Dataset Information

Genes regulated by Egr3 in primary myotubes. Tourtellotte-5R01NS040748-03

ABSTRACT: Muscle spindles are skeletal muscle stretch receptors that mediate axial and limb position sensation (proprioception) to the central nervous system. Defective proprioception, often characterized by gait ataxia and poor coordination, is present in a variety of human sensory and motor neuropathies having diverse etiologies. Spindles contain specialized intrafusal muscle fibers that are induced during development by sensory innervation. The molecular events mediating the morphogenetic induction process are poorly characterized but depend upon neuregulins and their cognate ErbB receptor signaling. Recently, we demonstrated that the transcription factor Egr3 is induced and regulated by intrafusal muscle fiber sensory innervation during spindle induction. Moreover, Egr3-deficient mice have impaired spindle morphogenesis and profound gait ataxia. Thus, Egr3 mediated transcription is critical for muscle stretch receptor development and potentially for myotube fate specification. Egr3 may mediate spindle morphogenesis induced by Neuregulin/ErbB signaling after myotubes are contacted by sensory axons during development. As a starting point for understanding the specific role for Egr3 in this process, we will use a genome wide expression analysis to identify genes regulated (either directly or indirectly) by Egr3 in primary myotubes. It is not practical to examine Egr3 mediated gene expression directly within spindles since they comprise less than 0.1% of the muscle mass. Therefore, gene expression analysis will be examined in primary myotubes grown in vitro. Primary myoblasts isolated from mice and differentiated in vitro are biochemically most similar to extrafusal muscle fibers which do not express Egr3. To identify genes regulated by Egr3 in myotubes, gene expression in myotubes enforced to express full length Egr3 will be compared to gene expression in myotubes enforced to express truncated (transcriptionally inactive) Egr3. Egr3-deficient mice lack muscle spindles and have profound proprioceptive deficits. Egr3 is specifically expressed in a subset of developing primary myotubes after innervation by sensory axons. Spindle induction is dependent upon sensory/myotube contact which involves sensory axon produced neuregulin and ErbB receptor signaling in the myotube. Shortly after or during induction, Egr3 expression is upregulated and maintained in the nascent myotube/spindle as one of the earliest molecular events know to occur during spindle morphogenesis. We hypothesize that Egr3 mediated transcription is critical for engaging gene programs specific to and essential for, muscle spindle formation. Moreover, Egr3 may be involved in fate specification of myotubes that develop into biochemically and physiologically distinct intrafusal muscle fibers within spindles. To date, the target genes regulated by Egr3 during spindle morphogenesis have not been characterized. Myoblasts from wild type newborn mice will be isolated and purified. Myoblasts will be plated on collagen coated plates and differentiated in vitro for 7 days. During in vitro differentiation, myoblasts fuse to form multinucleated myotubes that exhibit contractile properties. Egr3 is not expressed in myotubes in vitro, which is consistent with their similarity to extrafusal muscle fibers that also do not express Egr3 in vivo. Only intrafusal fibers of muscle spindles express high levels of Egr3 in this muscle fiber context. Adenoviruses that express full length Egr3 (Egr3wt; transcriptionally active) and truncated Egr3 (Egr3tr; transcriptionally inactive) have been generated and characterized in our laboratory. After 7 days of in vitro differentiation, primary myotubes will be infected with either Egr3wt or Egr3tr adenoviruses. 100% infection is routinely obtained which is confirmed by coexpression of enhanced green fluorescent protein (EGFP). Care will be taken to minimize viral induced toxicity of the myotubes after infection. Myotubes are maintained after infection for 24 hours, after which total RNA is extracted from the cell lysates. The two RNA samples will be used for microarray analysis to identify genes that are upregulated and downregulated by Egr3 in the myotube cellular context. We will repeat the infection, RNA isolation and microarray analysis three times to minimize identifying false positive differentially expressed genes. Differentially expressed genes will be confirmed using real-time PCR. Interesting differentially expressed genes will be examined by in situ hybridization to determine if they are specifically expressed in wild type spindles. Since the identified differentially expressed genes will represent both indirect and direct target genes, we will perform Chromatin Immunoprecipitation (ChIP) from Egr3wt infected myotubes and screen the ChIP DNA library by PCR to determine which gene promoters are directly bound by Egr3. Keywords: other

ORGANISM(S): Mus musculus

PROVIDER: GSE1998 | GEO | 2004/11/24

SECONDARY ACCESSION(S): PRJNA91019

REPOSITORIES: GEO

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Project description:Muscle spindles are skeletal muscle stretch receptors that mediate axial and limb position sensation (proprioception) to the central nervous system. Defective proprioception, often characterized by gait ataxia and poor coordination, is present in a variety of human sensory and motor neuropathies having diverse etiologies. Spindles contain specialized intrafusal muscle fibers that are induced during development by sensory innervation. The molecular events mediating the morphogenetic induction process are poorly characterized but depend upon neuregulins and their cognate ErbB receptor signaling. Recently, we demonstrated that the transcription factor Egr3 is induced and regulated by intrafusal muscle fiber sensory innervation during spindle induction. Moreover, Egr3-deficient mice have impaired spindle morphogenesis and profound gait ataxia. Thus, Egr3 mediated transcription is critical for muscle stretch receptor development and potentially for myotube fate specification.,Egr3 may mediate spindle morphogenesis induced by Neuregulin/ErbB signaling after myotubes are contacted by sensory axons during development. As a starting point for understanding the specific role for Egr3 in this process, we will use a genome wide expression analysis to identify genes regulated (either directly or indirectly) by Egr3 in primary myotubes. It is not practical to examine Egr3 mediated gene expression directly within spindles since they comprise less than 0.1% of the muscle mass. Therefore, gene expression analysis will be examined in primary myotubes grown in vitro. Primary myoblasts isolated from mice and differentiated in vitro are biochemically most similar to extrafusal muscle fibers which do not express Egr3. To identify genes regulated by Egr3 in myotubes, gene expression in myotubes enforced to express full length Egr3 will be compared to gene expression in myotubes enforced to express truncated (transcriptionally inactive) Egr3.,Egr3-deficient mice lack muscle spindles and have profound proprioceptive deficits. Egr3 is specifically expressed in a subset of developing primary myotubes after innervation by sensory axons. Spindle induction is dependent upon sensory/myotube contact which involves sensory axon produced neuregulin and ErbB receptor signaling in the myotube. Shortly after or during induction, Egr3 expression is upregulated and maintained in the nascent myotube/spindle as one of the earliest molecular events know to occur during spindle morphogenesis. We hypothesize that Egr3 mediated transcription is critical for engaging gene programs specific to and essential for, muscle spindle formation. Moreover, Egr3 may be involved in fate specification of myotubes that develop into biochemically and physiologically distinct intrafusal muscle fibers within spindles. To date, the target genes regulated by Egr3 during spindle morphogenesis have not been characterized.,Myoblasts from wild type newborn mice will be isolated and purified. Myoblasts will be plated on collagen coated plates and differentiated in vitro for 7 days. During in vitro differentiation, myoblasts fuse to form multinucleated myotubes that exhibit contractile properties. Egr3 is not expressed in myotubes in vitro, which is consistent with their similarity to extrafusal muscle fibers that also do not express Egr3 in vivo. Only intrafusal fibers of muscle spindles express high levels of Egr3 in this muscle fiber context. Adenoviruses that express full length Egr3 (Egr3wt; transcriptionally active) and truncated Egr3 (Egr3tr; transcriptionally inactive) have been generated and characterized in our laboratory. After 7 days of in vitro differentiation, primary myotubes will be infected with either Egr3wt or Egr3tr adenoviruses. 100% infection is routinely obtained which is confirmed by coexpression of enhanced green fluorescent protein (EGFP). Care will be taken to minimize viral induced toxicity of the myotubes after infection. Myotubes are maintained after infection for 24 hours, after which total RNA is extracted from the cell lysates. The two RNA samples will be used for microarray analysis to identify genes that are upregulated and downregulated by Egr3 in the myotube cellular context. We will repeat the infection, RNA isolation and microarray analysis three times to minimize identifying false positive differentially expressed genes. Differentially expressed genes will be confirmed using real-time PCR. Interesting differentially expressed genes will be examined by in situ hybridization to determine if they are specifically expressed in wild type spindles. Since the identified differentially expressed genes will represent both indirect and direct target genes, we will perform Chromatin Immunoprecipitation (ChIP) from Egr3wt infected myotubes and screen the ChIP DNA library by PCR to determine which gene promoters are directly bound by Egr3.

Project description:Egr3 is a zinc-finger transcription factor involved in growth and development. Egr3-deficient mice have severe sensory ataxia due to failed development of muscle spindle stretch receptors. Sensory and motor neurons that normally innervate spindles are absent in Egr3-deficient mice, presumably as a secondary consequence to the loss of trophic signals produced by spindles during development that are required for innervation and neuron survival. The molecular mechanisms involving motor neuron fate specification, target derived growth factor dependencies, and specification of target innervation have been difficult to study since select markers for functionally specific motor neurons are very poorly characterized. A more thorough understanding of the molecular mediators of motor neuron biology will be important to evaluate the efficacy of new strategies devised to thwart neuron death that occurs in a variety of human motor neuronopathies and neuropathies. To identify genes specifically expressed by spinal cord fusimotor neurons: Many motor neuron specific genes have been described over the years. However, none have been described that distinguish fusimotor neurons from skeletomotor neurons despite the fact that they have distinct muscle targets (muscle spindle stretch receptors) and comprise 25-30% of the spinal motor neuron populations. Since these motor neurons have remarkably different target innervation and function, we hypothesize that they express genes that establish their specific phenotypes during development. We hypothesize that fusimotor neurons can be distinguished in the spinal cord by characterizing fusimotor neuron specific gene expression. Once fusimotor neuron specific genes are identified, they will be used as markers to identify fusimotor neurons in complex neuroglial cell populations in vivo and in vitro. We hypothesize that by characterizing fusimotor neuron specific genes, unique marker molecules will be identified for in vivo and in vitro study of this functionally distinct and important motor neuron subtype. Moreover, we hypothesize that many of the genes that are specifically expressed by fusimotor neurons will be involved in mechanisms related to their fate specification, target innervation and growth factor dependent biology. We will use the Affymetrix microarray platform to identify genes that are specifically expressed by fusimotor neurons in mouse spinal cord. The differential expression analysis will be performed on microdissected segments of spinal cord (L3-L5) from wild type and Egr3-deficient mice. Postnatal Egr3-deficient mice lack muscle spindles and fusimotor neurons in their spinal cords. By comparing gene expression from microdissected segments of spinal cord (L3-L5) between wild type and Egr3-deficient mice, we hypothesize that fusimotor neuron selective genes can be identified. We will microdissect L3-L5 segments of spinal cord using precise anatomical landmarks to ensure that comparable spinal cord regions are anlayzed from each animal. For each microarray experiment, total RNA will be extracted from L3-L5 cords (approximately 2 mm length of spinal cord). The integrity of each RNA sample will be verified by gel electrophoresis. The intact RNA samples from mice of similar genotype will be pooled from three (3) 27-day old animals. The intact cord dissection is easier in young animals (eg: 27-day old) and the phenotype is known to exist at this developmental stage. The RNA from each animal of a similar genotype will be pooled into a single sample to minimize false positive gene calls that may represent genes related to the specific state of vigilance of a particular animal at the time of sacrifice (eg: activity dependent genes). Thus, each of the two RNA samples to be analyzed for a particular microarray experiment will represent RNA from three (3) spinal cords of each genotype. RNA amplification for probe synthesis should not be necessary since we will provide 7 ug of intact pooled total RNA for each sample. For statistical analysis, the experiment will be performed twice. Since the RNA samples are precious, they will be provided to the Array Consortium in two shipments with each of the experiments performed independently.

Project description:Skeletal muscle atrophy is dictated by the balance between protein synthesis and protein breakdown, known as proteostasis. With advanced age, muscle atrophy contributes to development of co-morbidities, loss of strength and independence, and all-cause mortality. Aging also coincides with the accumulation of senescent cells within skeletal muscle that produce inflammatory products, known as the senescence associated secretory phenotype. Previously, we found that a metformin + leucine (MET+LEU) combination treatment had synergistic effects in aged mice to improve skeletal muscle structure and function during disuse atrophy. Therefore, the objective of this study was to determine the mechanisms by which MET+LEU exhibits muscle atrophy protection in vitro and if this occurs through cellular senescence. C2C12 myoblasts differentiated into myotubes were used to determine MET+LEU mechanisms during serum-deprivation (SD) atrophy. Single myofibers were isolated from aged (22-23 mo) C57Bl6/J mice, and human primary myoblasts were extracted from a healthy older adult donor. 25 + 125 µM MET+LEU treatment increased differentiation of myotubes and prevented SD induced atrophy. Low concentration (0.1 + 0.5 µM) MET+LEU had unique effects to prevent myotube atrophy and cause transcriptional reprogramming compared to individual therapies. SD increased inflammatory and cellular senescence pathways that was reversed with MET+LEU treatment. SD resulted in dysregulated proteostasis that was rescued with MET+LEU, and proteasome inhibition with MG-132 also rescued myotube atrophy. Cellular senescence transcriptional pathways and markers increased by SD were reversed with MET+LEU treatment, and dasatinib + quercetin (D+Q) senolytic treatment prevented myotube atrophy similar to MET+LEU. In aged myofibers, MET+LEU prevented SD atrophy, and in human primary myotubes MET+LEU prevented reductions in myonuclei fusion. These data provide evidence that MET+LEU has muscle cell intrinsic properties to prevent atrophy by reversing senescence and improving proteostasis

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Genes regulated by Egr3 in primary myotubes. Tourtellotte-5R01NS040748-03

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