Genetic Screen And Mechanism Study Of Zebrafish Pronephric Mutants

Vertebrates have developed three forms of kidneys with increasing complexity during evolution:the pronephros, mesonephros, and metanephros. Despite of morphological difference, they are almost same in the cellular composition and distribution, the molecular mechanism of development are conserved. Thus, the study of pronephric development in zebrafish can be exploited to further our general understanding of renal development and biology. Zebrafish pronephros, with full physiological function, are simple in structure, and are very useful to investigate nephrogenic mesoderm differentiation, kidney cell type differentiation, nephron patterning, interactions between kidney and vasculature, glomerular function, and diseases model with defects in glomerular filtration and tubule lumen size, i.e., cystic kidney. As a classic animal model, Zebrafish is easy to feed,productive, and with fast sexual maturation, their embryos are translucent and externally developed,which provide convenience for observation. These characteristics makes zebrafish the best vertebrate model for a large-scale genetic screening. Mice are mostly used to study kidney development and function because both humans and mice are mammals, however, this animal model has some disadvantages, such as complicated kidney structure, developing inside mother’s body, and limitations of currently used methods. Thus, there are a lot of questions to be addressed in kidney development.We performed a genetic screen for zebrafish pronephric mutants to systematically study molecular mechanism of kidney development without genetic discrimination. It is expected to find new molecular mechanisms and regulatory factors involved in kidney development from the screen.We used the chemical mutagen ENU to introduce mutations into genomes, which was efficient and without site preference, and took a classic F2 screening strategy for recessive pronephros mutants. In previous reports, screens were carried out by morphological observation under dissection microscope, however, we utilized a substrate of alkaline phosphatase to clearly visualize pronephric tubules according to the high alkaline phosphatase activity in tubular epithelium. By this way we isolated 37 mutants from 212 mutated genomes screened, these mutants were grouped into four classes based on their phenotypes: renal cysts-like mutants, short and thick renal tubule mutants,edema mutants and other mutants. In order to detect glomerular development in mutants, we performed in situ hybridization using podocin RNA probe. Preliminary results showed that more than half of mutants’ glomeruli are normal. This suggests that pronephric tubules and glomeruli may take different developmental gene regulatory networks, although some of them are common.After completion of the screening, we selected V3-5(renal cysts-like mutants), V47(short and thick renal tubule mutants) and L27(edema mutants) for positional cloning mutations. These mutants with AB background were crossed with WIK strain to establish mapping families. Due to genetic background and other unknown reasons, V3-5 mutant from mapping families showed phenotypes at very low percentage, and it is difficult to collect mutant embryos. Through the bulked separation analysis(BSA), the mutation was located on chromosome 23. Due to the small number of mutant embryos, the progress of chromosome walking to locate the candidate region were very slow,then we tried a method for identification of the mutation based on RNA-seq, the sequencing data are being analyzed. As for L27, after analyzing the recombination of 3,497 mutant embryos, the mutation eventually was located in a region with 0.662 Mb on chromosome 13. This region contains15 coding genes, we will identify candidate genes by DNA sequencing one by one. We are collecting mutant embryos for the V47 mapping.Phenotype analysis of L27 mutant showed that the pronephric proximal tubule was normally differentiated, but it was parallel straight tube in shape and far away from the pectoral fin. The neck and podocyte-specific marker were down regulated or absent. These phenotypes are similar with wt1a/b or osr1 morphants. There are no reports about L27 candidate genes involved in regulating glomerular development. This suggests that L27 mutation is a new factor which regulates the development of pronephric glomerulus and neck.In addition to a forward genetic screen, we knocked-out lhx1 a in zebrafish by TALEN technology to study the role of lhx1 a in zebrafish pronephros. Through RNA in situ hybridization,we analyzed lhx1 a temporal and spatial expression pattern. The results showed that lhx1 a was a maternal gene, it also expressed in the early stage of pronephros and their precursors. Ater the completion of pronephros segmentation differentiation, the expression of lhx1 a in pronephros sharply declined and couldn’t be detected. Knockout of lhx1 a resulted in the gradual loss of wt1 a,complete loss of podocin and reduced or partial loss of pax2 a expression, but pronephric tubule is normal. These results suggest that lhx1 a is required for podocytes genesis, but not for pronephric tubule. We had attempted to rescue the defects by injection of capped in vitro transcribed lhx1 a mRNA. Surprisingly, it was not only unable to rescue lhx1 a mutant phenotypes, it caused a reduction even loss of podocin expression in wild type and heterozygous embryos. This result indicates that overexpression lhx1 a will inhibit the terminal differentiation of glomerular podocytes.Our work will find a novel pronephros development regulatory factor L27, and provide many materials to study kidney development to further our general understanding of renal development and biology.