Genetic Screening And Mutation Mapping Of Zebrafish Pronephric Mutants

Kidney is an essential organ in human body,and it is important to study its development.As a mammalian which is evolutionarily close to human,mouse is the most popular animal model for kidney development study,and many advances have been achieved in this model.However,the complicated structures of kidney and inconvenient observation of intrauterine embryogenesis are difficult to overcome in mouse.Zebrafish emerges as a classic vertebrate animal model due to the following advantages: easy to breed,short generation time,highly productive of offspring,transparent embryo and the external development.Moreover,zebrafish is the only vertebrate suitable for large scale genetic screen.There are three forms of kidney in vertebrates during evolution,pronephros,mesonephros and metanephros.In mammals,pronephros and mesonephros degenerate very quickly,and metanephros is the functional kidney in embryo and adult.In fish,pronephros is functional during embryogenesis,and mesonephros will replace pronephros in larva and adult.Zebrafish pronephros consists of only two nephrons which is conserved in cellar composition and developmental regulatory mechanism.Considering the advantages of zebrafish,its simple pronephros becomes an ideal model to study kidney development.To systematically investigate molecular mechanisms underlying pronephros development in zebrafish,we carried out a forward genetic screen for pronephric mutants.ENU(ethylnitrosourea)was used to introduce mutations into genomes,and a classic F2 screen strategy was applied to identify recessive mutants in F3 embryos.The pronephric tubule in 4 dpf embryo was marked by alkaline phosphatase staining,a method which had never been reported for a genetic screen for pronephric mutants.Two previous screens had been performed by direct observation under dissection microscope,in which only mutants with obvious pronephric defects,such as renal cyst,could been picked out.Since the improvement of our method,many unreported mutants had been identified in our screen.The scale of the screen was 212 mutagenized genomes,and we finally obtained 24 mutant lines after four rounds of screen and outcross to purify genetic background.According to results of alkaline phosphatase staining,we divided mutants into 4 types: thick pronephric tubule mutant,thin pronephric tubule mutant,short pronephric tubule mutant and renal cyst-like mutant.In order to confirm the reliability of alkaline phosphatase staining,we performed whole mount in situ hybridization(WISH)use cdh17 and pax2 a RNA probes(specific to pronephric tubule)to check mutant phenotypes in 22 lines using 2.5dpf embryos as samples.And I divided them into 5 types of phenotype,besides the above four types,some mutants belonged to a normal tubule type,in which the mutation affected pronephric tubule development between 2.5 to 4 dpf.The WISH results agreed with ones obtained by alkaline phosphatase staining.I also checked cilia in pronephric tubule in these mutants with immunohistochemistry using anti-acetyl tubulin,and found cilia in two cyst-like mutants were disorganized,which was consistent with other reports.The mutant morphology of pronephric tubule marked by cilia were the same to WISH results.In order to examine glomerular development of mutants,I carried out other WISH experiments using podocin and wt1 a RNA probes(specific to glomerulus)in 2.5 dpf embryos.I scored results and divided mutants into 5 groups: big glomerulus mutant,small glomerulus mutant,loss of glomerulus mutant,fusion defective mutant and normal type.Then,I analyzed glomerular and tubular phenotypes in mutants together to find out the relationship between them during pronephros development.Three groups were divided: tubule specific mutant,glomerulus specific mutant and double defects mutant.More than half mutant lines belonged to the last group,and other two groups had the same number of members.These results suggest that some genes are unique to regulate glomerulus and tubule development respectively,and quite a lot of them do both.Our final target is to identify mutant gene responsible for the phenotype,and we applied positional cloning to find the mutation.The phenotype of V47 mutant included thick pronephric tube with the loss of anterior PCT,retard development and severe body malformation.In initial mapping using bulk segregant analysis(BSA),the mutation of V47 was found located on chromosome 15,and then further to define into a region of 4.4Mb by chromosome walking.However,genetic markers in this regions did not match the genomic DNA sequence from NCBI in the following recombination frequency analysis.This is probably due to the mis-alignment of genomic sequence in the region.Another mutant,V43,showed renal cyst and dis-organized cilia in tubule cells.The mutation was also found located on chromosome 15,now it was defined into a 4.7 centimorgan region by chromosome walking.Taking together,we have obtained 24 stable zebrafish pronephric mutant lines,including two renal cyst mutants and other 22 lines with phenotypes that have not been identified in genetic screens.Following studies on these mutants will help us to understand molecular mechanisms in kidney development.