functional circuitry by acting as a terminal selector of functionally connected neuron types. We identify a number of additional transcription factors that are also expressed in synaptically connected neurons and propose that terminal selectors may also function as ‘circuit organizer transcription factors’ to control the assembly of functional circuitry throughout the nervous system. We hypothesize that such organizational properties of transcription factors may be reflective of not only ontogenetic, but perhaps also phylogenetic trajectories of neuronal circuit establishment.

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Neural Circuits of Sexual Behavior inCaenorhabditis elegans

Annual Review of Neuroscience ◽

10.1146/annurev-neuro-070815-014056 ◽

2018 ◽

Vol 41 (1) ◽

pp. 349-369 ◽

Cited By ~ 20

Author(s):

Scott W. Emmons

Keyword(s):

Nervous System ◽

Sex Differences ◽

Sexual Behaviors ◽

Neural Circuits ◽

Egg Laying ◽

Synaptic Connectivity ◽

Comprehensive Description ◽

C Elegans ◽

Shared Component ◽

Male Specific

The recently determined connectome of the Caenorhabditis elegans adult male, together with the known connectome of the hermaphrodite, opens up the possibility for a comprehensive description of sexual dimorphism in this species and the identification and study of the neural circuits underlying sexual behaviors. The C. elegans nervous system consists of 294 neurons shared by both sexes plus neurons unique to each sex, 8 in the hermaphrodite and 91 in the male. The sex-specific neurons are well integrated within the remainder of the nervous system; in the male, 16% of the input to the shared component comes from male-specific neurons. Although sex-specific neurons are involved primarily, but not exclusively, in controlling sex-unique behavior—egg-laying in the hermaphrodite and copulation in the male—these neurons act together with shared neurons to make navigational choices that optimize reproductive success. Sex differences in general behaviors are underlain by considerable dimorphism within the shared component of the nervous system itself, including dimorphism in synaptic connectivity.

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Temporal transitions in post-mitotic neurons throughout the C. elegans nervous system

10.1101/2020.10.06.328880 ◽

2020 ◽

Author(s):

HaoSheng Sun ◽

Oliver Hobert

Keyword(s):

Nervous System ◽

Transcription Factor ◽

Embryonic Development ◽

Model Organism ◽

Specific Gene ◽

Molecular Changes ◽

Functional Changes ◽

C Elegans ◽

Larval Stages ◽

Regulatory Strategies

SUMMARYIn most animals, the majority of the nervous system is generated and assembled into neuronal circuits during embryonic development. However, during juvenile stages, nervous systems still undergo extensive anatomical and functional changes to eventually form a fully mature nervous system by the adult stage. The molecular changes in post-mitotic neurons across post-embryonic development and the genetic programs that control these temporal transitions are not well understood. Using the model organism C. elegans, we comprehensively characterized the distinct functional states (locomotor behavior) and corresponding distinct molecular states (transcriptome) of the post-mitotic nervous system across temporal transitions from early post-embryonic periods to adulthood. We observed pervasive changes in gene expression, many of which are controlled by the developmental upregulation of the conserved heterochronic miRNA lin-4/mir-125 and the subsequent promotion of a mature neuronal transcriptional program through the repression of its target, the transcription factor lin-14. The functional relevance of these molecular transitions are exemplified by a temporally regulated target gene of the lin-14 transcription factor, nlp-45, a neuropeptide-encoding gene. We found that nlp-45 is required for temporal transitions in exploratory activity across larval stages, across sexual maturation, and into a diapause arrest stage. Our studies provide new insights into regulatory strategies that control neuron-type specific gene batteries to modulate distinct behaviors states across temporal, sex and environmental dimensions of post-embryonic development, and also provides a rich atlas of post-embryonic molecular changes to uncover additional regulatory mechanisms.

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Connectomes across development reveal principles of brain maturation in C. elegans

10.1101/2020.04.30.066209 ◽

2020 ◽

Cited By ~ 9

Author(s):

Daniel Witvliet ◽

Ben Mulcahy ◽

James K. Mitchell ◽

Yaron Meirovitch ◽

Daniel R. Berger ◽

...

Keyword(s):

Nervous System ◽

Brain Maturation ◽

Wiring Diagram ◽

Motor Pathways ◽

C Elegans ◽

Section Electron ◽

Synaptic Changes ◽

Serial Section Electron Microscopy ◽

Synaptic Change ◽

The Brain

AbstractFrom birth to adulthood, an animal’s nervous system changes as its body grows and its behaviours mature. However, the extent of circuit remodelling across the connectome is poorly understood. Here, we used serial-section electron microscopy to reconstruct the brain of eight isogenic C. elegans individuals at different ages to learn how an entire wiring diagram changes with maturation. We found that the overall geometry of the nervous system is preserved from birth to adulthood, establishing a constant scaffold upon which synaptic change is built. We observed substantial connectivity differences among individuals that make each brain partly unique. We also observed developmental connectivity changes that are consistent between animals but different among neurons, altering the strengths of existing connections and creating additional connections. Collective synaptic changes alter information processing of the brain. Across maturation, the decision-making circuitry is maintained whereas sensory and motor pathways are substantially remodelled, and the brain becomes progressively more modular and feedforward. These synaptic changes reveal principles that underlie brain maturation.

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Sexually dimorphic differentiation of a C. elegans hub neuron is cell-autonomously controlled by a conserved transcription factor

10.1101/081083 ◽

2016 ◽

Author(s):

Esther Serrano-Saiz ◽

Meital Oren-Suissa ◽

Emily A. Bayer ◽

Oliver Hobert

Keyword(s):

Transcription Factor ◽

Glutamate Transporter ◽

Sexually Dimorphic ◽

Neuronal Function ◽

Sexual Dimorphisms ◽

C Elegans ◽

Cellular Specificity ◽

Male Specific ◽

The Brain ◽

Canonical Pattern

SUMMARYFunctional 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 describe 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 master regulator of hermaphroditic cell identity, the Gli-like transcription factor tra-1, which transcriptionally represses dmd-3 in hermaphrodite PHC. Taken together, our studies provide mechanistic insights into how neurons are specified in a sexually dimorphic manner.

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Timing mechanism of sexually dimorphic nervous system differentiation

10.1101/416735 ◽

2018 ◽

Author(s):

Laura Pereira ◽

Florian Aeschimann ◽

Chen Wang ◽

Hannah Lawson ◽

Esther Serrano-Saiz ◽

...

Keyword(s):

Nervous System ◽

Transcription Factors ◽

Sexual Maturation ◽

Sexual Differentiation ◽

Rna Binding ◽

Receptor Expression ◽

Sexually Dimorphic ◽

Neuron Type ◽

C Elegans ◽

Male Specific

ABSTRACTIn all animals, sexual differentiation of somatic tissue is precisely timed, yet the molecular mechanisms that control the timing of sexual differentiation, particularly in the brain, are poorly understood. We have used sexually dimorphic molecular, anatomical and behavioral features of the C. elegans nervous system to decipher a regulatory pathway that controls the precise timing of sexual differentiation. We find that the sexually dimorphic differentiation of postmitotic neurons in the male nervous system is abrogated in animals that carry a mutation in the miRNA let-7 and prematurely executed in animals either lacking the let-7 inhibitor lin-28, or the direct let-7 target lin-41, an RNA-binding, posttranscriptional regulator. We show that an isoform of a phylogenetically conserved transcription factor, lin-29a, is a critical target of LIN-41 in controlling sexual maturation of sex-shared neurons. lin-29a is expressed in a male-specific manner in a subset of sex-shared neurons at the onset of sexual maturation. lin-29a acts cell-autonomously in these neurons to control the expression of sexually dimorphic neurotransmitter switches, sensory receptor expression, neurite anatomy and connectivity, and locomotor behavior. lin-29a is not only required but also sufficient to impose male-specific features at earlier stages of development and in the opposite sex. The temporal, sexual and spatial specificity of lin-29a expression is controlled intersectionally through the lin-28/let-7/lin-41 heterochronic pathway, sex chromosome configuration and neuron type-specific terminal selector transcription factors. Two Doublesex-like transcription factors represent additional neuron-type specific targets of LIN-41 and are regulated in a similar intersectional manner, indicating the existence of modular outputs downstream of the heterochronic pathway. In conclusion, we have provided insights into the molecular logic of the timing of sexual differentiation in the C. elegans nervous system. Remarkably, the lin28/let7 axis also controls the timing of sexual differentiation in mice and humans thereby hinting toward a striking universality of the control mechanisms of sexual differentiation.

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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 ◽

10.1242/dev.128.11.1951 ◽

2001 ◽

Vol 128 (11) ◽

pp. 1951-1969 ◽

Cited By ~ 8

Author(s):

Zeynep Altun-Gultekin ◽

Yoshiki Andachi ◽

Ephraim L. Tsalik ◽

David Pilgrim ◽

Yuji Kohara ◽

...

Keyword(s):

Nervous System ◽

Cell Fate ◽

Homeobox Genes ◽

Homeobox Gene ◽

Homeodomain Proteins ◽

Differentiation Markers ◽

Regulatory Relationship ◽

C Elegans ◽

Regulatory Cascade ◽

The Individual

The development of the nervous system requires the coordinated activity of a variety of regulatory factors that define the individual properties of specific neuronal subtypes. We report a regulatory cascade composed of three homeodomain proteins that act to define the properties of a specific interneuron class in the nematode C. elegans. We describe a set of differentiation markers characteristic for the AIY interneuron class and show that the ceh-10 paired-type and ttx-3 LIM-type homeobox genes function to regulate all known subtype-specific features of the AIY interneurons. In contrast, the acquisition of several pan-neuronal features is unaffected in ceh-10 and ttx-3 mutants, suggesting that the activity of these homeobox genes separates pan-neuronal from subtype-specific differentiation programs. The LIM homeobox gene ttx-3 appears to play a central role in regulation of AIY differentiation. Not only are all AIY subtype characteristics lost in ttx-3 mutants, but ectopic misexpression of ttx-3 is also sufficient to induce AIY-like features in a restricted set of neurons. One of the targets of ceh-10 and ttx-3 is a novel type of homeobox gene, ceh-23. We show that ceh-23 is not required for the initial adoption of AIY differentiation characteristics, but instead is required to maintain the expression of one defined AIY differentiation feature. Finally, we demonstrate that the regulatory relationship between ceh-10, ttx-3 and ceh-23 is only partially conserved in other neurons in the nervous system. Our findings illustrate the complexity of transcriptional regulation in the nervous system and provide an example for the intricate interdependence of transcription factor action.

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