Noncanonical Function Of Histidyl-tRNA Synthetase Inhibits Vascular Hyperbranching During Zebrafish Development

Vasculature is the earliest organ to form during the vertebrate embryonic development.A functional vascular system is essential for continued embryonic development and adult survival.We all know that formation of the complex vascular system occurs in two fundamentally distinct processes: vasculogenesis and angiogenesis.Vasculogenesis defines the formation of a primitive vascular network.Angiogenesis refers to the formation of new blood vessels by sprouting of endothelial cells(ECs)from preexisting vessels and subsequent proliferation,migration and remodeling.An aberrant vasculature contributes to the pathogenesis of numerous disease states including cancer.The treatment of vascular diseases are more complicated,especially cerebrovascular disease,cerebral cavernous malformations(CCMs)is one of the most typical disease,CCMs is a typical case of zebrafish as a model of vascular disease.Zebrafish as an ideal model has been for many years,the variety of research methods are already mature,which is the great advantage of zebrafish as a model for the study of vascular development..ENU large scale mutagenesis screening in zebrafish to search for new genes that affect the vascular development is an effective method to study the vascular development.We screened a novel mutant with vascular defects,and identified that the mutant gene was histidyl-tRNA synthetase(hars)which is one of the 20 aminoacyltRNA synthetase(AARSs).The canonical functions of aminoacyl-tRNAsynthetases(AARSs)are indispensable for protein synthesis,with specific AARSs catalyzing the ligation of amino acids to their cognate tRNAs.The canonical function transformed the genetic code from the base to protein.However,recent studies have found that some aminoacyl tRNA synthetases possess new biological functions,including immunity,inflammation,vascular development,and so on.However,most of the studies are limited in vitro,different AARSs play different new biological functions and the mechanisms are not the same,they function as transcription factors,involved in some signaling pathway,or involved in protein synthesis of related genes.So far,what aminoacyl tRNA synthetase play a role in the development of blood vessels have not yet been fully understood,and their mechanisms of action have been poorly studied.In this study,we found that Hars has a noncanonical function in regulating vascular development in zebrafish.Through the large-scale screening,we screened a zebrafish mutant with vascular defect during development.after 48 hours post fertilization(hpf),the mutant showed increased sprouting of angiogenic vessels in the head and began to exhibit ectopic intersegment vessels(ISVs)branching in the dorsal part of the trunk,the vascular connecting disordered,with a disorganized vascular pattern.The abnormal vascular is mainly sprouting after 36 hpf,the mutation mainly affected angiogenesis.We performed genome mapping and positional cloning to identify the defective locus in the mutant.We finally identified hars is the mutation gene.We use the protein synthesis inhibitor cycloheximide(CHX)inhibited zebrafish protein synthesis.Zebrafish does not appear the increased vascular sprouting phenotype as the mutant after protein synthesis inhibition.We detected the Hars activity directly by the ELISA kit and found no differences between wt and mutant.These indicate that the mutant increased vascular phenotype not caused by the lack of canonical function of protein synthesis,but the noncanonical function of Hars.We found that the expression of vascular endothelial adhesion protein(VE-cadherin,Cdh5)was upregulated in the mutant,the distribution of Cdh5 at the sprouting increased endothelial cell was abnormal,and endothelial cell polarity related proteins(Podocalyxin,Pdxl2)was irregular increased at the sprouting increased and connection disordered sites,this suggests that wrong polarity of endothelial cells in the mutant.After morpholino knockdown of cdh5 expression,the mutant phenotype of vascular disruption in the head was surpressed,indicating that cdh5 caused endothelial cell junction dysfunction.At the same time,we detected the expression of vascular endothelial growth factor A(vegfa)was increased in the mutant,Vegfa is an important growth factor required for vascular growth,the important function in mice and zebrafish and in other species have been proved.We use the Vegf signal pathway inhibitor SU5416 to teat the mutant embryos from 36 hpf and observe at 60 hpf.We found that the abnormal vascular sprouting in the head and trunk are rescued,suggesting that Hars regulate vascular growth by vegfa.In addition,Aligning Hars protein sequences in three species showed that zebrafish and mammalian HARS were highly homologous Furthermore,injection of human HARS mRNA potently abolished ectopic CtAs branching in zebrafish mutant.This suggests that the regulatory function of Hars in vascular development is conserved between zebrafish and human.Finally,we detected the expression of HARS in the nucleus of human umbilical vein endothelial cells(HUVECs),suggesting that HARS may be a novel transcription factor that directly regulates the transcription of related genes.In summary,we screened a new zebrafish mutant,identified the mutant gene is hars,we revealed that Hars possess function of regulating vascular development,lacking of noncanonical function will lead to the abnormal sprouting and disorder vascular connection,noncanonical function of this gene is conserved in zebrafish and humans.The noncanonical function of Hars regulates vascular development by modulating the expression of cdh5 and vegfa.This suggests that the gene mutation in humans may cause similar vascular defects.The mutants in zebrafish can be a research model for this disease.