Identification of the neuromuscular junction transcriptome of extraocular muscle by laser capture microdissection.
OBJECTIVE
To examine and characterize the profile of genes expressed at the synapses or neuromuscular junctions (NMJs) of extraocular muscles (EOMs) compared with those expressed at the tibialis anterior (TA).


METHODS
Adult rat eyeballs with rectus EOMs attached and TAs were dissected, snap frozen, serially sectioned, and stained for acetylcholinesterase (AChE) to identify the NMJs. Approximately 6000 NMJs for rectus EOM (EOMsyn), 6000 NMJs for TA (TAsyn), equal amounts of NMJ-free fiber regions (EOMfib, TAfib), and underlying myonuclei and RNAs were captured by laser capture microdissection (LCM). RNA was processed for microarray-based expression profiling. Expression profiles and interaction lists were generated for genes differentially expressed at synaptic and nonsynaptic regions of EOM (EOMsyn versus EOMfib) and TA (TAsyn versus TAfib). Profiles were validated by using real-time quantitative polymerase chain reaction (qPCR).


RESULTS
The regional transcriptomes associated with NMJs of EOMs and TAs were identified. Two hundred seventy-five genes were preferentially expressed in EOMsyn (compared with EOMfib), 230 in TAsyn (compared with TAfib), and 288 additional transcripts expressed in both synapses. Identified genes included novel genes as well as well-known, evolutionarily conserved synaptic markers (e.g., nicotinic acetylcholine receptor (AChR) alpha (Chrna) and epsilon (Chrne) subunits and nestin (Nes).


CONCLUSIONS
Transcriptome level differences exist between EOM synaptic regions and TA synaptic regions. The 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.
Ketterer
Caroline
C
Department of Cell and Developmental Biology, Pennsylvania Muscle Institute, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
Zeiger
Ulrike
U
Budak
Murat T
MT
Rubinstein
Neal A
NA
Khurana
Tejvir S
TS
D000367:Age Factors; D000818:Animals; D005260:Female; D020869:Gene Expression Profiling / Q000592:standards; D007381:Intermediate Filament Proteins / Q000235:genetics; D007834:Lasers; D008297:Male; D042282:Microdissection / Q000295:instrumentation / Q000379:methods*; D018485:Muscle Fibers, Skeletal / Q000502:physiology; D018482:Muscle, Skeletal / Q000502:physiology; D009419:Nerve Tissue Proteins / Q000235:genetics; D064231:Nestin; D009469:Neuromuscular Junction / Q000502:physiology*; D009801:Oculomotor Muscles / Q000502:physiology*; D051381:Rats; D011978:Receptors, Nicotinic / Q000235:genetics; D015203:Reproducibility of Results; D013569:Synapses / Q000502:physiology
D007381:Intermediate Filament Proteins; D009419:Nerve Tissue Proteins; C577486:Nes protein, rat; D064231:Nestin; D011978:Receptors, Nicotinic
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