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

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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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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Establishment and maintenance of motor neuron identity via temporal modularity in terminal selector function

10.1101/2020.05.06.080184 ◽

2020 ◽

Author(s):

Yinan Li ◽

Anthony Osuma ◽

Edgar Correa ◽

Munachiso A. Okebalama ◽

Pauline Dao ◽

...

Keyword(s):

Transcription Factors ◽

Motor Neuron ◽

Motor Neurons ◽

Genome Engineering ◽

Life Stages ◽

C Elegans ◽

Neuronal Identity ◽

Protein Classes ◽

Selector Function

ABSTRACTTerminal selectors are transcription factors (TFs) that establish during development and maintain throughout life post-mitotic neuronal identity. We previously showed that UNC-3/Ebf, the terminal selector of C. elegans cholinergic motor neurons (MNs), acts indirectly to prevent alternative neuronal identities (Feng et al., 2020). Here, we globally identify the direct targets of UNC-3. Unexpectedly, we find that the suite of UNC-3 targets in MNs is modified across different life stages, revealing “temporal modularity” in terminal selector function. In all larval and adult stages examined, UNC-3 is required for continuous expression of various protein classes (e.g., receptors, transporters) critical for MN function. However, only in late larvae and adults, UNC-3 is required to maintain expression of MN-specific TFs. Minimal disruption of UNC-3’s temporal modularity via genome engineering affects locomotion. Another C. elegans terminal selector (UNC-30/Pitx) also exhibits temporal modularity, supporting the potential generality of this mechanism for the control of neuronal identity.

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Establishment and maintenance of motor neuron identity via temporal modularity in terminal selector function

eLife ◽

10.7554/elife.59464 ◽

2020 ◽

Vol 9 ◽

Author(s):

Yinan Li ◽

Anthony Osuma ◽

Edgar Correa ◽

Munachiso A Okebalama ◽

Pauline Dao ◽

...

Keyword(s):

Transcription Factors ◽

Motor Neuron ◽

Motor Neurons ◽

Genome Engineering ◽

Life Stages ◽

C Elegans ◽

Neuronal Identity ◽

Protein Classes ◽

Selector Function

Terminal selectors are transcription factors (TFs) that establish during development and maintain throughout life post-mitotic neuronal identity. We previously showed that UNC-3/Ebf, the terminal selector of C. elegans cholinergic motor neurons (MNs), acts indirectly to prevent alternative neuronal identities (Feng et al., 2020). Here, we globally identify the direct targets of UNC-3. Unexpectedly, we find that the suite of UNC-3 targets in MNs is modified across different life stages, revealing ‘temporal modularity’ in terminal selector function. In all larval and adult stages examined, UNC-3 is required for continuous expression of various protein classes (e.g. receptors, transporters) critical for MN function. However, only in late larvae and adults, UNC-3 is required to maintain expression of MN-specific TFs. Minimal disruption of UNC-3’s temporal modularity via genome engineering affects locomotion. Another C. elegans terminal selector (UNC-30/Pitx) also exhibits temporal modularity, supporting the potential generality of this mechanism for the control of neuronal identity.

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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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LIM homeobox gene-dependent expression of biogenic amine receptors in restricted regions of the C. elegans nervous system

Developmental Biology ◽

10.1016/s0012-1606(03)00447-0 ◽

2003 ◽

Vol 263 (1) ◽

pp. 81-102 ◽

Cited By ~ 143

Author(s):

Ephraim L Tsalik ◽

Timothy Niacaris ◽

Adam S Wenick ◽

Kelvin Pau ◽

Leon Avery ◽

...

Keyword(s):

Nervous System ◽

Biogenic Amine ◽

Homeobox Gene ◽

C Elegans ◽

Amine Receptors

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The enteric nervous system of C. elegans is specified by the Sine Oculis-like homeobox gene ceh-34

10.1101/2021.11.30.470650 ◽

2021 ◽

Author(s):

Berta Vidal ◽

Burcu Gulez ◽

Wen Xi Cao ◽

Eduardo Leyva Diaz ◽

Tessa Tekieli ◽

...

Keyword(s):

Nervous System ◽

Enteric Nervous System ◽

Critical Role ◽

Terminal Differentiation ◽

Homeobox Gene ◽

Neuron Type ◽

Nervous Systems ◽

Sine Oculis ◽

C Elegans ◽

Circuit Assembly

Overarching themes in the terminal differentiation of the enteric nervous system, an autonomously acting unit of animal nervous systems, have so far eluded discovery. We describe here the overall regulatory logic of enteric nervous system differentiation of the nematode C. elegans that resides within the foregut (pharynx) of the worm. A Caenorhabditis elegans homolog of the Drosophila Sine Oculis homeobox gene, ceh-34, is expressed in all 14 classes of interconnected pharyngeal neurons from their birth throughout their life time, but in no other neuron type of the entire animal. Constitutive and temporally controlled ceh-34 removal shows that ceh-34 is required to initiate and maintain the neuron type-specific terminal differentiation program of all pharyngeal neuron classes, including their circuit assembly, without affecting panneuronal features. Through additional genetic loss of function analysis, we show that within each pharyngeal neuron class, ceh-34 cooperates with different homeodomain transcription factors to individuate distinct pharyngeal neuron classes. Our analysis underscores the critical role of homeobox genes in neuronal identity specification and links them to the control of neuronal circuit assembly of the enteric nervous system. Together with the pharyngeal nervous system simplicity as well as its specification by a Sine Oculis homolog, our findings invite speculations about the early evolution of nervous systems.

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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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Semaphorin signaling restricts neuronal regeneration in C. elegans

10.1101/2021.11.13.468479 ◽

2021 ◽

Author(s):

Maria Belen Harreguy ◽

Esha Shah ◽

Zainab Tanvir ◽

Blandine Simprevil ◽

Tracy S. Tran ◽

...

Keyword(s):

Nervous System ◽

Synapse Formation ◽

Neuronal Morphology ◽

Axonal Guidance ◽

Axon Pathfinding ◽

Neuronal Regeneration ◽

C Elegans ◽

Locomotion Behavior ◽

Neuronal Growth Cone ◽

Knockout Animals

Extracellular signaling proteins serve as neuronal growth cone guidance molecules during development and are well positioned to be involved in neuronal regeneration and recovery from injury. Semaphorins and their receptors, the plexins, are a family of conserved proteins involved in development that, in the nervous system, are axonal guidance cues mediating axon pathfinding and synapse formation. The Caenorhabditis elegans genome encodes for three semaphorins and two plexin receptors: the transmembrane semaphorins, SMP-1 and SMP-2, signal through their receptor, PLX-1, while the secreted semaphorin, MAB-20, signals through PLX-2. Here, we determined the neuronal morphology and locomotion behavior of knockout animals missing each of the semaphorins and plexins; we described the expression pattern of all plexins in the nervous system of C. elegans; and we evaluated their effect on the regeneration of motoneuron neurites and the recovery of locomotion behavior following precise laser microsurgery.

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

Development ◽

10.1242/dev.199687 ◽

2021 ◽

Author(s):

Tessa Tekieli ◽

Eviatar Yemini ◽

Amin Nejatbakhsh ◽

Chen Wang ◽

Erdem Varol ◽

...

Keyword(s):

Nervous System ◽

Just In Time ◽

Differentiation Markers ◽

C Elegans ◽

Larval Stages ◽

Neuronal Identity ◽

Fourth Larval Stage ◽

Developmental Patterning ◽

Male Specific ◽

The Brain

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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