Generation of novel models of kidney defects using the. The zebrafish is a powerful genetic model of vertebrate development, including kidney organogenesis. Previous genetic screens isolated zebrafish kidney mutants in which nephrons-the functional units of the kidney-evince cyst formation. Work by many labs has revealed that cystic mutants harbor defects in ciliogenesis genes, and thus are relevant to study polycystic kidney disease. In recent years, it has become appreciated that zebrafish nephrons are segmented into functionally discrete domains of proximal and distal epithelial cells, similar to mammals, and that nephron progenitors express similar genes across these species. Many processes in the specification and differentiation of nephron cells from renal stem cells are difficult to study, and today remain poorly understood, due to the complex nature of mammalian kidney development. We hypothesize that the genetic pathways of nephrogenesis will be conserved between vertebrates. The features of the zebrafish embryo enable genetic screens of mutagenized genomes and interrogation of genetic pathways with small molecules, which we have used to begin delineating the genetic recipe of nephrogenesis. However, implementing a systematic genetic analysis of nephron cell type formation would proffer a wealth of meaningful new insights ultimately applicable to the prevention and treatment of congenital kidney defects. We propose to conduct a forward screen for heritable mutations that disrupt zebrafish nephron formation, and to use chemical libraries to identify small molecules that can modulate zebrafish renal progenitor patterning. In a pilot haploid screen with ENU-mutagenized zebrafish, we isolated a cohort of unique nephrogenesis mutants. These findings establish the precedence that our screen enterprise can successfully uncover novel mutations affecting nephron cell type formation. We will conduct a large-scale screen, then complementation and gene expression analyses to chronicle the kidney defects. In a parallel approach, we will employ chemical genetics to screen libraries of bioactive molecules with known targets and identify compounds that alter nephrogenesis. We will also determine the epistatic relationship between nephron genes and the known renal progenitor signals, retinoic acid and Notch. Finally, we will clone and further study a subset of the kidney mutations. The overall goal of this project is to establish a genetic toolkit that can facilitate and energize the burgeoning use of zebrafish in nephrology research. A fully annotated interface with mutant descriptions, images, and chemical phenotypes will be made freely available on the Zebrafish Information Network (ZFIN), and kidney mutant distribution will be coordinated through the Zebrafish International Resource Center (ZIRC). These studies will produce a valuable commodity that will complement existing kidney research tools like the mammalian GUDMAP, and foster the use of zebrafish to formulate and test advanced hypotheses that can forge significant inroads into dissecting the genetic pathways of nephrogenesis.

Public Health Relevance

Congenital and acquired kidney diseases occur at epidemic proportions worldwide. Many of these kidney diseases arise from defects with the cellular composition of nephrons, the functional units within the kidney that cleanse the body of waste and regulate water balance. The proposed studies will help to determine how nephron cells are formed during development, which has the potential to provide insights that can facilitate the creation of new therapies to treat human kidney malformations and adult renal disorders.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK100237-02
Application #
8715803
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Hoshizaki, Deborah K
Project Start
2013-08-06
Project End
2018-05-31
Budget Start
2014-06-01
Budget End
2015-05-31
Support Year
2
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Notre Dame
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
City
Notre Dame
State
IN
Country
United States
Zip Code
46556
Marra, Amanda N; Wingert, Rebecca A (2016) Epithelial cell fate in the nephron tubule is mediated by the ETS transcription factors etv5a and etv4 during zebrafish kidney development. Dev Biol 411:231-45
Poureetezadi, Shahram Jevin; Wingert, Rebecca A (2016) Little fish, big catch: zebrafish as a model for kidney disease. Kidney Int 89:1204-10
Marra, Amanda N; Li, Yue; Wingert, Rebecca A (2016) Antennas of organ morphogenesis: the roles of cilia in vertebrate kidney development. Genesis 54:457-69
McCampbell, Kristen K; Springer, Kristin N; Wingert, Rebecca A (2015) Atlas of Cellular Dynamics during Zebrafish Adult Kidney Regeneration. Stem Cells Int 2015:547636
Cheng, Christina N; Wingert, Rebecca A (2015) Nephron proximal tubule patterning and corpuscles of Stannius formation are regulated by the sim1a transcription factor and retinoic acid in zebrafish. Dev Biol 399:100-16
Cheng, Christina N; Verdun, Valerie A; Wingert, Rebecca A (2015) Recent advances in elucidating the genetic mechanisms of nephrogenesis using zebrafish. Cells 4:218-33
Poureetezadi, Shahram Jevin; Donahue, Eric K; Wingert, Rebecca A (2014) A manual small molecule screen approaching high-throughput using zebrafish embryos. J Vis Exp :e52063
McCampbell, Kristen K; Springer, Kristin N; Wingert, Rebecca A (2014) Analysis of nephron composition and function in the adult zebrafish kidney. J Vis Exp :e51644
Miceli, R; Kroeger, Pt; Wingert, Ra (2014) Molecular Mechanisms of Podocyte Development Revealed by Zebrafish Kidney Research. Cell Dev Biol 3:
McKee, Robert; Gerlach, Gary F; Jou, Jonathan et al. (2014) Temporal and spatial expression of tight junction genes during zebrafish pronephros development. Gene Expr Patterns 16:104-13

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