The Study Of Klf6a Involved In Zebrafish Caudal Venous Plexus Pruning Regulated By Blood Flow

As an extremely important tissue,blood vessel is a complex and precise three-dimensional network,which is responsible for supplying oxygen and energy to the organism while discharging metabolic waste.Excessive growth,reduction,or dysfunction of the blood vessels may lead to multiple systemic lesions.In the course of development,although the vascular network is constantly forming,but its three-dimensional network structure is ceaselessly simplified,and the classification is gradually obvious.Scientists have found that the simplification process after the formation of vascular network is caused by vascular fusion or degradation in the complex local construction.This process is also called vascular pruning,attracting more and more researchers' attention recent years.The study of mouse retinal blood vessel model,zebrafish cerebral blood vessel model and zebrafish mesenteric blood vessel model show that the process of vascular pruning is accomplished by apoptosis or rearrangement of cells under the regulation of hemodynamic factors.However,till now,no group has studied the vascular pruning process and molecular mechanism involved in it by using zebrafish caudal blood vessel model.Based on the above scientific background,this paper uses a variety of transgenic zebrafish,laser confocal microscopy,morpholino knockdown technology,Crispr/Cas9 gene editing technology and a series of experiments to make a detailed study on the pruning process of zebrafish caudal vessels and genetic molecular involved in it,the main contents and results of this thesis are as follows.(1)Description of the pruning process in zebrafish caudal vessels.First,use confocal microscopy to observe the morphological changes of the zebrafish caudal vessels during its development(1dpf to 9.5dpf).Second,use Tg(fli:nGFP×flk1:mCherry)transgenic line to count the nuclear number of dosal artery,the caudal vein and the caudal venous plexus during its development(1dpf to 9.5dpf).According to the variation of the quantities,the pruning process is divided into caudal vein determination stage(1.5dpf to 2.5dpf),venous plexus degeneration stage with cell migration and apoptosis(2.5dpf later).Finally,use the whole mount immunofluorescence experiment to verify the above hypothesis.(2)Hemodynamic factors regulate the formation of zebrafish caudal vein.First,use tricaine as the blood flow inhibitor to treat zebrafish embryos from 1.5dpf to 2.5dpf,abnormal development of caudal vessels was observed by confocal microscope.Second,perform continuous observation of caudal vein(48hpf to 72hpf)by using the time-lapse method,and it is found that the blood flow is able to induce the fusion of adjacent vessels.Above results showed that hemodynamic factor can regulate the formation of zebrafish caudal vein.(3)klf6a can act as a mechanical sensor to regulate the formation of zebrafish caudal vein.First,carry on in vitro mechanical loading method to culture the human umbilical vein endothelial cells(HUVECs)under different shear stress for 12 hours,the expression of Klf6 is found to be increased significantly with the increase of shear stress by the qPCR.Then,verify its mechanical perception by western blot and cell immunofluorescence experiments.Secondly,use confocal microscope to observe the morphology of zebrafish caudal vein at 3dpf after specific knockdown by morpholino and knockout by CRISPR/Cas9 of the zebrafish klf6 a gene.The deletion of KLF6 a results in the abnormal vascular fusion of caudal venous plexus and the formation of caudal vein,thus leading to the disorder of blood flow.Based on the above results,it is concluded that klf6 a can act as a mechanical sensor to regulate the formation of zebrafish caudal vein.In this study,a detailed description of the pruning process of zebrafish caudal vessels is made for the first time so that it could be used as a new vascular pruning model for more research.In addition,klf6 a is screened as a mechanical sensor to participate in the process of vascular pruning,which provides a direction for further research.