Regulatory Logic of Pan-Neuronal Gene Expression in C. elegans

Stefanakis, Nikolaos; Carrera, Inés; Hobert, Oliver

doi:10.1016/j.neuron.2015.07.031

Cited by 157 publications

(224 citation statements)

References 46 publications

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“…We observed that both reporters were expressed in I4 and other MS-derived neurons (Figure 1F and unpublished data); observations by Stefanakis et al [11] similarly suggest that MS- and AB-derived neurons share basic neuronal molecular attributes. To determine whether I4 precursor cells express mesodermal characteristics, we examined the expression of an hlh-1 reporter during embryogenesis.…”

Section: Resultssupporting

confidence: 80%

The CDK8 Complex and Proneural Proteins Together Drive Neurogenesis from a Mesodermal Lineage

Luo

Horvitz

2017

Current Biology

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SUMMARY At least some animal species can generate neurons from mesoderm or endoderm, but the underlying mechanisms remain unknown. We screened for C. elegans mutants in which the presumptive mesoderm-derived I4 neuron adopts a muscle-like cell fate. From this screen, we identified HLH-3, the C. elegans homolog of a mammalian proneural protein (Ascl1) used for in vitro neuronal reprogramming, as required for efficient I4 neurogenesis. We discovered that the CDK-8 Mediator kinase module acts together with a second proneural protein, HLH-2, and in parallel to HLH-3 to promote I4 neurogenesis. Genetic analysis revealed that CDK-8 most likely promotes I4 neurogenesis by inhibiting the CDK-7/CYH-1 (CDK7/cyclin H) kinase module of the transcription initiation factor TFIIH. Ectopic expression of HLH-2 and HLH-3 together promoted expression of neuronal features in non-neuronal cells. These findings reveal that the Mediator CDK8 kinase module can promote non-ectodermal neurogenesis and suggest that inhibiting CDK7/cyclin H might similarly promote neurogenesis.

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Section: Resultssupporting

confidence: 80%

The CDK8 Complex and Proneural Proteins Together Drive Neurogenesis from a Mesodermal Lineage

Luo

Horvitz

2017

Current Biology

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“…In the absence of FLP expression, neither strain produced detectable levels of GFP in the nervous system ( Figure 4B and Figure S5). Upon pan-neuronal expression of FLP, both strains exhibited strong GFP expression throughout the nervous system, as expected since both of these genes are expressed in all neurons (Stefanakis et al 2015). We conclude that the FLP-on connectors permit recombination-dependent tag expression for both N-and C-terminal tags.…”

supporting

confidence: 73%

SapTrap, a Toolkit for High-Throughput CRISPR/Cas9 Gene Modification in Caenorhabditis elegans

2016

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In principle, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 allows genetic tags to be inserted at any locus. However, throughput is limited by the laborious construction of repair templates and guide RNA constructs and by the identification of modified strains. We have developed a reagent toolkit and plasmid assembly pipeline, called "SapTrap," that streamlines the production of targeting vectors for tag insertion, as well as the selection of modified Caenorhabditis elegans strains. SapTrap is a high-efficiency modular plasmid assembly pipeline that produces single plasmid targeting vectors, each of which encodes both a guide RNA transcript and a repair template for a particular tagging event. The plasmid is generated in a single tube by cutting modular components with the restriction enzyme SapI, which are then "trapped" in a fixed order by ligation to generate the targeting vector. A library of donor plasmids supplies a variety of protein tags, a selectable marker, and regulatory sequences that allow cellspecific tagging at either the N or the C termini. All site-specific sequences, such as guide RNA targeting sequences and homology arms, are supplied as annealed synthetic oligonucleotides, eliminating the need for PCR or molecular cloning during plasmid assembly. Each tag includes an embedded Cbr-unc-119 selectable marker that is positioned to allow concurrent expression of both the tag and the marker. We demonstrate that SapTrap targeting vectors direct insertion of 3-to 4-kb tags at six different loci in 10-37% of injected animals. Thus SapTrap vectors introduce the possibility for high-throughput generation of CRISPR/Cas9 genome modifications.KEYWORDS CRISPR/Cas9; high-throughput; gene tagging; plasmid toolkit T HE clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has revolutionized genome editing in nearly all model systems, including Caenorhabditis elegans (Frøkjaer-Jensen 2013;Doudna and Charpentier 2014;Xu 2015). The Cas9 protein cuts genomic DNA at sites that match an 20-nucleotide guide RNA sequence (Gasiunas et al. 2012;Jinek et al. 2012). Error-prone repair of the break can create point mutations, small indels, or large deletions at the cut site (Cho et al. 2013;Cong et al. 2013;Friedland et al. 2013;Jinek et al. 2013;van Schendel et al. 2015). However, the true power of CRISPR/Cas9 for genome editing lies in the insertion of exogenous DNA sequences, such as genetically encoded protein tags (Katic and Grosshans 2013;Lo et al. 2013;Mali et al. 2013;Tzur et al. 2013). To insert an exogenous sequence, one must simply supply a repair template with homology arms that flank the cut site. The cell uses homologybased repair to heal the break and copy the exogenous DNA into the cut site.Although in theory CRISPR/Cas9 makes it easy to insert exogenous sequences into a genome, practical limitations have prevented high-throughput implementation. Two critical limiting factors are (1) the time and expense required to build both the repair template a...

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“…Because shn-1 is expressed in muscles (Stefanakis et al, 2015), we assayed EGL-19/Ca v 1 channel function by recording voltage-activated calcium currents in body muscle, using salines containing potassium channel blockers (TEA and 4AP). In these conditions, the remaining voltage-activated inward current is entirely blocked by the EGL-19 antagonist nemadipine (Lainé et al, 2011).…”

Section: Resultsmentioning

confidence: 99%

Shank is a dose-dependent regulator of Cav1 calcium current and CREB target expression

Pym

Sasidharan

Thompson-Peer

et al. 2017

eLife

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Shank is a post-synaptic scaffolding protein that has many binding partners. Shank mutations and copy number variations (CNVs) are linked to several psychiatric disorders, and to synaptic and behavioral defects in mice. It is not known which Shank binding partners are responsible for these defects. Here we show that the C. elegans SHN-1/Shank binds L-type calcium channels and that increased and decreased shn-1 gene dosage alter L-channel current and activity-induced expression of a CRH-1/CREB transcriptional target (gem-4 Copine), which parallels the effects of human Shank copy number variations (CNVs) on Autism spectrum disorders and schizophrenia. These results suggest that an important function of Shank proteins is to regulate L-channel current and activity induced gene expression.DOI: http://dx.doi.org/10.7554/eLife.18931.001

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Regulatory Logic of Pan-Neuronal Gene Expression in C. elegans

Cited by 157 publications

References 46 publications

The CDK8 Complex and Proneural Proteins Together Drive Neurogenesis from a Mesodermal Lineage

The CDK8 Complex and Proneural Proteins Together Drive Neurogenesis from a Mesodermal Lineage

SapTrap, a Toolkit for High-Throughput CRISPR/Cas9 Gene Modification in Caenorhabditis elegans

Shank is a dose-dependent regulator of Cav1 calcium current and CREB target expression

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