View Project Tourtellotte-5R01NS040748-03

Project Summary
Status:	Public
Publications:	2 Published 1 In Preparation
Project Detail		Data Detail
Platform:	Affymetrix	MIAME Areas	Compliance
Species:	Mouse	Array Design Detail	true
Organ/Tissue Type:	skeletal muscle	Experiment Detail	false
Organ Region:	myotubes	Sample Detail	false
Cell Type:	Myoblasts	Hybridization Detail	true
Study Type:	subclassification	Measurement Detail	true
Disease/Condition:	Defective proprioception
Replicates:	2
Expected Samples:

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Investigator Contact Detail
Name	Warren G Tourtellotte
Street Address:	303 E. Chicago Ave.
City, State/Province:	Chicago , IL
Zip/Postal Code:	60611
Country:	United States
Work Phone:	312-503-2415
Fax:	312-503-2459
E-mail:	warren@northwestern.edu

Proposal Detail

Grant:

5R01NS040748-03

Status:

Public

Service Type:

Start to Finish Profiling

IACUC:

02-11-359-A

IACUC date:

2002-12-27

Study Relevance:

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.

Hypothesis:

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.

Specific Aim:

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.

Experimental Procedure and Design:

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.

Experimental Factors:

Conditions that are tested in the experiment. At least one is required. Experimental factors are the independent variables in the experiment.


Experimental Factors is empty.

Project Samples

Samples associated with this project.

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Name	Description	Bio-Source	Extracts
RNA275	Murine myotubes	BioSource4	1
RNA276	Murine myotubes	BioSource5	1
RNA419	Murine myotubes	BioSource6	1
RNA420	Murine myotubes	BioSource7	1

Project Hybridizations

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Name	Array	Labeled Extract	Hybridization Protocol
Mouse Expression Set 430 Array MOE430A_1_hyb	Mouse Expression Set 430 Array MOE430A_1	RNA276_e1_le1	Affymetrix
Mouse Expression Set 430 Array MOE430B_1_hyb	Mouse Expression Set 430 Array MOE430B_1	RNA276_e1_le2	Affymetrix
Mouse Expression Set 430 Array MOE430A_2_hyb	Mouse Expression Set 430 Array MOE430A_2	RNA275_e1_le1	Affymetrix
Mouse Expression Set 430 Array MOE430B_2_hyb	Mouse Expression Set 430 Array MOE430B_2	RNA275_e1_le2	Affymetrix
Mouse Expression Set 430 Array MOE430A_3_hyb	Mouse Expression Set 430 Array MOE430A_3	RNA419_e1_le1	Affymetrix
Mouse Expression Set 430 Array MOE430B_3_hyb	Mouse Expression Set 430 Array MOE430B_3	RNA419_e1_le2	Affymetrix
Mouse Expression Set 430 Array MOE430A_4_hyb	Mouse Expression Set 430 Array MOE430A_4	RNA420_e1_le1	Affymetrix
Mouse Expression Set 430 Array MOE430B_4_hyb	Mouse Expression Set 430 Array MOE430B_4	RNA420_e1_le2	Affymetrix

Project Reference Files

File Name	Size
NINDS-TMM-276-vs-275-1aMOEA.CHP	5.48 Mb
NINDS-TMM-276-vs-275-1aMOEB.CHP	5.46 Mb
NINDS-TMM-276-vs-420-1aMOEA.CHP	5.48 Mb
NINDS-TMM-276-vs-420-1aMOEB.CHP	5.46 Mb
NINDS-TMM-420-vs-275-1aMOEA.CHP	5.48 Mb
NINDS-TMM-420-vs-275-1aMOEB.CHP	5.46 Mb
NINDS-TMM-420-vs-419-1aMOEA.CHP	5.48 Mb
NINDS-TMM-420-vs-419-1aMOEB.CHP	5.46 Mb
Signal Values all four arrays MOEA.TXT	6.44 Mb
Signal Values all four arrays MOEB.TXT	5.35 Mb

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