A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

Summary

We describe a method of generating a possible zebrafish model of polycystic kidney disease. We used Tg(wt1b:GFP) fish to visualize kidney structure. Knockdown of wnt5a was by morpholino injection. Pronephric cyst formation after wnt5a knockdown was observed in this GFP transgenic zebrafish.

Abstract

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The molecular mechanisms leading to cyst formation in PKD remain somewhat unclear, but many genes are thought to be involved. Wnt5a is a non-canonical glycoprotein that regulates a wide range of developmental processes. Wnt5a works through the planar cell polarity (PCP) pathway that regulates oriented cell division during renal tubular cell elongation. Defects of the PCP pathway have been found to cause kidney cyst formation. Our paper describes a method for developing a zebrafish cystic kidney disease model by knockdown of the wnt5a gene with wnt5a antisense morpholino (MO) oligonucleotides. Tg(wt1b:GFP) transgenic zebrafish were used to visualize kidney structure and kidney cysts following wnt5a knockdown. Two distinct antisense MOs (AUG – and splice-site) were used and both resulted in curly tail down phenotype and cyst formation after wnt5a knockdown. Injection of mouse Wnt5a mRNA, resistant to the MOs due to a difference in primary base pair structure, rescued the abnormal phenotype, demonstrating that the phenotype was not due to “off-target” effects of the morpholino. This work supports the validity of using a zebrafish model to study wnt5a function in the kidney.

Introduction

Zebrafish (Danio rerio) embryos have been widely used as a model for studying kidney development and polycystic kidney disease. There are many advantages to using zebrafish as an animal model: the feasibility of studying genetic interactions, the ability to use antisense morpholinos (MO) for protein knockdown, the opportunity to quickly assay large numbers of embryos, and the ease of viewing organ phenotypes in living larvae ¹. The pronephros is the first kidney to develop in vertebrates and is functional in larval zebrafish ². The structure of the zebrafish pronephros is relatively simple compared to the mammalian metanephros, the third and final kidney to develop in mammals. The nephron is the working unit of the kidney, with each human kidney containing between 500,000-1,000,000 nephrons^3,4 and each mouse kidney having approximately 13,000 nephrons⁵, making it difficult to observe single nephron structure in human or mouse kidneys. The zebrafish has only two nephrons, and each zebrafish nephron contains all the major components found in the glomerulus and tubules of mice and humans⁶and similar specialized renal cell types. Compared to other vertebrate models such as Xenopus, the zebrafish nephron more closely resembles the mammalian nephron because it has a closed system⁷.

In recent years, the zebrafish genome has been sequenced, allowing the wide introduction of genetic tools, extensive mutant resources, and collections of transgenic reporter lines in zebrafish models. The zebrafish pronephros forms between 12-72 hr post fertilization (hpf) and can be visualized easily in the transparent embryos. The Wilm’s tumor protein WT1 is an essential factor for kidney development. Transgenic zebrafish lines expressing green fluorescent protein (GFP) under the control of the wt1b promoter Tg(wt1b:GFP) show GFP expression specifically located in pronephric regions in zebrafish embryos, starting from 17 hpf ⁸. Nephronophthisis (NPHP), an autosomal recessive cystic kidney disease, is caused by mutations of NPHP genes ⁹. NPHP4 knockdown by morpholino caused cyst formation in the Tg(wt1b:GFP) fish. ¹⁰ Therefore, this transgenic fish is a suitable model for observing kidney structures and cyst formation during kidney development. Importantly, the influence of modulators of kidney development can be studied using this strain in a time and labor efficient manner.

Our paper describes the use of Tg(wt1b:GFP) fish as a model to visualize kidney cyst formation after gene modulation. We used start- and splice-site anti-sense MOs to knock down the wnt5a gene in zebrafish. Wnt5a is a non-canonical secreted glycoprotein of the Wnt family that plays an important role in the development of various organs and postnatal cellular function¹¹. Wnt5a works through non-canonical Wnt pathways, including the planar cell polarity (PCP) pathway, which has been found to play a role in oriented cell division during renal tubular elongation. Wnt5a regulates the Wnt/PCP pathway by forming a complex with the receptor like tyrosine kinase (Ryk), which further transduces Wnt5a signaling by forming a complex with the VANGL planar cell polarity protein 2 (Vangl2), thereby promoting Vangl2 stability ¹². Defects in the PCP pathway can result in random cell division and cause renal cyst formation. We used the Tg(wt1b:GFP) zebrafish line to observe kidney cyst formation following wnt5a knockdown. The Tg(wt1b:GFP) zebrafish model allows live imaging and timely observation of kidney structure. After wnt5a knockdown, kidney cyst formation was found beginning at 24 hpf; at 72 hpf, cysts could be found in the glomeruli and the proximal tubules. This method could also be used to screen other genes that might cause kidney cyst formation.

Protocol

NOTE: Ethics Statement: All zebrafish experiments were approved by the Institutional Animal Care and Use Committee at the Eastern Virginia Medical School. 1. Morpholino Preparation Design and synthesize translation-blocking (AUG-) and splice-inhibiting (Splice-) anti-sense morpholino (MO) oligonucleotides for the gene of interest as per manufacturer’s instructions (Figure 1A). Please see manufacturer’s information in Table 1. NOTE: MOs are shipp…

Representative Results

Wnt5a knock down was achieved by introducing translation blocking MO (AUG-MO) or exon/intron border splice MO (splice-MO) to zebrafish embryos at the one cell stage. The AUG-MO targets the start codon and, therefore, inhibits both maternal and zygotic wnt5a message. The splice-MO targets the third splice donor site and inhibits only the zygotic transcript of wnt5a (Figure 1A). The AUG- and splice- morphants phenocopied each other with multiple defects, including curly tail down bod…

Discussion

Polycystic kidney disease (PKD) is one of the leading causes of end-stage renal disease in humans and is characterized by progressive cyst formation, renal enlargement, and abnormal tubule development ¹⁴. Autosomal dominant PKD (ADPKD) is a genetic disease in which mutation of either PKD1, encoding polycystin-1 (PC1), or PKD2, encoding polycystin-2 (PC2), results in polycystic kidneys. Many other genes, especially those encoding proteins found in the primary cilium, are thought to be involved in the developmen…

Materials

0.5% Phenol-Red	Sigma	P0290	Color indicator for injection
Morpholino	Genn Tools, LLC	(customized)	Customized designed to the gene of interest
Pre-pulled needle	Tritech Research	MINJ-PP	If large amount of needle is required, you can also purchase a needle puller and prepare the needle in the lab
T7 mMessage Kit	Ambion	1344	For in vitro transcription to make capped mRNA for rescue experiment
N-Phenylthiourea	Sigma	P7629	For prevention of melanization
Tricaine	Sigma	A5040	Also called ethyl 3-aminobenzoate, for zebrafish anesthesia
Methyl Cellulose	Sigma	M-0387	For position of zebrafish
QIAquick PCR Purification Kit	Qiagen	28104	For purification of PCR product for cap RNA synthesis.
dNTP mix	Promega	U1511	For PCR
Tag DNA Polymerase	Invitrogen	10342-053	For PCR
Equipment
NanoDrop sepctrophotometer	Thermo Scientific	ND-1000	For measure morpholino and RNA concentration
Air Compressor	Werther International, Inc.	Panther Compact 106	Air source for injection
Pico micro-injection pump	World Precision Instruments Inc	PV830 Pnematic PicoPump	Other types of microinjection system can be used.
Micro-manuplator	World Precision Instruments Inc	MMJR	Right-handed (MMJL for left handed)
Needle holder	World Precision Instruments Inc	5430-ALL	To hold needle for micromanipulation
Dumont Tweezers	Fine Surgical Tools	11253-20	For breaking off the needle tip
Dissecting microscope	Leica M205C		For observing and imaging zebrafish embryos
Fluorscence microscope	Zeiss Axio Obserer D1m		For imaging zebrafish pronephros
Capillary tube (I.D 0.15 mm)	VitroCom	CV1525Q-100	For measure the volume of each injected drop