Detecting The Potential Molecules Regulating Development And Regeneration Of The NMJ By Using High-throughput Deep Sequencing

Neuromuscular junction(NMJ)is a classical peripheral cholinergic chemical synapse,which consists three types of cells: the motoneurons,skeletal muscle fibers and Schwann cells.The development,maturation and regeneration of NMJ are exquisitely regulated by many molecules.Lots of molecules can be secreted by motoneurons to promote the development of the postsynaptic membrane,such as neuronal AGRIN which can promote postsynaptic acetylcholine receptor(ACHR)clustering.There are many postsynaptic signaling molecules which can be activated by signaling from motoneurons or schwann cells,to regulate the expression of downstream genes by activation or inhibition of the intracellular signaling pathways in muscle cells.In this project,we first set up three kinds of model to study the gene expression pattern during the development and regeneration of the NMJ.First,C2C12 murine myoblasts as a model of skeletal muscle development and postsynaptic AChR cluster formation by neuronal AGRIN stimulation.Second,comparison of the gene expression between synaptic and non-synaptic region from neonatal and mature murine diaphragm as a model to study the development and differentiation of the NMJ.Third,murine brachial plexus denervation as a model to study the degeneration and regeneration of the NMJ.Based on these three kinds of models,we adopted Illumina's high-throughput RNA-sequencing technology to screen and compare the gene expression patterns between different groups.Base on the quality assessments of the clean reads and the sequencing saturation and the randomness,we make sure that all the samples meet the requirement.After the quality assessment ofthe sequencing data,the resuls were analyzed on Gene Ontology and KEGG pathway.By doing these,we have analyzed the cellular component,biological process,molecular function and pathway of these genes.After these analysis,we found that most of these genes distribute on cytomembrane,organelle and extracellular region;most of these genes participate metabolic process,biological regulation and regulation of biological process;most of these genes plays an important role on binding and enzyme regulator activity;these genes participate many signals,such as Focal adhesion metabolic pathways,ECM-receptor interaction,WNT signaling pathway.By doing these,we have identified several molecules which may play potential roles during the development,maturation and regeneration of the NMJ respectively.Note that some of the molecules in our lists have been reported and proved in previous studies,such as ACHRs,and majority of them are still elusive and need further functional studies,such as Smads?Ccnd1&Ccnd3 and Sfrps.Together,in this thesis,by discovery and analysis of differential expression genes from three models of NMJ including development,maturation and regeneration respectively,we have identified many potential genes which might be crucial for the development and regeneration of the NMJ,such as Laminin ?2.Our work not only provided important clues for understanding the neurobiological mechanisms of the NMJ development,but also provided potential targets for the treatment of neural regeneration and neuromuscular diseases.