A cellular and regulatory map of the GABAergic nervous system of C. elegans

Neurotransmitter maps are important complements to anatomical maps and represent an invaluable resource to understand nervous system function and development. We report here a comprehensive map of neurons in the C. elegans nervous system that contain the neurotransmitter GABA, revealing twice as many GABA-positive neuron classes as previously reported. We define previously unknown glia-like cells that take up GABA, as well as 'GABA uptake neurons' which do not synthesize GABA but take it up from the extracellular environment, and we map the expression of previously uncharacterized ionotropic GABA receptors. We use the map of GABA-positive neurons for a comprehensive analysis of transcriptional regulators that define the GABA phenotype. 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

Modular Organization of Cis-regulatory Control Information of Neurotransmitter Pathway Genes in Caenorhabditis elegans

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.This work was supported by the Howard Hughes Medical Institute. E.S.-S. has been supported by the Ramon y Cajal program (RYC-2016-20537), and M.G. was supported by the European Molecular Biology Organization and Human Frontier Science Program postdoctoral fellowships.Peer reviewe

Optimizing the Design of Oligonucleotides for Homology Directed Gene Targeting

BACKGROUND: Gene targeting depends on the ability of cells to use homologous recombination to integrate exogenous DNA into their own genome. A robust mechanistic model of homologous recombination is necessary to fully exploit gene targeting for therapeutic benefit. METHODOLOGY/PRINCIPAL FINDINGS: In this work, our recently developed numerical simulation model for homology search is employed to develop rules for the design of oligonucleotides used in gene targeting. A Metropolis Monte-Carlo algorithm is used to predict the pairing dynamics of an oligonucleotide with the target double-stranded DNA. The model calculates the base-alignment between a long, target double-stranded DNA and a probe nucleoprotein filament comprised of homologous recombination proteins (Rad51 or RecA) polymerized on a single strand DNA. In this study, we considered different sizes of oligonucleotides containing 1 or 3 base heterologies with the target; different positions on the probe were tested to investigate the effect of the mismatch position on the pairing dynamics and stability. We show that the optimal design is a compromise between the mean time to reach a perfect alignment between the two molecules and the stability of the complex. CONCLUSION AND SIGNIFICANCE: A single heterology can be placed anywhere without significantly affecting the stability of the triplex. In the case of three consecutive heterologies, our modeling recommends using long oligonucleotides (at least 35 bases) in which the heterologous sequences are positioned at an intermediate position. Oligonucleotides should not contain more than 10% consecutive heterologies to guarantee a stable pairing with the target dsDNA. Theoretical modeling cannot replace experiments, but we believe that our model can considerably accelerate optimization of oligonucleotides for gene therapy by predicting their pairing dynamics with the target dsDNA

EFFETS DES SEQUENCES MICROSATELLITES DINUCLEOTIDIQUES (CA/GT)N SUR LA RECOMBINAISON MEIOTIQUE CHEZ SACCHAROMYCES CEREVISIAE

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

A Neurotransmitter Atlas of the Caenorhabditis elegans Male Nervous System Reveals Sexually Dimorphic Neurotransmitter Usage

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.This work was supported by the National Institutes of Health (2R37-NS039996) and the Howard Hughes Medical Institute. M.G. was supported by the European Molecular Biology Organization and Human Frontier Science Program postdoctoral fellowships.Peer reviewe