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SUMMARYThe plasma membrane of excitable cells is highly structured and molecular scaffolds recruit proteins to specific membrane compartments. Here, we show that potassium channels and proteins belonging to the dystrophin-associated protein complex define multiple types of planar-polarized membrane compartments at the surface ofC. elegansmuscle cells. Surprisingly, conserved planar cell polarity proteins are not required for this process. However, we implicate a Wnt signaling module involving the Wnt ligand EGL-20, the Wnt receptor CAM-1, and the intracellular effector DSH-1/disheveled in the formation of this cell polarity pattern. Moreover, using time-resolved and tissue-specific protein degradation, we demonstrate that muscle cell polarity is a dynamic state, requiring continued presence of DSH-1 throughout post-embryonic life. Our results reveal the intricate, highly reproducible, and entirely unsuspected complexity of the worm’s sarcolemma. This novel case of planar cell polarity in a tractable genetic model organism may provide valuable insight into the molecular and cellular mechanisms that regulate cellular organization, allowing specific functions to be compartmentalized within distinct plasma membrane domains.

Here, Serrano-Saiz et al. describe the cis-regulatory logic of how neurotransmitter identity is imposed onto individual, distinct neuron types... We explore here the cis-regulatory logic that dictates gene expression in specific cell types in the nervous system. We focus on a set of eight genes involved in the synthesis, transport, and breakdown of three neurotransmitter systems: acetylcholine (unc-17/VAChT, cha-1/ChAT, cho-1/ChT, and ace-2/AChE), glutamate (eat-4/VGluT), and γ-aminobutyric acid (unc-25/GAD, unc-46/LAMP, and unc-47/VGAT). These genes are specifically expressed in defined subsets of cells in the nervous system. Through transgenic reporter gene assays, we find that the cellular specificity of expression of all of these genes is controlled in a modular manner through distinct cis-regulatory elements, corroborating the previously inferred piecemeal nature of specification of neurotransmitter identity. This modularity provides the mechanistic basis for the phenomenon of “phenotypic convergence,” in which distinct regulatory pathways can generate similar phenotypic outcomes (i.e., the acquisition of a specific neurotransmitter identity) in different neuron classes. We also identify cases of enhancer pleiotropy, in which the same cis-regulatory element is utilized to control gene expression in distinct neuron types. We engineered a cis-regulatory allele of the vesicular acetylcholine transporter, unc-17/VAChT, to assess the functional contribution of a “shadowed” enhancer. We observed a selective loss of unc-17/VAChT expression in one cholinergic pharyngeal pacemaker motor neuron class and a behavioral phenotype that matches microsurgical removal of this neuron. Our analysis illustrates the value of understanding cis-regulatory information to manipulate gene expression and control animal behavior.

Neurobeachin (NBEA) was initially identified as a candidate gene for autism. Recently, variants in NBEA have been associated with neurodevelopmental delay and childhood epilepsy. Here, we report on a novel NBEA missense variant (c.5899G > A, p.Gly1967Arg) in the Domain of Unknown Function 1088 (DUF1088) identified in a child enrolled in the Undiagnosed Diseases Network (UDN), who presented with neurodevelopmental delay and seizures. Modeling of this variant in the Caenorhabditis elegans NBEA ortholog, sel-2, indicated that the variant was damaging to in vivo function as evidenced by altered cell fate determination and trafficking of potassium channels in neurons. The variant effect was indistinguishable from that of the reference null mutation suggesting that the variant is a strong hypomorph or a complete loss-of-function. Our experimental data provide strong support for the molecular diagnosis and pathogenicity of the NBEA p.Gly1967Arg variant and the importance of the DUF1088 for NBEA function.

We explore here the cis-regulatory logic that dictates gene expression in specific cell types in the nervous system. We focus on a set of eight genes involved in the synthesis, transport, and breakdown of three neurotransmitter systems: acetylcholine (unc-17/VAChT, cha-1/ChAT, cho-1/ChT, and ace-2/AChE), glutamate (eat-4/VGluT), and γ-aminobutyric acid (unc-25/GAD, unc-46/LAMP, and unc-47/VGAT). These genes are specifically expressed in defined subsets of cells in the nervous system. Through transgenic reporter gene assays, we find that the cellular specificity of expression of all of these genes is controlled in a modular manner through distinct cis-regulatory elements, corroborating the previously inferred piecemeal nature of specification of neurotransmitter identity. This modularity provides the mechanistic basis for the phenomenon of “phenotypic convergence,” in which distinct regulatory pathways can generate similar phenotypic outcomes (i.e., the acquisition of a specific neurotransmitter identity) in different neuron classes. We also identify cases of enhancer pleiotropy, in which the same cis-regulatory element is utilized to control gene expression in distinct neuron types. We engineered a cis-regulatory allele of the vesicular acetylcholine transporter, unc-17/VAChT, to assess the functional contribution of a “shadowed” enhancer. We observed a selective loss of unc-17/VAChT expression in one cholinergic pharyngeal pacemaker motor neuron class and a behavioral phenotype that matches microsurgical removal of this neuron. Our analysis illustrates the value of understanding cis-regulatory information to manipulate gene expression and control animal behavior.

In this study, Serrano-Saiz et al. map the neurotransmitter identity of neurons in the Caenorhabditis elegans male nervous system, thereby providing... The nervous system of most animals is sexually dimorphic but such dimorphisms are generally poorly mapped on an anatomical, cellular, and molecular level. The adult nervous system of the nematode Caenorhabditis elegans displays a number of clearly defined anatomical sexual dimorphisms, but molecular features of sexually dimorphic neurons remain sparse. In this resource paper, we provide a comprehensive atlas of neurotransmitters used in the nervous system of the male and compare it to that of the hermaphrodite. Among the three major neurotransmitter systems, acetylcholine (ACh) is the most frequently used, followed by glutamate (Glu), and lastly γ-aminobutyric acid (GABA). Many male-specific neurons utilize multiple neurotransmitter systems. Interestingly, we find that neurons that are present in both sexes alter their neurotransmitter usage depending on the sex of the animal. One neuron scales up its usage of ACh, another becomes serotonergic in males, and another one adds a new neurotransmitter (glutamate) to its nonsex-specific transmitter (ACh). In all these cases, neurotransmitter changes are correlated with substantial changes in synaptic connectivity. We assembled the neurotransmitter maps of the male-specific nervous system into a comprehensive atlas that describes the anatomical position of all the neurons of the male-specific nervous system relative to the sex-shared nervous system. We exemplify the usefulness of the neurotransmitter atlas by using it as a tool to define the expression pattern of a synaptic organizer molecule in the male tail. Taken together, the male neurotransmitter atlas provides an entry point for future functional and developmental analysis of the male nervous system.

The nervous system of most animals is sexually dimorphic but such dimorphisms are generally poorly mapped on an anatomical, cellular, and molecular level. The adult nervous system of the nematode

The nervous system of the nematode C. elegans is the only nervous system with a complete parts list and a complete wiring diagram. Neurotransmitter maps are important complements to such anatomical maps and represent an invaluable resource that will help to understand how signals are transmitted throughout the nervous system and how a nervous system is developmentally patterned. In this resource paper, we report a comprehensive map of neurons in the hermaphroditic and male C. elegans nervous system that contain the neurotransmitter GABA, the most prominent inhibitory neurotransmitter in mammalian nervous systems. We reveal twice as many GABA-positive neuron classes as previously reported, several of them also cotransmitting acetylcholine. We define previously unknown glia-like cells that reuptake GABA, as well as 'GABA reuptake neurons' which do not synthesize GABA but take it up from the extracellular environment. One GABA reuptake neuron type may clear GABA thereby possibly modulating GABAergic neurotransmission ('GABA clearance' neuron), while another GABA reuptake neuron type may re-release GABA (termed 'GABA recycling' neuron). We used the map of GABA-positive neurons for a comprehensive analysis of transcriptional regulators that define the GABA phenotype. We show that nine transcription factors operate in specific combinations to control GABAergic identity of most GABAergic neuron classes. Among these factors is elt-1, the C. elegans ortholog of GATA2/3, a selector gene of GABAergic identity of several distinct types of GABAergic neurons in vertebrates. Other factors that control GABAergic identity include the Tlx-type nuclear hormone receptor nhr-67 which controls the identity of four distinct GABAergic neuron classes, in which, depending on neuron type, nhr-67 cooperates with three different types of homeobox genes. We also identified homeobox genes that specify GABA recycling neurons and GABA clearance neurons. The regulators of GABAergic neuron identity do not only specify neurotransmitter identity, but multiple other identity features as well. We synthesize our findings of specification of GABAergic neurons with previous reports on the specification of glutamatergic and cholinergic neurons into a nervous system-wide regulatory map which defines neurotransmitter specification mechanisms for more than half of all neuron classes in C. elegans.

The dystrophin-associated protein complex (DAPC) consists of several transmembrane and intracellular scaffolding elements that have been implicated in maintaining the structure and morphology of the vertebrate neuromuscular junction (NMJ). Genetic linkage analysis has identified loss-of-function mutations in DAPC genes that give rise to degenerative muscular dystrophies. Although much is known about the involvement of the DAPC in maintaining muscle integrity, less is known about the precise contribution of the DAPC in cell signaling events. To better characterize the functional role of the DAPC at the NMJ, we used electrophysiology, immunohistochemistry, and fluorescent labeling to directly assess cholinergic synaptic transmission, ion channel localization, and muscle excitability in loss-of-function (lf) mutants of Caenorhabditis elegans DAPC homologues. We found that all DAPC mutants consistently display mislocalization of the Ca(2+)-gated K(+) channel, SLO-1, in muscle cells, while ionotropic acetylcholine receptor (AChR) expression and localization at the NMJ remained unaltered. Synaptic cholinergic signaling was also not significantly impacted across DAPC(lf) mutants. Consistent with these findings and the postsynaptic mislocalization of SLO-1, we observed an increase in muscle excitability downstream of cholinergic signaling. Based on our results, we conclude that the DAPC is not involved in regulating AChR architecture at the NMJ, but rather functions to control muscle excitability, in an activity-dependent manner, through the proper localization of SLO-1 channels.

For nerves to communicate efficiently with their target cells, the sites of neurotransmitter release must be properly aligned with zones of higher receptor densities. Working on the genetically tractable worm Caenorhabditis elegans, Gendrel et al. show that muscle cells secrete the protein LEV-9, which in turn interacts with LEV-10 to control proper clustering of the acetylcholine receptor. Surprisingly, LEV-9's synaptic function relies on 'control complement protein' (CCP) domains, known until now for their possible involvement in the mammalian immune system. The many CCP proteins that are produced in the mammalian brain might similarly control synapse organization, rather than immune functions. For efficient neurotransmission at chemical synapses to occur, there must be spatial congruence between the presynaptic area where synaptic vesicles fuse and the postsynaptic area where neurotransmitter receptors concentrate. An extracellular scaffold is now described that is necessary for the clustering of acetylcholine receptors at neuromuscular junctions in the nematode Caenorhabditis elegans; it involves the protein LEV-9, the function of which relies on complement control protein domains. Efficient neurotransmission at chemical synapses relies on spatial congruence between the presynaptic active zone, where synaptic vesicles fuse, and the postsynaptic differentiation, where neurotransmitter receptors concentrate. Diverse molecular systems have evolved to localize receptors at synapses, but in most cases, they rely on scaffolding proteins localized below the plasma membrane1,2,3. A few systems have been suggested to control the synaptic localization of neurotransmitter receptors through extracellular interactions, such as the pentraxins that bind AMPA receptors and trigger their aggregation4. However, it is not yet clear whether these systems have a central role in the organization of postsynaptic domains in vivo or rather provide modulatory functions5. Here we describe an extracellular scaffold that is necessary to cluster acetylcholine receptors at neuromuscular junctions in the nematode Caenorhabditis elegans. It involves the ectodomain of the previously identified transmembrane protein LEV-10 (ref. 6) and a novel extracellular protein, LEV-9. LEV-9 is secreted by the muscle cells and localizes at cholinergic neuromuscular junctions. Acetylcholine receptors, LEV-9 and LEV-10 are interdependent for proper synaptic localization and physically interact based on biochemical evidence. Notably, the function of LEV-9 relies on eight complement control protein (CCP) domains. These domains, also called ‘sushi domains’, are usually found in proteins regulating complement activity in the vertebrate immune system7. Because the complement system does not exist in protostomes, our results suggest that some of the numerous uncharacterized CCP proteins expressed in the mammalian brain might be directly involved in the organization of the synapse, independently from immune functions.

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