En mulig Zebrafisk Model af polycystisk nyresygdom: Knockdown af<em> Wnt5a</em> Årsager cyster i zebrafisk Nyrer
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
Zebrafisk (Danio rerio) embryoner er ofte blevet brugt som model for at studere nyre udvikling og polycystisk nyresygdom. Der er mange fordele ved at bruge zebrafisk som en dyremodel: muligheden for at studere genetiske interaktioner, evnen til at anvende antisense morpholinos (MO) for protein knockdown, mulighed for hurtigt at analysere et stort antal embryoner, og den lethed visning orgel fænotyper i levende larver 1. De pronephros er den første nyre at udvikle sig i hvirveldyr og er funktionel i larvernes zebrafisk 2. Strukturen af zebrafisk pronephros er relativt simpel i forhold til de mammale metanephros, at den tredje og sidste nyre udvikles i pattedyr. Den nephron er arbejdsmiljøet enhed i nyrerne, med hvert menneske nyre indeholder mellem 500.000-1.000.000 nefroner 3,4 og hver mus nyre har cirka 13.000 nefroner 5, hvilket gør det vanskeligt at observere enkelt nephron struktur in humane eller muse nyrer. Zebrafisken har kun to nefroner, og hver zebrafisk nephron indeholder alle de vigtige komponenter fundet i glomerulus og tubuli i mus og mennesker 6 og lignende specialiserede renale celletyper. Sammenlignet med andre modeller hvirveldyr såsom Xenopus, zebrafisk nephron mere ligner pattedyrs nefron, fordi det har et lukket system 7.
I de seneste år har zebrafisk genom blevet sekventeret, så den brede introduktion af genetiske værktøjer, omfattende mutant ressourcer og samlinger af transgene reporter linier i zebrafisk modeller. Zebrafisken pronephros dannes mellem 12-72 timer efter befrugtning (HPF) og kan visualiseres let i de transparente embryoner. Den Wilms tumor protein WT1 er en væsentlig faktor for nyre udvikling. Transgene zebrafisk, der udtrykker grønt fluorescerende protein (GFP) under kontrol af den wt1b promotoren Tg (wt1b: GFP) viser GFP EXPREfission specifikt placeret i pronephric regioner i zebrafisk embryoner, startende fra 17 HPF 8. Nephronophthisis (NPHP), en autosomal recessiv cystisk nyresygdom, er forårsaget af mutationer i NPHP gener 9. NPHP4 knockdown af morpholino forårsagede cyste dannelse i Tg (wt1b: GFP). Fisk 10 Derfor er denne transgene fisk er en egnet model til at observere nyre strukturer og cyste dannelse under nyre udvikling. Vigtigere, kan indflydelsen af modulatorer af nyre udvikling undersøges ved hjælp af denne stamme i en tid og arbejdskraft effektiv måde.
Vores papir beskriver brugen af Tg (wt1b: GFP) fisk som en model til at visualisere nyre cyste dannelse efter gen graduering. Vi brugte start- og splejse-site anti-sense Mos at banke ned wnt5a genet i zebrafisk. Wnt5a er en ikke-kanoniske secerneret glycoprotein af Wnt familie, som spiller en vigtig rolle i udviklingen af forskellige organer og postnatal cellulærefunktion 11. Wnt5a virker gennem ikke-kanoniske Wnt veje, herunder plane cellepolaritet (PCP) pathway, som har vist sig at spille en rolle i orienteret celledeling under tubulær forlængelse. Wnt5a regulerer Wnt / PCP pathway ved at danne et kompleks med receptoren lignende tyrosinkinase (Ryk), hvilket yderligere transducerer Wnt5a signalering ved at danne et kompleks med VANGL plane cellepolaritet protein 2 (Vangl2) og derved fremme Vangl2 stabilitet 12. Defekter i PCP pathway kan resultere i tilfældig celledeling og forårsage nyrecyste formation. Vi brugte Tg (wt1b: GFP) zebrafisk linje at observere nyre cyste dannelse efter wnt5a knockdown. Tg (wt1b: GFP) zebrafisk model giver mulighed direkte billedvisning og rettidig observation af nyre struktur. Efter wnt5a knockdown blev nyre cyste dannelse fundet begyndende ved 24 HPF; ved 72 HPF kunne cyster findes i glomeruli og den proksimale tubuli. Denne metode kan også anvendes til at screen andre gener, der kan forårsage nyre cyste dannelse.
Protocol
Representative Results
Discussion
Polycystisk nyresygdom (PKD) er en af de førende årsager til end-stage renal sygdom hos mennesker og er kendetegnet ved progressiv cystedannelse, renal udvidelsen, og unormal tubulus udvikling 14. Autosomal dominant PKD (ADPKD) er en genetisk sygdom, hvor mutation af enten pKD1, der koder polycystin-1 (PC1) eller PKD2 kodende polycystin-2 (PC2), resulterer i polycystiske nyrerne. Mange andre gener, især dem, der koder for proteiner, der findes i den primære cilium, menes at være involveret i udviklingen …
Disclosures
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev støttet af NIH (DK093625 at LH og DK069909 og DK047757 til JHL) og VA (Merit Award I01BX000820 til JHL). Vi vil gerne takke Dr. Michael Pack og Dr. Jie Han ved University of Pennsylvania zebrafisk Core for at yde væsentlig støtte.
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 |
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