Research On Mechanisms Of The Molecules Involved In Regulating Zebrafish Brain Vascular Development

The brain vasculature is among the main contributors of the brain,in that it occupys about 15-25 percent the brain volume.The brain vasculature plays important regulatory roles in the development,function and disease of the brain.During brain development,the brain vasculature surves as a“scaffold”for the migration of developing neurons.Physiologically,the brain vasculature supplies oxygen and nutrients to the brain and carries metabolic waste.What's more,it has been found in animal models such as Alzheimer's disease and Parkinson's disease that cerebrovascular diseases occur before abnormalities in neuronal morphology and function develop.Thus,it is of great importance to explore the mechanism of brain vascular development and regulatory factors involved in angiogenesis.Aim:To explore the role of hydrogen sulfide?H₂S?and interferon??IFN-??in zebrafish brain vascular development.Method:We used the zebrafish as a model organism for studying brain vasculardevelopment.We knocked down and knocked out the two H₂S-producing enzymes,cystathionine?-synthase?CBS?and cystathionine?-lyase?CSE?,through morpholino oligomers?MOs?and CRISPR/Cas9.We then observed the brain vasular development of the larvae through confocal imaging.We conduct rescue experiments on CBS and CSE morphants through a H₂S donor.Analysis of quantitative charactristics of themidbrain vasculature morphology was conducted through a special software.Wedetected the downstream of H₂S using western bloting and qPCR.Also,We knocked down the zebrafish IFN-?receptor,crfb17,through MO,and observed the brainvascular development during 3 days post fertilization?dpf?and 5 dpf as well as the trunk vascular development during 28 hours post fertilization?hpf?and 31 hpfthrough confocal imaging.We further analyzed and compared the midbrain vascular network characteristics between the crfb17 MO group and the control MO group.Result:Knockdown and knockout CBS and CSE impaired brain vascular development of larval zebrafish.Incubation with slow-releasing H₂S donor,GYY4137,alleviated the brain vascular developmental defects in CBS and CSE morphants.Analysis of quantitative charactristics of the midbrain vasculaturemorphology showed that H₂S functions in angiogenesis without affecting topological structure formation of the brain vasculature.Nitric oxide synthase 2a?nos2a?expression and NO production were decreased in both CBS and CSE morphants while ERK 1/2 phosphorylation and vascular endothelial growth factor?VEGF?expression showed no significant difference,implicating the nos2a/NO pathway may be involved in the H₂S-induced brain angiogenesis.In our next study,we found that knockdown of zebrafish IFN-?receptor,crfb17,impaired both brain and trunk vasculardevelopment of larval zebrafish.Further analysis of quantitative charactristics of the midbrain vasculature morphology revealed that the total segment length,segment number and vessel density of midbrain in the crfb17 MO group were significantly lower than those in the control MO group,indicating the pro-angiogenic effect ofIFN-?in brain vasucular development.Conclusion:Our study found that endogenous H₂S and IFN-?are both important in zebrafish brain vascular development.