Functional Study Of Stk3 In Zebrafish Microglia Development

A collection of diseases,including Alzheimer’s disease and Parkinson’s disease,neurotropic viral infections,stroke,paraneoplastic disorders,traumatic brain injury and multiple sclerosis,are classified as neurodegenerative diseases.The main pathological features of neurodegenerative diseases are slowly progressive dysfunction and loss of neurons and axons in the central nervous system.Although the triggering events are different,they all share a common feature: chronic immune activation,especially microglial activation.Microglia are macrophages that reside in the central nervous system.In addition to removing pathogens and apoptotic cells,microglia are also involved in the regulation of neural development and function.Microglia are closely related to neurodegenerative diseases and are an important target for the treatment of these diseases.Therefore,it is of great significance to understand the molecular regulatory network of microglia.Zebrafish is an important vertebrate model organism with many advantages,such as rapid development,large spawning volume,and easy genetic manipulation.In vitro fertilization and embryogenesis,translucency of embryos,such characters make zebrafish an ideal model for studying macrophage development and tissue colonization.In the zebrafish embryonic brain,the most microglia were distributed in the optic tectum region of the midbrain.The colonization of microglia precursors in the optic tectum region of zebrafish is regulated by the Il34-Csf1 ra signaling pathway and neuronal apoptosis signaling in the brain.In addition,the deletion of Slc7a7,Nlrc3-like,Xpr1 b,Mcoln1a and Pdcd11 also affects the number of microglia.However,the regulatory mechanism of microglia colonization in the brain has yet to be further refined.Hippo signaling pathway is a highly conserved signaling pathway that mainly regulates cell proliferation and apoptosis,and controls organ size.In recent years,more and more articles have reported that Hippo signaling pathway components are involved in hematopoiesis and immune regulation.MST1/2 are key kinases in the Hippo signaling pathway,and MST2 expression increases in the brain’s hippocampus during aging.Experiments in hippocampal primary cell cultures showed that oxidative stress activates the MST2 homolog,MST1,to mediate cell death through the MST1-FOXO pathway,suggesting that MST1/2 may play a role in the neurodegenerative process in Alzheimer’s disease and potentially other neurodegenerative diseases.Furthermore,in a model of cerebral ischemia-reperfusion injury,MST2 is phosphorylated at tyrosine 433 by Src to mediate the activation of microglia.However,the role of MST1/2 in microglial colonization remains to be explored.Mst2,but not Mst1 has homologues in zebrafish,which is called stk3.We observed that Stk3 was enriched in zebrafish microglia.In order to study its function,we constructed stk3 mutant zebrafish by CRISPR / Cas9 technology.The number of microglia in the optic tectum region was significantly reduced in stk3 mutant.Then,we constructed transgenic zebrafish lines Tg(cryyaa: cerulean-huc: stk3)(neuron specific)and Tg(cryyaa: cerulean-coro1a: stk3)(leukocyte specific),only the latter was able to rescue the microglial phenotype in stk3 mutants.Further observations revealed that at 30 hpf,the number of early primitive macrophages in stk3 mutants was less than siblings;at 3-5 dpf,primitive macrophages in stk3 mutant showed an abnormal distribution pattern with more macrophages in the ventral PBI and fewer in the dorsal spinal cord region.Although primitive macrophages in stk3 mutants were able to migrate to caudal hematopoietic tissue,they responded poorly to tail injuries Furthermore,macrophages in stk3 mutants have abnormal morphology with less protrusions,which is an important characteristic of mature macrophages.As an upstream regulator of Hippo signaling pathway,Stk3 may participate in the development of zebrafish microglia through the classical Hippo signaling pathway.We examined microglia in the mutants of other core molecular of Hippo signaling pathway,but found no abnormalities.Moreover,Yap did not accumulate significantly in the stk3 mutant at 3dpf,and Yap knockout could not rescue the number of microglia in the stk3 mutant.These results suggest that the regulation of microglia development by Stk3 may not depend on the canonical Hippo signaling pathway.Interestingly,previous studies reported that the morphology and distribution of macrophages in csf1 ra mutant were abnormal,the response of macrophages to injury was weakened,and the number of microglia in optic tectum was reduced,which was highly consistent with our observations in stk3 mutant.More importantly,we found that there was a genetic interaction between stk3 and csf1 ra,and Tg(cryyaa: cerulean-coro1a: stk3)could not rescue the number of microglia in csf1 ra mutant.The expression of csf1 ra at m RNA level was normal in stk3 mutant,and Co-IP showed that there was no interaction between Stk3 and Csf1 ra,but Stk3 helped to stabilize Csf1 ra in transfected cell lines.Taken together,we have revealed that Stk3 may regulate zebrafish primitive macrophage development and microglial colonization in a cell-autonomous manner by stabilizing Csf1 ra,and our study enriches the molecular regulatory network of microglial colonization.