Functional Study Of Dync2h1 In Zebrafish Pronephrons

The kidney is responsible for removing metabolic wastes,regulating osmotic pressure and maintaining water and electrolyte homeostasis.With millions of nephrons within the kidney,the structure of human kidney is intricate.Zebrafish pronephrons only consist of two nephrons,while the cell type,gene expression and basic function of zebrafish pronephrons are highly similar to that of mammalian metanephros,making them a model for studying kidney development and disease.Cilia are evolutionarily conserved organelles.Motile cilia generate fluid flow over the surface of epithelial cells,while immotile primary cilia sense and transmit extracellular signals.The maintenance of cilia structure and function relays on the intraflagellar transport?IFT?.Ciliary dysfunction are the major cause for cystic kidney in human and zebrafish.DYNC2H1 encodes the heavy chain of Cytoplasmic dynein 2 complex,which drives the reverse IFT along cilia.A recent study has shown that DYNC2H1 mutation also causes fetal kidney dysplasia,suggesting an important role of DYNC2H1 in kidney development,however,the relationship between DYNC2H1 and cystic kidney is still unclear.In this paper,we explored the pathomechanism of cystic kidney caused by of dync2h1 mutation in zebrafish,which provides a theoretical basis for the diagnosis and treatment of human cystic kidney disease.Forward genetic study is an important method to explore genes'function.Based on the genetic screening in ENU mutagenic zebrafish,we got the V43 mutant,which exhibited enlarged nephric tubule shown by alkaline phosphatase staining.To study the function of mutation gene in kidney development,we conducted our exploration through four steps:positional cloning,phenotype analysis,mechanism exploration and gene knockout.Firstly,to find out the mutation gene,we performed positional cloning and mapped the mutation to the dync2h1 in chromosome15.The cDNA sequencing and genomic DNA sequencing revealed a nonsense mutation of C to T in dync2h1 exon10,leading to a premature stop codon.Prediction the protein products of dync2h1^V4343 showed that Dync2h1^V43 was a truncated protein which lost almost all functional domains.We conclude that the V43 mutant habours a dync2h1 mutation.Secondly,to explore the effect of dync2h1 mutation on zebrafish kidney development,we conducted phenotype analysis.In appearance,dync2h1^V43/V43 mutant developed glomerular cyst,body curvature and cardiac edma from 3dpf.The morphological structure of glomerular was abnormal in mutant,but the podocytes differentiation and the glomerular filtration were unaffected.Besides,the mutant nephric tubule became enlarged at 3dpf,and the proximal tubules failed to form convolution.For dync2h1playing role in IFT,we detected the ciliary phenotype,and the results showed the cilia was disorganized with morphological abnormality in mutant nephric tubule,while the cilia docking was unchanged.Moreover,the number and the length of cilia in Kupffer's vesicle did not altered,indicating that the ciliogrenesis was unaffected.We conclude that the dync2h1^V43/V43 is a mutant of cystic kidney with ciliary dysfunction.Thirdly,to further explore the mechanism of renal cyst formation in dync2h1^V43/V43mutant,we detected the cell proliferation,apical-basal cell polarity and distribution of multiciliated cells?MCCs?in renal epithelia.The results showed that the renal tubular cell proliferation rate in mutant was significantly increased,while the glomerular proliferation was not changed.In addition,the mutant showed mislocalization of Na⁺/K⁺ATPase and?PKC in tubular cells,which both are markers of cell polarity.Finally,the number of MCCs was increased in the posterior tubule of dync2h1^V43/V43 mutant.We conclude that the increased cell proliferation rate and altered apical-basal cell polarity are the potential causes for the renal cyst formation in dync2h1^V43/V43 mutant,on the other hand,the abnormal distribution of MCCs may be related to the fluid accumulation and tubular expansion.Lastly,to verify the role of dync2h1 in kidney development.we knocked out dync2h1by CRISPR/Cas9,and got a mutation of four bases-pair deletion in dync2h1 exon12,which termed dync2h1^?4.Similar to dync2h1^V43/V43 mutant,dync2h1^?4/?4 mutant developed glomerular cyst,body curvature,and cardiac edma from 3dpf.Furtherly,the genetic complementary assay was carried out using dync2h1^WT/V43T/V43 and dync2h1^WT/?4,and the results showed that dync2h1^V43/?443/?4 mutant exhibited glomerular malformation and enlarged nephric tubule,indicating that the positional cloning of V43 were correct.We conclude that dync2h1 is essential for kidney development,whose mutation causes cystic kidney in zebrafish.