| Kidney is an important metabolic organ of human,so it is of great significance to study renal development and disease model.Zebrafish is an important vertebrate model for the study of organ development.Its short generation time makes it amenable to genetic manipulation and analysis,and its small size and high fecundity make it especially well suited for large-scale forward genetic and chemical screens.Fast-developing zebrafish embryos are transparent,facilitating live imaging of a variety of developmental processes in wild-type and mutant animals.The kidneys of vertebrates have three periods in the development process:anterior kidney,middle kidney and posterior kidney.The kidneys of adult mammals developed from the posterior kidney.The kidneys of mice contain tens of thousands of nephrons,while the kidneys of humans have nearly one million nephrons.Vertebrate kidneys play an important role in regulating life activities and excreting metabolic wastes,mainly in metabolite reabsorption,regulating acid-base balance and secreting hormones.The basic functional unit of the kidney is the renal unit,which consists of the glomeruli and renal tubules connected to the collecting duct.The kidneys function by first filtering metabolites from the blood through the glomeruli and then recycling ions and small molecules through epithelial cells such as renal tubules and collecting tubes.The kidney of adult zebrafish developed from the middle kidney,containing only a few hundred nephrons,while the anterior kidney is composed of only two nephrons.The former kidney of zebrafish is very similar to the nephrons of mammals,which are divided into glomeruli and renal tubules,and the molecular regulatory mechanism and cell type is also highly conserved.Therefore,the anterior kidney of zebrafish is an ideal model to study the development of kidney.Positive genetic screening is an effective method to find new genes affecting normal development of tissues and organs.Induced method by means of chemical or physical mutagenesis method makes the male fish spermatagonial cells mutation,and the wild type females crossbred progeny for phenotypic selection,with abnormal phenotype mutation of genealogy,by map-based cloning or whole genome sequencing method,find the mutation,a study on molecular mechanism of further.ENU is a kind of alkylation agent,which can be artificially synthesized to lead to a variety of random mutations,and the mutation efficiency is very high.In this laboratory,male adult spermatogonial cells were mutated by ENU mutagenesis,and their offspring were screened for phenotype after hybridization with wild-type females,and mutant families with abnormal phenotypes were obtained.By means of map-based cloning,the mutant genes were found and the molecular mechanism was further studied.The phenotype of the V3-202-A mutant was alkaline phosphatase stained renal tubular anterior segment thickened,the glomerulus could not fuse normally,but the cilia developed normally,and the pericardium was enlarged from 2dpf,followed by systemic edema and transparent vesicles behind otolites.The homozygous mutant could not survive into adulthood,and began to die at about 7dpf.Initial Mapping was performed to locate the mutation on chromosome 3,and the mutation site was subsequently narrowed down to about 2.20 mb by Chromosomal Walking.Gene knockout is an important way for zebrafish to acquire mutants.At present,endonuclease is the most commonly used gene editing tool,including ZFN,TALENs and CRISPR/Cas.CRISPR/Cas9 gene editing techniques is after ZFN and TALENs,the third generation of gene editing techniques,more simple and efficient,its working principle is on the purpose gene selection gRNA gumming,Cas9 mRNA in gRNA,which can realize the genome specific sites of shear,using DNA sequences can happen when the self-healing mismatch,the introduction of insertion loss mutation,gene reading frame changes,thus to knockout.WT1?Wilms'tumor?encoding zinc finger transcription protein is a highly conserved regulator of renal development in vertebrates and mammals.Mutations in the human WT1 gene lead to abnormal gonadal and renal development,and are prone to childhood malignant solid tumors.The knockout of wt1 gene in mice resulted in renal necrosis,gonadal and splenic defects.There are two homologous genes of wt1,wt1a and wt1b,in zebrafish.In zebrafish,the anterior renal glomerulus develops from the middle mesoderm at the third body segment.wt1a plays an important role in glomerular development and plays a role in inducing podocyte differentiation in early renal development.wt1a and wt1b genes express in kidney,heart,spleen,and gonads.In this paper,by using in situ hybridization method to observe the wt1a,wt1b expression pattern of space and time,and found two genes expression of parts overlap but not completely consistent,wt1b is a little later than wt1a,both originated from mesoderm,among with the embryonic development will gradually focused on glomerular area.We used CRISPR/Cas9 technology to knock out wt1a and wt1b genes,and observed that homozygous wt1a mutants began to have pericardial swelling at 3.5dpf,gradually began to have systemic edema,and 8dpf began to die.Through in situ hybridization experiment,it was found that wt1a was almost not expressed in homozygous mutants,podocin and nephrin of podocyte markers were also not expressed.Therefore,it was known that the knockout of wt1a gene in zebrafish could lead to damage of glomerular development and loss of podocyte development.Meanwhile,we performed in situ hybridization experiments of each tubular segmentation,with marker and cdh17 of the whole renal tubular respectively.Marker of the anterior segment of renal tubular ae2;Marker of renal tubular DE region,slc12a1;Marker in renal tubular DL region,slc12a3,was expressed normally.The above data showed that glomerular development was absent and renal tubular development was normal in zebrafish embryos of wt1a-/-. |
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