Boulin T et al. (2007) Nat Protoc "Mos1-mediated insertional mutagenesis in Caenorhabditis elegans."

Bessereau JL, Boulin T

[Nat Protoc, 2007]

We describe a protocol for mutating genes in the nematode Caenorhabditis elegans using the Mos1 transposon of Drosophila mauritiana. Mutated genes containing a Mos1 insertion are molecularly tagged by this heterologous transposable element. Mos1 insertions can therefore be identified in as little as 3 weeks using only basic molecular biology techniques. Mutagenic efficiency of Mos1 is tenfold lower than classical chemical mutagens. However, the ease and speed with which mutagenic insertions can be mapped compares favorably with the vast amount of work involved in classical genetic mapping. Therefore, Mos1 could be the tool of choice when screening procedures are efficient. In addition, Mos1 mutagenesis can greatly simplify the mapping of mutations that exhibit low penetrance, subtle or synthetic phenotypes. The recent development of targeted engineering of C. elegans loci carrying Mos1 insertions further increases the attractiveness of Mos1-mediated mutagenesis.

Boulin T et al. (2012) Wiley Interdiscip Rev Dev Biol "From genes to function: the C. elegans genetic toolbox."

Boulin T, Hobert O

[Wiley Interdiscip Rev Dev Biol, 2012]

This review aims to provide an overview of the technologies which make the nematode Caenorhabditis elegans an attractive genetic model system. We describe transgenesis techniques and forward and reverse genetic approaches to isolate mutants and clone genes. In addition, we discuss the new possibilities offered by genome engineering strategies and next-generation genome analysis tools.

Boulin T et al. (2012) Nat Neurosci "Positive modulation of a Cys-loop acetylcholine receptor by an auxiliary transmembrane subunit."

Richmond JE, Stigloher C, Boulin T, Rapti G, Paoletti P, Bessereau JL, Briseno-Roa L

[Nat Neurosci, 2012]

Auxiliary subunits regulate the trafficking, localization or gating kinetics of voltage- and ligand-gated ion channels by associating tightly and specifically with pore-forming subunits. However, no auxiliary subunits have been identified for members of the Cys-loop receptor superfamily. Here we identify MOLO-1, a positive regulator of levamisole-sensitive acetylcholine receptors (L-AChRs) at the Caenorhabditis elegans neuromuscular junction. MOLO-1 is a one-pass transmembrane protein that contains a single extracellular globular domain-the TPM domain, found in bacteria, plants and invertebrates, including nonvertebrate chordates. Loss of MOLO-1 impairs locomotion and renders worms resistant to the anthelmintic drug levamisole. In molo-1 mutants, L-AChR-dependent synaptic transmission is reduced by half, while the number and localization of receptors at synapses remain unchanged. In a heterologous expression system, MOLO-1 physically interacts with L-AChRs and directly enhances channel gating without affecting unitary conductance. The identification of MOLO-1 expands the mechanisms for generating functional and pharmacological diversity in the Cys-loop superfamily.

Boulin T et al. (2006) Curr Biol "A novel Eph receptor-interacting IgSF protein provides C. elegans motoneurons with midline guidepost function."

Boulin T, Hobert O, Pocock R

[Curr Biol, 2006]

BACKGROUND: The ventral midline is a prominent structure in vertebrate and invertebrate nervous systems that provides crucial topological information for guiding axons to their appropriate target destinations. Rather than being composed of specialized midline glia cells as in many other species, the embryonic midline of the nematode Caenorhabditis elegans is physically defined by motoneuron cell bodies that separate the left from the right ventral cord fascicles. Their function during development, if any, is not known. RESULTS: We show here that besides being components of the postembryonic locomotory circuit, these embryonic motoneurons (eMNs) actively provide midline guidance information for a specific subset of ventral midline axons. This information is provided in the form of a novel, cell-surface-anchored immunoglobulin superfamily (IgSF) member, WRK-1. WRK-1 acts in eMNs to prevent follower axons from inappropriately crossing the ventral midline. We describe the function of the Eph receptor vab-1 and multiple ephrin ligands at the midline, and we show by double mutant analysis and physical interaction tests that WRK-1 functionally interacts with the Eph receptor system. This interaction appears to occur exclusively in the context of axon guidance at the ventral midline but not in other cellular contexts, thereby suggesting that Eph receptor signaling is mechanistically distinct in different tissue types. CONCLUSIONS: Our studies reveal cellular and molecular components of axon midline patterning and suggest that Ephrin signaling relies on previously unknown accessory components.

Boulin T et al. (2021) Mol Genet Metab "Functional analysis of a de novo variant in the neurodevelopment and generalized epilepsy disease gene NBEA."

Shimada S, Boulin T, Malicdan MC, Itani O, Murphy JL, Macnamara E, Silverman GA, Turner D, Soldatos A, Leclercq-Blondel A, Gorman MP, Gendrel M, Pak SC, Schedl T, Lindsey A, Baldridge D, El Mouridi S

[Mol Genet Metab, 2021]

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.

Boulin T et al. (2008) Proc Natl Acad Sci U S A "Eight genes are required for functional reconstitution of the Caenorhabditis elegans levamisole-sensitive acetylcholine receptor."

Gielen M, Paoletti P, Bessereau JL, Boulin T, Richmond JE, Williams DC

[Proc Natl Acad Sci U S A, 2008]

Levamisole-sensitive acetylcholine receptors (L-AChRs) are ligand-gated ion channels that mediate excitatory neurotransmission at the neuromuscular junctions of nematodes. They constitute a major drug target for anthelminthic treatments because they can be activated by nematode-specific cholinergic agonists such as levamisole. Genetic screens conducted in Caenorhabditis elegans for resistance to levamisole toxicity identified genes that are indispensable for the biosynthesis of L-AChRs. These include 5 genes encoding distinct AChR subunits and 3 genes coding for ancillary proteins involved in assembly and trafficking of the receptors. Despite extensive analysis of L-AChRs in vivo, pharmacological and biophysical characterization of these receptors has been greatly hampered by the absence of a heterologous expression system. Using Xenopus laevis oocytes, we were able to reconstitute functional L-AChRs by coexpressing the 5 distinct receptor subunits and the 3 ancillary proteins. Strikingly, this system recapitulates the genetic requirements for receptor expression in vivo because omission of any of these 8 genes dramatically impairs L-AChR expression. We demonstrate that 3 alpha- and 2 non-alpha-subunits assemble into the same receptor. Pharmacological analysis reveals that the prototypical cholinergic agonist nicotine is unable to activate L-AChRs but rather acts as a potent allosteric inhibitor. These results emphasize the role of ancillary proteins for efficient expression of recombinant neurotransmitter receptors and open the way for in vitro screening of novel anthelminthic agents.

Boulin T et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "MOLO-1: a novel acetylcholine receptor-associated protein that modulates cholinergic transmission at neuromuscular junctions in C. elegans"

Bessereau J, Richmond J E, Boulin T, Stigloher C, Martin A

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

Nicotinic acetylcholine receptors (AChRs) are ligand-gated ion channels that have been widely conserved throughout evolution and are implicated in numerous physiological and pathological processes in humans. In order to identify new regulators of nicotinic neurotransmission, we have performed a genetic screen for mutants which survive chronic exposure to lethal doses of the nematode-specific nicotinic agonist levamisole. We report here the identification and characterization of the novel protein MOLO-1 (modulator of levamisole receptor-1), which is required for efficient nicotinic neurotransmission at the neuromuscular synapse. MOLO-1 is a small type 1 transmembrane protein. MOLO-1 is expressed in muscle cells where it localizes specifically to synapses which contain the levamisole-sensitive AChR (L-AChR). Electrophysiological analysis of molo-1 mutant animals shows that the synaptic currents associated with L-AChRs are reduced by over 60%. In order to measure the surface expression of L-AChR in vivo, we have developed a novel C. elegans strain in which an affinity tag is recombined into the unc-29 L-AChR subunit locus. Surprisingly, the reduction in synaptic current is not associated with a reduction in the number of receptors at synapses in molo-1 mutants. In contrast, in animals which do not express the receptor, MOLO-1 is no longer detectable at synapses, suggesting an association between MOLO-1 and the L-AChR. We have recently been able to functionally reconstitute the levamisole-sensitive AChR in Xenopus oocytes (Boulin, 2008). Taking advantage of this system, we have now shown that co-expression of MOLO-1 with L-AChR leads to a two fold increase of the measured current compared to L-AChR alone. This result is fully consistent with our in vivo findings and strongly suggests that MOLO-1 has a direct effect on the receptor. MOLO-1 could function by modifying the biophysical properties of the receptor (gating, open probability, conductance). In order to test this hypothesis, we are conducting single channel recordings in primary cultured C. elegans muscle cells. In addition we are using single molecule fluorescence imaging (Ulbrich, 2007) in Xenopus oocytes to establish the relative stoechiometry of MOLO-1 and L-AChR. Taken together, our results suggest that the transmembrane protein MOLO-1 could directly modulate the electrophysiological properties of the AChR making it the first positive modulator of a nicotinic receptor.

Samuel ADT et al. (2003) J Neurosci "Synaptic activity of the AFD neuron in Caenorhabditis elegans correlates with thermotactic memory."

Murthy VN, Samuel ADT, Silva RA

[J Neurosci, 2003]

Thermotactic behavior in Caenorhabditis elegans is sensitive to both a worm's ambient temperature (T-amb) and its memory of the temperature of its cultivation (T-cult). The AFD neuron is part of a neural circuit that underlies thermotactic behavior. By monitoring the fluorescence of pH-sensitive green fluorescent protein localized to synaptic vesicles, we measured the rate of the synaptic release of AFD in worms cultivated at temperatures between 15 and 25degreesC, and subjected to fixed, ambient temperatures in the same range. We found that the rate of AFD synaptic release is high if either T-amb > T-cult or T-amb > T-cult, but AFD synaptic release is low if T-amb congruent to T-cult. This suggests that AFD encodes a direct comparison between T-amb and T-cult.

Aurelio O et al. (2003) Development "Identification of spatial and temporal cues that regulate postembryonic expression of axon maintenance factors in the C. elegans ventral nerve cord."

Hobert O, Boulin T, Aurelio O

[Development, 2003]

Patterns of gene expression are under precise spatial and temporal control. A particularly striking example is represented by several members of the zig gene family, which code for secreted immunoglobulin domain proteins required for maintaining ventral nerve cord organization in Caenorhabditis elegans. These genes are coordinately expressed in a single interneuron in the ventral nerve cord, known as PVT. Their expression is initiated at a precise postembryonic stage, long after PVT has been generated in mid-embryogenesis. We define spatial and temporal cues that are required for the precise regulation of zig gene expression. We find that two LIM homeobox genes, the Lhx3-class gene ceh-14 and the Lmx-class gene lim-6 are coordinately required for zig gene expression in PVT. Temporal control of zig gene expression is conferred by the heterochronic gene lin-14, a nuclear factor previously implicated in developmental timing in various contexts. Loss of the lim-6 and ceh-14 transcription factors and the developmental timer lin-14 cause not only a loss of zig gene expression but also lead to defects in the maintenance of ventral nerve cord architecture. Overriding the normal spatiotemporal control of zig gene expression through expression of one of the zig genes under control of heterologous promoters also causes axon patterning defects in the ventral nerve cord. Our findings illustrate the importance of spatial and temporal control of gene expression in the nervous system and, furthermore, implicate heterochronic genes in postmitotic neural

Bulow HE et al. (2004) Neuron "Differential functions of the C. elegans FGF receptor in axon outgrowth and maintenance of axon position."

Bulow HE, Hobert O, Boulin T

[Neuron, 2004]

Wiring of the nervous system requires that axons navigate to their targets and maintain their correct positions in axon fascicles after termination of axon outgrowth. We show here that the C. elegans fibroblast growth factor receptor (FGFR), EGL-15, affects both processes in fundamentally distinct manners. FGF-dependent activation of the EGL-15 tyrosine kinase and subsequently the GTPase LET-60/ras is required within epidermal cells, the substratum for most outgrowing axon, for appropriate outgrowth of specific axon classes to their target area. In contrast, genetic elimination of the FGFR isoform EGL-15(5A), defined by the inclusion of an alternative extracellular interimmunoglobulin domain, has no consequence for axon outgrowth but leads to a failure to postembryonically maintain axon position within defined axon fascicles. An engineered, secreted form of EGL-15(5A) containing only its ectodomain is sufficient for maintenance of axon position, thus providing novel insights into receptor tyrosine kinase function and the process of maintaining axon position.