Transcription profiling of mouse skeletal muscle to detect genes that regulate motor axon growth and differentiation

These experiments are designed to discover genes that are expressed selectively by synaptic nuclei in skeletal muscle with the particular goal of identifying genes that regulate motor axon growth and differentiation. We plan to isolate RNA from the dissected synaptic region of skeletal muscle and from the non-synaptic region of skeletal muscle and to identify the genes that are expressed at higher levels in the synaptic than non-synaptic region. Previously, we showed that motor axons fail to stop and differentiate in mice lacking MuSK, a receptor tyrosine kinase that is activated by motor neuron-derived Agrin. We hypothesize that MuSK activation normally leads to the production of a retrograde stop/differentiation signal that is encoded by a gene that is expressed preferentially in synaptic nuclei. In the absence of MuSK signaling, the retrograde signaling is not produced by synaptic nuclei, and consequently motor axons wander aimlessly over the muscle. We obtain 6 to 8 micrograms of total RNA from the dissected synaptic or non-synaptic region from a single P21 mouse diaphragm muscle. This is a standard procedure in the lab, and we have used these methods to analzye gene expression and to generate high-quality cDNA libraries. Because the synaptic zone is narrower in the left hemi-diaphragm, we will isolate RNA from this half of the diaphragm. In order to isolate sufficient RNA (5 micrograms from each sample), we will pool the synaptic and non-synaptic regions from two hemi-diaphragms. In order to reduce experimental variability, we wish to analzye expression in six samples: three samples of synaptic RNA and three samples of non-synaptic RNA. We will ship the isolated RNA samples to the Consortium in order to generate labeled cDNA, to screen Affymetrix mouse oligo arrays and to assist in the analysis. Several genes, including the subunits of the acetylcholine receptor, MuSK, acetylcholinesterase, and utrophin are known to be expressed preferentially in synaptic nuclei; thus, these genes serve as internal controls for the reliability and effectiveness of the screen. Most other genes, several of which we have analyzed in previous studies, including actin, GAPDH, runx1, nogoC, creatine kinase, etc. are expressed uniformly in skeletal muscle; thus, expression of these genes should be equally represented in synaptic and non-synaptic regions. Experiment Overall Design: as above本实验旨在筛选骨骼肌突触核选择性表达的基因，核心目标为鉴定调控运动轴突生长与分化的相关基因。我们计划从解剖获得的骨骼肌突触区域与非突触区域中分离核糖核酸（RNA），并筛选在突触区域表达水平高于非突触区域的基因。既往研究显示，在缺失MuSK（一种由运动神经元源性Agrin激活的受体酪氨酸激酶）的小鼠体内，运动轴突无法停止生长并完成分化。我们提出如下假说：正常情况下，MuSK激活会诱导产生一种逆行性停止/分化信号，该信号由突触核优先表达的基因编码；当缺乏MuSK信号转导时，突触核无法产生该逆行信号，进而导致运动轴突在肌肉表面无定向游走。我们可从单只P21小鼠膈肌解剖得到的突触或非突触区域中获取6~8微克总核糖核酸（RNA）。该实验流程为实验室标准操作，我们曾利用此类方法分析基因表达并构建高质量互补脱氧核糖核酸（cDNA）文库。由于左侧半膈肌的突触区域更狭窄，我们将从该侧半膈肌中分离RNA。为获取足量RNA（每个样本5微克），我们将混合两个半膈肌的突触区域与非突触区域样本。为降低实验变异性，我们计划分析6组样本：3组突触RNA样本与3组非突触RNA样本。我们将把分离得到的RNA样本送至该联盟，以完成标记cDNA的制备、Affymetrix小鼠寡核苷酸芯片的筛选，并协助后续数据分析。已知若干基因（包括乙酰胆碱受体亚基、MuSK、乙酰胆碱酯酶以及肌营养不良蛋白（utrophin））可在突触核中优先表达，因此这些基因可作为本次筛选可靠性与有效性的内参对照。其余多数基因（包括我们在既往研究中分析过的肌动蛋白（actin）、甘油醛-3-磷酸脱氢酶（GAPDH）、runx1、nogoC、肌酸激酶（creatine kinase）等）在骨骼肌中均匀表达，因此此类基因在突触区域与非突触区域的表达占比应趋于一致。实验总体设计如上所述。