The Role Of CRMP4 In Axon Growth And Guidance

There is nothing more subtle than the formation of the entire neural circuits during the development of the nervous system.To construct a functional neural circuit needs lots of axons to reach the target sites and establish synaptic connection with other neurons.If the axons fail to establish proper connection with other neurons,the neuronal information won't be delivered to other neurons accurately,which may disrupt nervous system function and induce a series of neurological diseases.Axon guidance cues are a family of proteins that are important for axon growth and guidance.Classic axon guidance cues can be divided into four categories,namely Slits,Netrins,Ephrins and Semaphorins.They mainly interact with corresponding protein receptors on growth cone surface,sense axonal external environment,regulate cytoskeleton assembly and depolymerization.As a result,the growth cone extends,collapses,branches and steers axon to its target position,and establishes synaptic connection.Semaphorins belong to an important axon guidance protein family.One of its members,Semaphorin 3A,was discovered to be able to mediate axonal growth cone collapse and thus named collapsin.Collaspin(Semaphorin 3A)transfers extracellular signals through its receptor Plexin and co-receptor neuropilin and then initiates relevant intracellular signaling pathways.Further studies showed that a class of proteins mediate above process and was named CRMPs(Collapsin Response Mediator Proteins).CRMPs are a family of cellular phosphoproteins,which have specific expression pattern in the brain of mice in early embryonic development.Five members of CRMPs(CRMP1,CRMP2,CRMP3,CRMP4,CRMP5)have been identified.Among the five members,crmp1,crmp2 and crmp4 genes have alternative splicing isoforms,resulting in the proteins with different N-terminals.Currently,there are some studies about CRMPs structure and function in vitro.Little is known about CRMPs function in vivo,especially CRMPs role in axon guidance and the role of alternative splicing isoforms.In this study,we used zebrafish as our experimental animal to study CRMPs,particularly CRMP4 function in neural circuit development.Firstly,we acquired CRMPs cDNA by reverse transcription PCR and TA cloning.Then,we prepared different CRMPs in situ hybridization probes which were labeled with DIG.Later,we analyzed CRMPs expression pattern through whole-mount in situ hybridization.Such results helped us learn more about CRMPs function.To distinguish CRMP4 alternative splicing,we prepared three kinds of specific probes-CRMP4,CRMP4a and CRMP4b.Based on the result of in situ hybridization,we found that there was a very strong expression of CRMP4a during early zebrafish neural development,and its expression pattern was very similar with CRMP4.However,CRMP4b showed no evident expression.That indicated CRMP4 could exercise its function mainly through CRMP4a during the nervous system development.In addition,we also found CRMP4 was abundantly expressed in zebrafish retinal ganglion cells(RGC).This suggested that CRMP4 might be involved in axonal growth and guidance.Lastly,we knocked down CRMP4a in vivo by microinjecting CRMP4 morpholino into zebrafish zygotes.Using the classic model of retinal axon,we found that knocking down of CRMP4a leaded to RGC axons projection obstacles.To further study the underlying mechanism,we designed double knockdown experiments by co-injection Nrp1a and CRMP4a in half dose,which proved that CRMP4a might be involved in Semaphorin signaling pathway for its function on axon growth and guidance.