ABSTRACT: Using Affymetrix GeneChips, we analyzed expression profiles of SP cells from EOM and TA. 348 differentially expressed transcripts defined the EOM-SP transcriptome: 229 upregulated in EOM-SP and 119 in TA-SP. Keywords: Expression Profiling
Project description:Using Affymetrix GeneChips, we analyzed expression profiles of SP cells from EOM and TA. 348 differentially expressed transcripts defined the EOM-SP transcriptome: 229 upregulated in EOM-SP and 119 in TA-SP. Experiment Overall Design: Six independently separated EOM and six TA SP cell preparations were used for microarray analysis using the Affymetrix® Mouse 430 ver 2.0 GeneChip arrays.
Project description:Purpose: To examine and characterize the expression profile of genes expressed at the neuromuscular junctions (NMJs) of extraocular muscles (EOMs) in comparison to the NMJs of tibialis anterior muscle (TA). Methods: Adult rat rectus EOMs and TAs were dissected, flash-frozen, serially sectioned and stained for acetylcholinesterase to identify NMJs. Approximately 6000 NMJs for EOM (EOMsyn) and 6000 NMJs for TA (TAsyn) and equal amounts of NMJ-free fiber regions (EOMfib, TAfib) and underlying myonuclei were captured using laser capture microdissection (LCM). RNA was isolated, processed and used for microarray-based expression profiling. Profiles were generated for genes differentially expressed at synaptic and non-synaptic regions of TA (TAsyn vs TAfib) and EOM (EOMsyn vs EOMfib) using a false discovery rate (FDR) of 5% as well as an “interaction list” revealing the most significantly differentially expressed genes at an FDR of 1%. We validated the profiles by real-time quantitative reverse transcription-polymerase chain reaction (qPCR). Results: The regional transcriptomes associated with NMJ of EOMs and TAs were identified. We found 275 genes that were preferentially expressed in EOMsyn and 230 known transcripts that were preferentially expressed in TAsyn; 288 of the transcripts were common to both synapses; these included well-known, evolutionarily conserved, synaptic markers (e.g. nicotinic Acetylcholine receptor (ACHR) alpha and epsilon subunits, nestin) as well as a large number of novel genes. Conclusion: Transcriptome level differences exist between EOM synaptic regions and TA synaptic regions. Our definition of the synaptic transcriptome provides insight into the mechanism of formation and functioning of the unique synapses of EOM and their differential involvement in diseases noted in the EOM allotype.
Project description:Purpose: To examine and characterize the expression profile of genes expressed at the neuromuscular junctions (NMJs) of extraocular muscles (EOMs) in comparison to the NMJs of tibialis anterior muscle (TA). Methods: Adult rat rectus EOMs and TAs were dissected, flash-frozen, serially sectioned and stained for acetylcholinesterase to identify NMJs. Approximately 6000 NMJs for EOM (EOMsyn) and 6000 NMJs for TA (TAsyn) and equal amounts of NMJ-free fiber regions (EOMfib, TAfib) and underlying myonuclei were captured using laser capture microdissection (LCM). RNA was isolated, processed and used for microarray-based expression profiling. Profiles were generated for genes differentially expressed at synaptic and non-synaptic regions of TA (TAsyn vs TAfib) and EOM (EOMsyn vs EOMfib) using a false discovery rate (FDR) of 5% as well as an 'interaction list' revealing the most significantly differentially expressed genes at an FDR of 1%. We validated the profiles by real-time quantitative reverse transcription-polymerase chain reaction (qPCR). Results: The regional transcriptomes associated with NMJ of EOMs and TAs were identified. We found 275 genes that were preferentially expressed in EOMsyn and 230 known transcripts that were preferentially expressed in TAsyn; 288 of the transcripts were common to both synapses; these included well-known, evolutionarily conserved, synaptic markers (e.g. nicotinic Acetylcholine receptor (ACHR) alpha and epsilon subunits, nestin) as well as a large number of novel genes. Conclusion: Transcriptome level differences exist between EOM synaptic regions and TA synaptic regions. Our definition of the synaptic transcriptome provides insight into the mechanism of formation and functioning of the unique synapses of EOM and their differential involvement in diseases noted in the EOM allotype. Tissue preparation: A total of 4 rats were killed by CO2 inhalation. The bony orbit was removed from the skull and opened at the lamina cribrosa. The globe with the four recti EOMs still attached was carefully dissected from the bony orbit. The eyeball with muscles was placed on cryomolds, covered with OCT tissue embedding medium (Tissue-Tek: Sakura Finetek, Tokyo, Japan) and flash-frozen in isopentane, cooled in liquid nitrogen and stored at -80 degreeC. The tibialis anterior (TA) muscles of all rats were dissected and frozen in the same way. The EOM and TA were then cut transversely into 10 um sections using a Microm HM 500 cryostat (Zeiss, Oberkochen, Germany), mounted on PEN (poly-ethylene-naphthalene) Membrane Slides (Arcturus) and refrozen immediately. Unfixed sections were stored at -80 degreeC until needed. Section staining: Sections for LCM were stained for acetylcholinesterase based on the method of Karnowsky and Roots to visualize NMJ. Palm microdissection: The PALM MicroBeam System was used for microdissection and for catapulting isolated tissue into a microfuge cap containing 80 ul RLT-Lysis Buffer (Quiagen). Approximately 1000 NMJ and equal amount of non-synaptic regions were collected for each muscle.
Project description:Skeletal muscle stem cells (MuSCs) are recognised as functionally heterogeneous. Cranial MuSCs are reported to have greater proliferative and regenerative capacity when compared with those in the limb. A comprehensive understanding of the mechanisms underlying this functional heterogeneity is lacking. Here, we have used clonal analysis, live imaging and scRNA-seq to identify crucial features that distinguish extraocular muscle (EOM) from limb muscle stem cell populations. A MyogenintdTom reporter showed that the increased proliferation capacity of EOM MuSCs correlates with deferred differentiation and lower expression of the myogenic commitment gene Myod. Unexpectedly, EOM MuSCs activated in vitro expressed a large array of extracellular matrix components typical of mesenchymal non-muscle cells. Computational analysis underscored a distinct co-regulatory module, which is absent in limb MuSCs, as the driver of these features. The EOM transcription factor network, with Foxc1 as key player, appears to be hardwired to EOM identity as it persists during growth, disease and in vitro after several passages. Our findings shed light on how high-performing MuSCs regulate myogenic commitment by remodelling their local environment and adopting properties not generally associated with myogenic cells.
Project description:This SuperSeries is composed of the following subset Series: GSE16536: Using pooled earthworm RNA to test 244K probes on TA-1 test array design 2520022 sense 1 GSE16548: Using pooled earthworm RNA to test 244K probes on TA-2 test array design 2520023 antisense 1 GSE16549: Using pooled earthworm RNA to test 244K probes on TA-3 test array design 2520024 sense 2 GSE16550: Using pooled earthworm RNA to test 244K probes on TA-4 test array design 2520025 antisense 2 Refer to individual Series
Project description:The extraocular muscles (EOMs) are a unique group of muscles that are anatomically and physiologically distinct from other skeletal muscles. Previously, we and others have shown that EOMs have a unique transcriptome and proteome. Here, we investigated the expression pattern of microRNAs (miRNAs) in EOM, as they may play a role in generating the unique EOM allotype. We screened LC Sciences miRNA microarrays covering the sequences of miRBase 10.0 to define the microRNAome of normal mouse EOM and tibialis anterior (TA) limb muscle. 74 miRNAs were found to be differentially regulated (p-value < 0.05) and 31 miRNAs (14 up-regulated and 17 down-regulated) were found to be differentially regulated at a signal strength > 500 including the muscle-specific miR-206, miR-1, miR-133a, miR-133b and miR-499. qPCR analysis was used to validate the differential expression. Bioinformatic tools were used to identify potential miRNA-mRNA-protein interactions and integrate data with previous transcriptome and proteomic profiling data. Luciferase assays using co-transfection of precursor miRNAs (pre-miRNAs) along with reporter constructs containing the 3â-untranslated region (3âUTR) of their predicted target genes were used to validate targeting by identified miRNAs. The definition of the EOM microRNAome complements existing transcriptome and proteome data about the molecular make-up of EOM and provides further insight into regulation of muscle genes. These data will also help to further explain the unique EOM muscle allotype and its differential sensitivity to diseases such as Duchenne's muscular dystrophy (DMD) and may assist in development of therapeutic strategies. Total RNA from four EOM and four TA tissue samples dissected from four adult male C57/Bl10 mice were used (TA served as control) to screen four LC Sciences microRNA Microarray chips. The chips contained microRNA sequences based on miRBase content 10.0 totalling 568 different miRNAs. Samples were labelled with Cy3 and Cy5 using dye-swap. Relative differences of miRNA expression was expressed as fold-changes EOM/TA, which were calculated after normalization across all four arrays.