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The Notch target E(spl)m? is a muscle-specific gene involved in methylmercury toxicity in motor neuron development.

ABSTRACT: Methylmercury (MeHg) is a ubiquitous environmental toxin that has a selective and potent impact on the nervous system, particularly during neural development yet, the mechanisms for its apparent neurodevelopmental specificity are unknown. The Notch receptor pathway has been implicated as a MeHg target in several studies. Notch signaling mediates cell-cell signals in a number of developmental contexts including neurogenesis and myogenesis, where it fundamentally acts to repress differentiation. Previous work in our lab has shown that MeHg causes preferential upregulation of a canonical Notch response gene, E(spl)m?, in Drosophila embryos. In parallel, MeHg is seen to disrupt outgrowth of embryonic intersegmental motor nerves (ISN), which can be mimicked by expression of activated Notch in embryonic neurons. However, overexpression of E(spl)m? in developing neurons fails to elicit motor neuron outgrowth defects, pointing to a non-autonomous role for E(spl)m? in motor axon development. In this study we investigate a role for E(spl)m? in conveying the toxicity of MeHg in the embryo. We find that endogenous expression of the E(spl)m? gene localizes to developing somatic muscles in embryos. Notably, E(spl)m? expression is seen in several muscles that are known synaptic targets for both the ISN and the segmental motor nerve (SN). We also demonstrate that the SN, similar to the ISN, exhibits disrupted axon outgrowth in response to MeHg. E(spl)m? can induce a SN motor neuron phenotype, similar to MeHg treatment; but, only when E(spl)m? expression is targeted to developing muscles. E(spl)m? overexpression in developing muscles also results in aberrant muscle morphology, which is not apparent with expression of the closely related E(spl)m? in developing muscles. Our data point to a role for the Notch target E(spl)m? in mediating MeHg toxicity in embryonic development by disrupting the coordinated targeting of motor neurons to their muscle targets.

SUBMITTER: Engel GL

PROVIDER: S-EPMC4043954 | biostudies-literature | 2014 May-Jun

REPOSITORIES: biostudies-literature

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The Notch target E(spl)mδ is a muscle-specific gene involved in methylmercury toxicity in motor neuron development.

Engel Gregory L GL Rand Matthew D MD

Neurotoxicology and teratology 20140313

Methylmercury (MeHg) is a ubiquitous environmental toxin that has a selective and potent impact on the nervous system, particularly during neural development yet, the mechanisms for its apparent neurodevelopmental specificity are unknown. The Notch receptor pathway has been implicated as a MeHg target in several studies. Notch signaling mediates cell-cell signals in a number of developmental contexts including neurogenesis and myogenesis, where it fundamentally acts to repress differentiation. P ...[more]

PMID: 24632433

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Project description:Methylmercury (MeHg) is a ubiquitous environmental contaminant and neurotoxicant that has long been known to cause a variety of motor deficits. These motor deficits have primarily been attributed to MeHg targeting of developing neurons and induction of oxidative stress and calcium dysregulation. Few studies have looked at how MeHg may be affecting fundamental signaling mechanisms in development, particularly in developing muscle. Studies in Drosophila recently revealed that MeHg perturbs embryonic muscle formation and upregulates Notch target genes, reflected predominantly by expression of the downstream transcriptional repressor Enhancer of Split mdelta [E(spl)m?]. An E(spl)m? reporter gene shows expression primarily in the myogenic domain, and both MeHg exposure and genetic upregulation of E(spl)m? can disrupt embryonic muscle development. Here, we tested the hypothesis that developing muscle is targeted by MeHg via upregulation of E(spl)m? using genetic modulation of E(spl)m? expression in combination with MeHg exposure in developing flies. Developmental MeHg exposure causes a decreased rate of eclosion that parallels gross disruption of indirect flight muscle (IFM) development. An increase in E(spl) expression across the pupal stages, with preferential E(spl)m? upregulation occurring at early (p5) stages, is also observed. E(spl)m? overexpression in myogenic lineages under the Mef2 promoter was seen to phenocopy eclosion and IFM effects of developmental MeHg exposure; whereas reduced expression of E(spl)m? shows rescue of eclosion and IFM morphology effects of MeHg exposure. No effects were seen on eclosion with E(spl)m? overexpression in neural and gut tissues. Our data indicate that muscle development is a target for MeHg and that E(spl)m? is a muscle-specific mediator of this myotoxicity. This research advances our knowledge of the target pathways that mediate susceptibility to MeHg toxicity, as well as a potential muscle development-specific role for E(spl)m?.

Project description:The motor neuron (MN)–hexamer complex consisting of LIM homeobox 3, Islet-1, and nuclear LIM interactor is a key determinant of motor neuron specification and differentiation. To gain insights into the transcriptional network in motor neuron development, we performed a genome-wide ChIP-sequencing analysis and found that the MN–hexamer directly regulates a wide array of motor neuron genes by binding to the HxRE (hexamer response element) shared among the target genes. Interestingly, STAT3-binding motif is highly enriched in the MN–hexamer–bound peaks in addition to the HxRE. We also found that a transcriptionally active form of STAT3 is expressed in embryonic motor neurons and that STAT3 associates with the MN–hexamer, enhancing the transcriptional activity of the MN–hexamer in an upstream signal-dependent manner. Correspondingly, STAT3 was needed for motor neuron differentiation in the developing spinal cord. Together, our studies uncover crucial gene regulatory mechanisms that couple MN–hexamer and STAT-activating extracellular signals to promote motor neuron differentiation in vertebrate spinal cord. To explain our experimental scheme briefly, we are interested in finding target sites for the dimer of transcription factors Isl1 and Lhx3. To mimic the biological activity of Isl1/Lhx3 dimer, we made Isl1-Lhx3 fusion and found that Isl1-Lhx3 has a potent biological activity in multiple systems (i.e. generation of ectopic motor neurons). Then we made ES cell line that induces Flag-tagged Isl1-Lhx3 expression upon Dox treatment. These *mouse* ES cells differentiate to motor neurons (iMN-ESCs) when treated with Dox following EB formation. To identify genomic binding sites of Isl1-Lhx3 (Flag-tagged), we performed ChIP with Flag antibody (pull down of Flag-Isl1-Lhx3) in ES cells treated with Dox. ChIP with Flag antibody in ES cells treated with vehicle (no Dox) was done as a negative control in parallel, and sequenced along with +Dox sample. We have done these experiments twice (two sets).

Dataset Information

The Notch target E(spl)m? is a muscle-specific gene involved in methylmercury toxicity in motor neuron development.

Publications

The Notch target E(spl)mδ is a muscle-specific gene involved in methylmercury toxicity in motor neuron development.

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