A semi-automated high-throughput approach to the generation of transposon insertion mutants in the nematode Caenorhabditis elegans

The generation of a large collection of defined transposon insertion mutants is of general interest to the Caenorhabditis elegans research community and has been supported by the European Union. We describe here a semi-automated high-throughput method for mutant production and screening, using the heterologous transposon Mos1. The procedure allows routine culture of several thousand independent nematode strains in parallel for multiple generations before stereotyped molecular analyses. Using this method, we have already generated >17 500 individual strains carrying Mos1 insertions. It could be easily adapted to forward and reverse genetic screens and may influence researchers faced with making a choice of model organism

Dynamic expression of the pro-dopaminergic transcription factors Pax6 and Dlx2 during postnatal olfactory bulb neurogenesis

Olfactory bulb (OB) neurogenesis generates neurons that use GABA or dopamine as their neurotransmitters throughout life. Regionalized stem cell populations in the periventricular zone (PVZ) of the lateral ventricles (LVs) have been shown to be at the basis of neuronal diversity in the system. For example dopaminergic neurons arise predominantly from neural stem cells (NSCs) residing in the dorsal PVZ and depend on the expression of the transcription factors Pax6 and Dlx2 for their specification. In addition, Dlx2 is required for neurogenesis in general. Using targeted in vivo electroporation combined with immuno-fluorescence imaging and microarray analysis, we provide here detailed spatial and temporal expression data with cellular resolution in this system. We find that all along the neurogenic process Pax6 expression remains restricted to the dorsal PVZ, whereas nearly all neuroblasts express Dlx2, including those of the dorsal lineage, which are switched on for Dlx2 when they enter the rostral migratory stream (RMS). These data allow to explain and precise the functions of these two genes in postnatal OB neurogenesis

MiR-200 family controls late steps of postnatal forebrain neurogenesis via Zeb2 inhibition

During neurogenesis, generation, migration and integration of the correct numbers of each neuron sub-Type depends on complex molecular interactions in space and time. MicroRNAs represent a key control level allowing the flexibility and stability needed f

Recherche d'une methode d'obtention d'individus androgenetique in situ chez le chou (Brassica oleracea L.)

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Post-transcriptional gene silencing in plants

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Potential use of the aux2 gene from Agrobacterium rhizogenes as a conditional negative marker in transgenic cabbage

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Potential use of the aux2 gene from Agrobacterium rhizogenes as a conditional negative marker in transgenic cabbage

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Infection of Tobacco or Arabidopsis plants by CMV counteracts systemic post-transcriptional silencing of nonviral (trans)genes

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Neuronal Subtype Generation During Postnatal Olfactory Bulb Neurogenesis

International audienceNew neurons are generated throughout life in two specific regions of the mammalian brain: the hippocampus and the olfactory bulb (OB). In the OB, large numbers of interneurons are permanently issued from stem cells residing in the ven-tricular/subventricular zone (V/SVZ) lining the forebrain ven-tricles. From here, they migrate via the rostral migratory stream (RMS) into the OB where they differentiate as interneurons. These postnatal and adult generated interneurons exhibit considerable phenotypic diversity at several levels. First, they are heterogeneous at the neurotransmitter level. Indeed, it has been shown that neurons using exclusively GABA, neurons that use both GABA and dopamine, and also very few gluta-matergic neurons are generated and integrated. Second, they show varying final locations in the OB. Although most OB interneurons remain in the deep positioned granule cell layer, a substantial fraction integrates in the superficial glomerular layer. Finally, adult-born neurons show a wide spectrum of morphologies, projection patterns, and targets. 1 Lineage studies demonstrated that the diversity of OB interneurons is closely tied to their spatial origin in the stem cell compartment. For example, interneurons generated by progenitors of the dorsal part of the V/SVZ will predominantly integrate in the superficial layers of the OB and express sub-type markers such as calretinin (CR), tyrosine hydroxylase, or the transcription factors (TFs) TBR1/2. In contrast, OB neu-rons produced along the lateral aspect of the ventricle are purely GABAergic and integrate in deeper layers of the OB. The discovery that neuronal heterogeneity is determined by their site of origin led to the notion that the stem cell niche represents a cellular mosaic in which populations of stem cells in defined dorsoventral and anteroposterior positions are predetermined to produce specific neurons for the OB. 2 This, in turn, implies that molecular determinants, for example, differentially expressed TFs, underlie early fate specification and ultimately neuronal function and connectivity. Gene Expression in Space and Time Tiveron et al. 3 set out to systematically identify and functionally characterize such fate determinants based on high-resolution gene expression analyses. Up to now, gene expression analyses performed in the V/SVZ-RMS-OB neurogenic system relied either on microdissection of tissues 4 or on cell sorting based on expression of a limited set of membrane markers 5,6 that define specific differentiation stages. However, in this neurogenic system , such approaches present several limitations. Although the stem cell compartments of the dorsal and lateral lineages are physically separated, these regions harbor progenitors at different differentiation stages (neural stem cells [NSCs], transit amplifying progenitors and migrating neuroblasts). In addition, in the RMS and the OB, both lineages are intermingled and cannot be easily distinguished, let alone isolated. To overcome these limitations, Tiveron et al. performed a lineage tracing approach based on targeted brain electropora-tion. Previous work demonstrated that in vivo brain electropo-ration can be used to transfect DNA-based vectors, 7 or even messenger RNA, 8 into the different stem cell compartments ABSTRACT: In the perinatal and adult forebrain, regionalized neural stem cells lining the ventricular walls produce different types of olfactory bulb interneurons. Although these postnatal stem cells are lineage related to their embryonic counterparts that produce, for example, cortical, septal, and striatal neurons, their output at the level of neuronal phenotype changes dramatically. Tiveron et al. investigated the molecular determinants underlying stem cell regionalization and the gene expression changes inducing the shift from embryonic to adult neuron production. High-resolution gene expression analyses of different lineages revealed that the zinc finger proteins, Zic1 and Zic2, are postnatally induced in the dorsal olfactory bulb neuron lineage. Functional studies demonstrated that these factors confer a GABAergic and calretinin-positive phenotype to neural stem cells while repressing dopaminergic fate. Based on these findings, we discuss the molecular mechanisms that allow acquisition of new traits during the transition from embryonic to adult neurogenesis. We focus on the involvement of epigenetic marks and emphasize why the identification of master transcription factors, that instruct the fate of postnatally generated neurons, can help in deciphering the mechanisms driving fate transition from embryonic to adult neuron production

DNA methylation and chromatin structure affect transcriptional and post-transcriptional transgene silencing in Arabidopsis

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