Oliver hobert

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The auxin-inducible degron (AID) system is a broadly used tool for spatiotemporal and reversible control of protein depletion in multiple experimental model systems. AID2 technology relies on a synthetic ligand, 5-phenyl-indole-3-acetic acid (5-Ph-IAA), for improved specificity and efficiency of protein degradation. Here, we provide a protocol for cost-effective 5-Ph-IAA synthesis utilizing the Suzuki coupling of 5-chloroindole and phenylboronic acid. We describe steps for evaluating the quality of lab-synthesized 5-Ph-IAA using a C...

To facilitate cell identification for expression pattern analysis in C. elegans , an SL2::GFP::H2B fluorescent reporter cassette has become a popular and widely used choice to generate nuclear localized reporter alleles by CRISPR/Cas9 genome engineering. When added at the 3' end of a locus of interest, this cassette concentrates GFP into the nucleus and permits the identification of expressing cells, for example with the help of the NeuroPAL tool. However, there are instances in which it is desirable to visualize the complete morphology of a cell that expresses an SL2::GFP::H2B reporter cassette...

Visualizing synaptic connectivity has traditionally relied on time-consuming electron microscopy-based imaging approaches. To scale the analysis of synaptic connectivity, fluorescent protein-based techniques have been established, ranging from the labeling of specific pre- or post-synaptic components of chemical or electrical synapses to transsynaptic proximity labeling technology such as GRASP and iBLINC. In this paper, we describe WormPsyQi, a generalizable image analysis pipeline that automatically quantifies synaptically localized fluorescent signals in a high-throughput and robust manner, with reduced human bias...

Recent single-cell transcriptome analysis has revealed a tremendous breadth and specificity of neuropeptide-encoding gene expression in the nervous system of C. elegans. To analyze the dynamics of neuropeptide gene expression, as well as to dissect the regulatory mechanism by which their expression is controlled, reporter genes remain an important tool. Using CRISPR/Cas9 genome-engineering, we generate here reporter alleles for 6 different neuropeptide encoding genes (3 flp genes, 1 nlp and 2 insulin genes)...

Efforts are ongoing to map synaptic wiring diagrams, or connectomes, to understand the neural basis of brain function. However, chemical synapses represent only one type of functionally important neuronal connection; in particular, extrasynaptic, "wireless" signaling by neuropeptides is widespread and plays essential roles in all nervous systems. By integrating single-cell anatomical and gene-expression datasets with biochemical analysis of receptor-ligand interactions, we have generated a draft connectome of neuropeptide signaling in the C...

Sec1/Munc18 (SM) proteins are important regulators of SNARE complex assembly during exocytosis throughout all major animal tissue types. However, expression of a founding member of the SM family, UNC-18, is mostly restricted to the nervous system of the nematode C. elegans, where it is important for synaptic transmission. Moreover, unc-18 null mutants do not display the lethality phenotype associated with (a) loss of all Drosophila and mouse orthologues of unc-18 and (b) with elimination of synaptic transmission in C...

Axons must project to particular brain regions, contact adjacent neurons, and choose appropriate synaptic targets to form a nervous system. Multiple mechanisms have been proposed to explain synaptic partnership choice. In a "lock-and-key" mechanism, first proposed by Sperry's chemoaffinity model,1 a neuron selectively chooses a synaptic partner among several different, adjacent target cells, based on a specific molecular recognition code.2 Alternatively, Peters' rule posits that neurons indiscriminately form connections with other neuron types in their proximity; hence, neighborhood choice, determined by initial neuronal process outgrowth and position, is the main predictor of connectivity...

Exposure to adverse nutritional and metabolic environments during critical periods of development can exert long-lasting effects on health outcomes of an individual and its descendants. Although such metabolic programming has been observed in multiple species and in response to distinct nutritional stressors, conclusive insights into signaling pathways and mechanisms responsible for initiating, mediating, and manifesting changes to metabolism and behavior across generations remain scarce. By using a starvation paradigm in Caenorhabditis elegans , we show that starvation-induced changes in dauer formation-16/forkhead box transcription factor class O (DAF-16/FoxO) activity, the main downstream target of insulin/insulin-like growth factor 1 (IGF-1) receptor signaling, are responsible for metabolic programming phenotypes...

Heterochrony is a foundational concept in animal development and evolution, first introduced by Ernst Haeckel in 1875 and later popularized by Stephen J. Gould1 . A molecular understanding of heterochrony was first established by genetic mutant analysis in the nematode C. elegans, revealing a genetic pathway that controls the proper timing of cellular patterning events executed during distinct postembryonic juvenile and adult stages2 . This genetic pathway is composed of a complex temporal cascade of multiple regulatory factors, including the first-ever discovered miRNA, lin-4, and its target gene, lin-14, which encodes a nuclear, DNA-binding protein2 , 3 , 4 ...

Are there common mechanisms of neurogenesis used throughout an entire nervous system? We explored to what extent canonical proneural class I/II bHLH complexes are responsible for neurogenesis throughout the entire Caenorhabditis elegans nervous system. Distinct, lineage-specific proneural class II bHLH factors are generally thought to operate via interaction with a common, class I bHLH subunit, encoded by Daughterless in flies, the E proteins in vertebrates and HLH-2 in C. elegans. To eliminate function of all proneuronal class I/II bHLH complexes, we therefore genetically removed maternal and zygotic hlh-2 gene activity...

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The neurotransmitter serotonin plays a central role in animal behavior and physiology, and many of its functions are regulated via evolutionarily conserved biosynthesis and degradation pathways. Here we show that in Caenorhabditis elegans, serotonin is abundantly produced in nonneuronal tissues via phenylalanine hydroxylase, in addition to canonical biosynthesis via tryptophan hydroxylase in neurons. Combining CRISPR-Cas9 genome editing, comparative metabolomics and synthesis, we demonstrate that most serotonin in C...

Homeobox genes are prominent regulators of neuronal identity, but the extent to which their function has been probed in animal nervous systems remains limited. In the nematode Caenorhabditis elegans, each individual neuron class is defined by the expression of unique combinations of homeobox genes, prompting the question of whether each neuron class indeed requires a homeobox gene for its proper identity specification. We present here progress in addressing this question by extending previous mutant analysis of homeobox gene family members and describing multiple examples of homeobox gene function in different parts of the C...

The classification of neurons into distinct types reveals hierarchical taxonomic relationships that reflect the extent of similarity between neuronal cell types. At the base of such taxonomies are neuronal cells that are very similar to one another but differ in a small number of reproducible and select features. How are very similar members of a neuron class that share many features instructed to diversify into distinct subclasses? We show here that the six very similar members of the Caenorhabditis elegans IL2 sensory neuron class, which are all specified by a homeobox terminal selector, unc-86/BRN3 , differentiate into two subtly distinct subclasses, a dorsoventral subclass and a lateral subclass, by the toggle switch-like action of the sine oculis/SIX homeobox gene unc-39 ...

After the generation, differentiation and integration into functional circuitry, post-mitotic neurons continue to change certain phenotypic properties throughout postnatal juvenile stages until an animal has reached a fully mature state in adulthood. We will discuss such changes in the context of the nervous system of the nematode C. elegans, focusing on recent descriptions of anatomical and molecular changes that accompany postembryonic maturation of neurons. We summarize the characterization of genetic timer mechanisms that control these temporal transitions or maturational changes, and discuss that many but not all of these transitions relate to sexual maturation of the animal...

The maturation of genome editing techniques dramatically broadens the range of organisms amenable to mechanistic investigation. Caenorhabditis briggsae is a nematode species related to C. elegans and a favored target for comparative studies. Here, we expand the repertoire of co-conversion markers to facilitate the screening and isolation of CRISPR/Cas9-edited lines in C. briggsae . Similar to its homologous C. elegans mutation, Cbr-dpy-10(Arg92Cys) is phenotypically easy to detect in its heterozygous form and is distinguishable from other combinations of Cbr-dpy- 10 alleles, a valuable feature for the reliable isolation of marker-free CRISPR/Cas9-edited animals...

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 Caenorhabditis elegans that resides within the foregut (pharynx) of the worm. A C. 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...

No abstract text is available yet for this article.

No abstract text is available yet for this article.