Next-Generation Sequencing-Based Approaches for Mutation Mapping and Identification in Caenorhabditis elegans

The use of next-generation sequencing (NGS) has revolutionized the way phenotypic traits are assigned to genes. In this review, we describe NGS-based methods for mapping a mutation and identifying its molecular identity, with an emphasis on applications in Caenorhabditis elegans. In addition to an overview of the general principles and concepts, we discuss the main methods, provide practical and conceptual pointers, and guide the reader in the types of bioinformatics analyses that are required. Owing to the speed and the plummeting costs of NGS-based methods, mapping and cloning a mutation of interest has become straightforward, quick, and relatively easy. Removing this bottleneck previously associated with forward genetic screens has significantly advanced the use of genetics to probe fundamental biological processes in an unbiased manner

Modelling the signal delivered by a population of first-order neurons in a moth olfactory system

A statistical model of the population of first-order olfactory receptor neurons (ORNs) is proposed and analysed. It describes the relationship between stimulus intensity (odour concentration) and coding variables such as rate and latency of the population of several thousand sex-pheromone sensitive ORNs in male moths. Although these neurons likely express the same olfactory receptor, they exhibit, at any concentration, a relatively large heterogeneity of responses in both peak firing frequency and latency of the first action potential fired after stimulus onset. The stochastic model is defined by a multivariate distribution of six model parameters that describe the dependence of the peak firing rate and the latency on the stimulus dose. These six parameters and their mutual linear correlations were estimated from experiments in single ORNs and included in the multidimensional model distribution. The model is utilized to reconstruct the peak firing rate and latency of the message sent to the brain by the whole ORN population at different stimulus intensities and to establish their main qualitative and quantitative properties. Finally, these properties are shown to be in agreement with those found previously in a vertebrate ORN population

Mating-induced transient inhibition of responses to sex pheromone in a male moth is not mediated by octopamine or serotonin

In the male moth, Agrotis ipsilon, mating induces a transient inhibition of behavioural and central nervous responses to sex pheromone. Newly mated males are not attracted to sex pheromone, and the sensitivity of their antennal lobe (AL) neurons is lower than in virgin males. This rapid transient olfactory inhibition prevents them from re-mating unsuccessfully until they have refilled their sex glands. We hypothesized that this olfactory ‘switch off’ might be controlled by neuromodulators such as biogenic amines. To test our hypothesis, we studied the effects of octopamine (OA) and serotonin (5-hydroxytryptamine, 5-HT) on the coding properties of pheromone-sensitive AL neurons in virgin and newly mated males. We show that AL neuron sensitivity increased in newly mated males after injection of OA or 5-HT, but only OA treatment affected certain response characteristics of AL neurons in virgin males. Whereas all measured AL neuron response characteristics were different between virgin and newly mated males, amine treatment in newly mated males restored only the latency and spike frequency, but not the duration of excitatory and inhibitory phases, which were initially found in virgin males. Additionally, we investigated the behavioural effects of OA and 5-HT treatments in virgin and mated males. Although OA and 5-HT enhanced the general flight activity of newly mated males, amine treatments did not restore the behavioural pheromone response of mated moths. Altogether, these results show that, although biogenic amines modulate the olfactory system in moths, OA and 5-HT are probably not involved in the post-mating inhibition of responses to sex pheromone in A. ipsilon males.Fil: Barrozo, Romina. Institut National de la Recherche Agronomique; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Jarriault, David. Institut National de la Recherche Agronomique; FranciaFil: Simeone, Xenia. Institut National de la Recherche Agronomique; FranciaFil: Gaertner, Cyril. Institut National de la Recherche Agronomique; FranciaFil: Gadenne, Christophe. Institut National de la Recherche Agronomique; FranciaFil: Anton, Sylvia. Institut National de la Recherche Agronomique; Franci

A robust system for RNA interference in the chicken using a modified microRNA operon

AbstractRNA interference (RNAi) provides an effective method to silence gene expression and investigate gene function. However, RNAi tools for the chicken embryo have largely been adapted from vectors designed for mammalian cells. Here we present plasmid and retroviral RNAi vectors specifically designed for optimal gene silencing in chicken cells. The vectors use a chicken U6 promoter to express RNAs modelled on microRNA30, which are embedded within chicken microRNA operon sequences to ensure optimal Drosha and Dicer processing of transcripts. The chicken U6 promoter works significantly better than promoters of mammalian origin and in combination with a microRNA operon expression cassette (MOEC), achieves up to 90% silencing of target genes. By using a MOEC, we show that it is also possible to simultaneously silence two genes with a single vector. The vectors express either RFP or GFP markers, allowing simple in vivo tracking of vector delivery. Using these plasmids, we demonstrate effective silencing of Pax3, Pax6, Nkx2.1, Nkx2.2, Notch1 and Shh in discrete regions of the chicken embryonic nervous system. The efficiency and ease of use of this RNAi system paves the way for large-scale genetic screens in the chicken embryo

Molecular basis for Jagged-1/Serrate ligand recognition by the Notch receptor.

We have mapped a Jagged/Serrate-binding site to specific residues within the 12th EGF domain of human and Drosophila Notch. Two critical residues, involved in a hydrophobic interaction, provide a ligand-binding platform and are adjacent to a Fringe-sensitive residue that modulates Notch activity. Our data suggest that small variations within the binding site fine-tune ligand specificity, which may explain the observed sequence heterogeneity in mammalian Notch paralogues, and should allow the development of paralogue-specific ligand-blocking antibodies. As a proof of principle, we have generated a Notch-1-specific monoclonal antibody that blocks binding, thus paving the way for antibody tools for research and therapeutic applications

Differential Interactions of Sex Pheromone and Plant Odour in the Olfactory Pathway of a Male Moth

Most animals rely on olfaction to find sexual partners, food or a habitat. The olfactory system faces the challenge of extracting meaningful information from a noisy odorous environment. In most moth species, males respond to sex pheromone emitted by females in an environment with abundant plant volatiles. Plant odours could either facilitate the localization of females (females calling on host plants), mask the female pheromone or they could be neutral without any effect on the pheromone. Here we studied how mixtures of a behaviourally-attractive floral odour, heptanal, and the sex pheromone are encoded at different levels of the olfactory pathway in males of the noctuid moth Agrotis ipsilon. In addition, we asked how interactions between the two odorants change as a function of the males' mating status. We investigated mixture detection in both the pheromone-specific and in the general odorant pathway. We used a) recordings from individual sensilla to study responses of olfactory receptor neurons, b) in vivo calcium imaging with a bath-applied dye to characterize the global input response in the primary olfactory centre, the antennal lobe and c) intracellular recordings of antennal lobe output neurons, projection neurons, in virgin and newly-mated males. Our results show that heptanal reduces pheromone sensitivity at the peripheral and central olfactory level independently of the mating status. Contrarily, heptanal-responding olfactory receptor neurons are not influenced by pheromone in a mixture, although some post-mating modulation occurs at the input of the sexually isomorphic ordinary glomeruli, where general odours are processed within the antennal lobe. The results are discussed in the context of mate localization

YAP and TAZ regulate adherens junction dynamics and endothelial cell distribution during vascular development

© Copyright Neto et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.Formation of blood vessel networks by sprouting angiogenesis is critical for tissue growth, homeostasis and regeneration. How endothelial cells arise in adequate numbers and arrange suitably to shape functional vascular networks is poorly understood. Here we show that YAP/TAZ promote stretch-induced proliferation and rearrangements of endothelial cells whilst preventing bleeding in developing vessels. Mechanistically, YAP/TAZ increase the turnover of VE-Cadherin and the formation of junction associated intermediate lamellipodia, promoting both cell migration and barrier function maintenance. This is achieved in part by lowering BMP signalling. Consequently, the loss of YAP/TAZ in the mouse leads to stunted sprouting with local aggregation as well as scarcity of endothelial cells, branching irregularities and junction defects. Forced nuclear activity of TAZ instead drives hypersprouting and vascular hyperplasia. We propose a new model in which YAP/TAZ integrate mechanical signals with BMP signaling to maintain junctional compliance and integrity whilst balancing endothelial cell rearrangements in angiogenic vessels.FN was financially supported by the Fundação para a Ciência e a Tecnologia (FCT), CRUK-CRICK and the MDC. ACV, AKB and EBK were supported by the DZHK (German Centre for Cardiovascular Research), AS was supported by the EMBO (European Molecular Biology Organization), JRC was supported by the FCT. CAF is supported by the FCT, EC-ERC Starting Grant, Portugal2020 program. MP is supported by the Max Planck Society, the ERC Starting Grant ANGIOMET, the Deutsche Forschungsgemeinschaft, the Excellence Cluster Cardiopulmonary System, the LOEWE grant Ub-Net, the DZHK, the Stiftung Charité and the EMBO Young Investigator Program. HG is supported by the DZHK and ERC Consolidator Grant Reshape 311719.info:eu-repo/semantics/publishedVersio