The Study Of Dual Mechanism That Stat3 Regulates Axon Outgrowth And Regeneration In Zebrafish Posterior Lateral Line

Axon is an important part of the nervous system.It's connected with target cells by forms synaptic and plays the role of sending information.The axon outgrowth and regeneration will directly affect the length of axon and keep establishing synaptic connections with target cells.The Jak-Stat pathway of Signal transduction and transcriptional activation factor 3?Stat3?,regulates a number of physiological processes,such as inflammatory response,stem cell stem maintenance,which is dependent on the Y705 phosphorylation of Stat3.In addition,Stat3 also regulates microtubule stability,cell migration,and mitochondrial activity through non-transcription-dependent pathways and different protein modifications.The posterior lateral line?PLL?system of zebrafish is composed of mechanoreceptor to feel water flow and the nerve to send information.Stat3 is specifically expressed in the neuromast and ganglion of zebrafish,so zebrafish is a good model to study the role of Stat3 in nerve development and regeneration.We successfully knocked out the Stat3 gene in zebrafish by using CRISPR/Cas9.Then,using Tg?Sq ET20:GFP?transgenic fish,found that the stat3 homozygous mutant has more neuromast in caudal than wildtype after 4 dpf.In situ hybridization results showed that the mbp signal on the posterior lateral line of the Stat3 homozygous mutant was less than wildtype.Further,we found that axon of posterior lateral line of stat3homozygous mutant stopped at the side of cloaca before 48 hpf,and no longer touched the caudal neuromast by using anti-acetylated-tubulin antibody and Tg BAC?Neurod1:EGFP?transgenic fish.This indicates that stat3 indirectly inhibits the lateral line progenitor proliferation by positively regulating axon outgrowth.In order to study how stat3 regulates nerve outgrowth,we used Tg BAC?Neurod1:EGFP?and Tg?ET189b:GFP?transgenic zebrafish to find that the shorter aoxn in mutant is not due to lack neurons which will touch the caudal neuromasts.And in situ hybridization showed that the sox10 signal labeled schwann cell precursor did not decrease in mutant at 32 hpf;transmission electron microscopy?TEM?showed that the myelin sheath was intact at 72 hpf,but axon diameter was generally thinne.After excluding the possibility that stat3 indirectly cause short axon in mutant by regulates other cells,we overexpressed Stat3 in the PLL's neurons of the mutant by injecting plasmids that specifically expressed in neurons.The results showed that the specific expression of stat3 in the neurons of the posterior lateral line of the mutant could save the shorter axon.This suggests that stat3 promotes nerve outgrowth by cell automatically regulate.To further explore the mechanism of stat3 in regulating nerve outgrowth,we used inhibitors of Jak-Stat,S3i-201,Pyridone6 and Stattic,as well as overexpression of Socs3a,Stat3^Y708F and Stat3^EE435-436AA,not only found that did not cause short axon in the wildtype,but also overexpress Stat3^Y708F and Stat3^EE435-436AA did not save the shorter axon in mutant.Therefore,it is shown that the axon outgrowth does not depend on Jak-Stat,but Y708and EE435-436 on Stat3 are necessary in the process.Finally,we measured the mitochondrial ATP synthase activity of 24 hpf and 48 hpf and found that the ATP synthase activity decreased in mutant;and the shorter axon was saved by injecting MTSStat3 m RNA.And then,the mitochdrial movement in the PLL's nerve is slow in mutant at 48 hpf and 72 hpf,by injecting the plasmid ptol2-neurod:tdtomato-cox8a.These showed that stat3 regulates the energy metabolism of mitochondria,thus affecting the speed of mitochondrial movement in the nerve,and regulates axon outgrowth in PLL.After clarifying the mechanism of stat3 regulates axon outgrowth in PLL,we ablated axon in wildtype and then feeding in inhibitor S3i-201 or Pyridone6;and ablating axon after injecting Socs3a m RNA,Stat3^Y708F m RNA or Stat3^EE435-436AAm RNA,which showed that Jak-Stat was necessary for nerve regeneration.In addition,we ablated axon after saving axon outgrowth by injecting MTSStat3 m RNA,and found no rescue regeneration.Therefore,Jak-Stat is necessary to regulate nerve regeneration.Mitochondrial Stat3alone is not enough to regulate nerve regeneration.Based on the above experiments,we conclude that the non-transcription-dependent pathway of stat3 is the key for regulate axon outgrowth.And the transcription-dependent pathway of stat3 is the key for axon regeneration.