Post-transcriptional regulation of the E/Daughterless ortholog HLH-2, negative feedback, and birth order bias during the AC/VU decision in <i>C. elegans</i>

Karp, Xantha; Greenwald, Iva

doi:10.1101/gad.1160803

Cited by 85 publications

(124 citation statements)

References 61 publications

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“…Thus, the same trans-acting factors (including HLH-2 that binds to the E boxes in the ACEL enhancer and is required for LIN-3 expression in the AC) (Hwang and Sternberg, 2004) appear to be responsible for LIN-3 expression in the pre-AC/pre-VU cells before AC/VU cell-fate specification. Since hlh-2 but not lin-3 is involved in the AC/VU specification (Karp and Greenwald, 2003), this finding also suggests that other genes expressed from the ACEL-like elements are likely to play important roles during the AC/VU specification process.…”

Section: Research Articlementioning

confidence: 64%

C. elegansEVI1 proto-oncogene, EGL-43, is necessary for Notch-mediated cell fate specification and regulates cell invasion

2007

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During C. elegans development, LIN-12 (Notch) signaling specifies the anchor cell (AC) and ventral uterine precursor cell (VU) fates from two equivalent pre-AC/pre-VU cells in the hermaphrodite gonad. Once specified, the AC induces patterned proliferation of vulva via expression of LIN-3 (EGF) and then invades into the vulval epithelium. Although these cellular processes are essential for the proper organogenesis of vulva and appear to be temporally regulated, the mechanisms that coordinate the processes are not well understood. We computationally identified egl-43 as a gene likely to be expressed in the pre-AC/pre-VU cells and the AC, based on the presence of an enhancer element similar to the one that transcribes lin-3 in the same cells. Genetic epistasis analyses reveal that egl-43 acts downstream of or parallel to lin-12 in AC/VU cell fate specification at an early developmental stage, and functions downstream of fos-1 as well as upstream of zmp-1 and him-4 to regulate AC invasion at a later developmental stage. Characterization of the egl-43regulatory region suggests that EGL-43 is a direct target of LIN-12 and HLH-2(E12/47), which is required for the specification of the VU fate during AC/VU specification. EGL-43 also regulates basement membrane breakdown during AC invasion through a FOS-1-responsive regulatory element that drives EGL-43 expression in the AC and VU cells at the later stage. Thus, egl-43integrates temporally distinct upstream regulatory events and helps program cell fate specification and cell invasion.

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Section: Research Articlementioning

confidence: 64%

C. elegansEVI1 proto-oncogene, EGL-43, is necessary for Notch-mediated cell fate specification and regulates cell invasion

2007

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“…RNAi can be similarly controlled by tissue-specific or tissue-restricted expression or activity (e.g., Tavernarakiset al 2000; Sijen et al 2001) or by timing of RNAi feeding (Karp and Greenwald 2003). These strategies permit temporal or spatial control of gene expression, but not both.…”

Section: Discussionmentioning

confidence: 99%

A “FLP-Out” System for Controlled Gene Expression in Caenorhabditis elegans

Voutev

Hubbard

2008

Genetics

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We present a two-part system for conditional FLP-out of FRT-flanked sequences in Caenorhabditis elegans to control gene activity in a spatially and/or temporally regulated manner. Using reporters, we assess the system for efficacy and demonstrate its use as a cell lineage marking tool. In addition, we construct and test a dominant-negative form of hlh-12, a gene that encodes a basic helix-loop-helix (bHLH) transcription factor required for proper distal tip cell (DTC) migration. We show that this allele can be conditionally expressed from a heat-inducible FLP recombinase and can interfere with DTC migration. Using the same DTC assay, we conditionally express an hlh-12 RNAi-hairpin and induce the DTC migration defect. Finally, we introduce a set of traditional and Gateway-compatible vectors to facilitate construction of plasmids for this technology using any promoter, reporter, and gene/hairpin of interest.T WO-COMPONENT gene expression systems are indispensable tools to probe molecular mechanisms underlying development. Because control can be exerted by each component independently, exquisite temporal and spatial control of gene activation or repression can be achieved. Using different promoter combinations to drive each component of these systems, additional control can be obtained beyond that afforded by heat-inducible or tissue-specific promoters alone. Site-specific recombination systems such as the FLP/FRT system have been used to control gene expression by ''FLP-out'': a recombinase-catalyzed intramolecular excision of spacer DNA that lies between tandemly oriented FRT sites. The spacer includes a transcriptional stop so that prior to activation of the FLP recombinase (and subsequent FLP-out) the gene downstream of the spacer is not transcribed (Golic and Lindquist 1989;Struhl and Basler 1993; Figure 1A). After the FRT-containing cassette is excised by the FLP recombinase, the downstream gene is brought into proximity to the promoter and is expressed (reporter 2 in Figure 1A). This system and related systems have proven quite powerful and flexible in model organisms including Drosophila and mouse (see Branda and Dymecki 2004, for review;McGuire et al. 2004). However, prior to our study presented here, and a recently published study (Davis et al. 2008), these systems had not been developed for use in Caenorhabditis elegans.An ideal FLP-out system provides the means to generate both loss-and gain-of-function effects in a spatially and temporally controlled manner. In addition, wild-type gene expression can be turned on in particular cells at particular times in an otherwise mutant background. In organisms where transgenes can be reliably inserted in single copy, FLP-out can also be used to eliminate wild-type gene expression by excision of an FRT-flanked wild-type cassette in a mutant background. In C. elegans, the most common methods for generating transgenic C. elegans introduce multiple copies of transgenes on extrachromosomal arrays (Stinchcomb et al. 1985;Mello et al. 1991;Kelly et al. 1997). ...

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“…Most previous studies of lag-2 regulation utilized transcriptional reporters containing only 3.4 kb of 59 flanking region. These reporters were based on the amount of 59 sequence sufficient to achieve rescue of the lag-2(0) lethal phenotype in a genomic context (e.g., Henderson et al 1994;Wilkinson et al 1994;Karp and Greenwald 2003), and most such reporters are not expressed in VPCs. Another previous reporter, consisting of 6.2 kb of 59 flanking region driving nuclearly localized b-galactosidase, displayed strong expression in P6.p after vulval induction (Chen and Greenwald 2004), suggesting that sequences located between 3.4 kb and 6.2 kb upstream of the protein coding region would mediate transcription of lag-2 in response to the inductive signal.…”

Section: Identification Of a Regulatory Module That Confers Correct Vmentioning

confidence: 99%

Spatial Regulation of lag-2 Transcription During Vulval Precursor Cell Fate Patterning in Caenorhabditis elegans lag-2

Zhang

Greenwald

2011

Genetics

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lag-2 encodes a ligand for LIN-12/Notch and is a component of the lateral signal that activates LIN-12/Notch during Caenorhabditis elegans vulval precursor cell (VPC) fate patterning. lag-2 is specifically transcribed in one VPC, named P6.p, in response to activation of EGFR/Ras/MAPK by the inductive signal that initiates vulval development. Here, we show that a critical molecular event linking inductive and lateral signaling is the relief of VPC-wide lag-2 repression in P6.p. We find that the lag-2 promoter contains an element, VPCrep, which mediates repression in all VPCs when the inductive signal is absent, and another promoter element, VPCact, which is required for activation when repression is relieved by the inductive signal. We show that repression through VPCrep is mediated by the Elk1 ortholog LIN-1, and that the level and subcellular accumulation of a functional LIN-1::GFP protein is similar in all six VPCs before and after vulval induction, suggesting that relief of LIN-1-mediated repression in P6.p is likely due to the known MAPK-dependent phosphorylation of LIN-1. We also provide evidence that the factor(s) acting through VPCact is present in all VPCs but is not modulated by the inductive signal, and that transcription of lag-2 requires the Hth/Meis ortholog UNC-62 and the Mediator complex component SUR-2. Relief of repression of lag-2 in P6.p offers a plausible mechanistic basis for spatial restriction of lag-2 in generating the precise spatial pattern of VPC fates.

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Post-transcriptional regulation of the E/Daughterless ortholog HLH-2, negative feedback, and birth order bias during the AC/VU decision in C. elegans

Cited by 85 publications

References 61 publications

C. elegansEVI1 proto-oncogene, EGL-43, is necessary for Notch-mediated cell fate specification and regulates cell invasion

C. elegansEVI1 proto-oncogene, EGL-43, is necessary for Notch-mediated cell fate specification and regulates cell invasion

A “FLP-Out” System for Controlled Gene Expression in Caenorhabditis elegans

Spatial Regulation of lag-2 Transcription During Vulval Precursor Cell Fate Patterning in Caenorhabditis elegans lag-2

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