Name: a possible zebrafish model of polycystic kidney disease: knockdown of wnt5a causes cysts in zebrafish kidneys
Uploaded: 2014-12-02T11:00:00.000+00:00
Duration: 10 min 51 s
Description: Watch this Scientific Journal Video about A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys at JoVE.com

This work was supported by the NIH (DK093625 to L.H., and DK069909 and DK047757 to J.H.L.) and the VA (Merit Award I01BX000820 to J.H.L.). We would like to thank Dr. Michael Pack and Dr. Jie He at the University of Pennsylvania Zebrafish Core for providing essential support.

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
<ol>
	<li>Design and synthesize translation-blocking (AUG-) and splice-inhibiting (Splice-) anti-sense morpholino (MO) oligonucleotides for the gene of interest as per manufacturer&#8217;s instructions (Figure&#160;1A). Please see manufacturer&#8217;s information in Table 1. 
	NOTE: MOs are shipped as lyophilized stocks in glass bottles.</li>
	<li>Add high-grade sterile water to the glass bottles to re-suspend MOs to a final concentration of 25 &#956;g/&#956;l. Make sure that the oligonucleotide is completely dissolved. If some solid remains, heat the vial containing the stock oligonucleotide solution at 65 &#176;C for 5 to 10 min and vortex briefly.</li>
	<li>Store the MO stock solution at RT. Do not store them at 4 &#176;C or -20 &#176;C because lower temperatures could cause the MO oligonucleotide to bind to the container wall. Measure the concentration of the stock solution using a spectrophotometer (please refer to Table1) each time a new MO stock solution is made.</li>
	<li>Prepare the MO working solution on the day of injection by diluting the stock solution with high-grade sterile water to the desired dose. Add 0.5% phenol-red to reach a concentration of 0.05%. For example, to make 5 &#956;l working solution with a concentration of 15 ng/nL, add 3 &#956;l MO stock solution and 0.5 &#956;l 0.5% phenol-red to 1.5 &#956;l of water. 
	NOTE: With this working concentration, each drop (500 pl) of injection contains 7.5 ng of MO and two drops contain 15 ng of MO.</li>
</ol>
2. Preparation of the Injection Apparatus
<ol>
	<li>Purchase glass pre-pulled needles (please see Table 1 for detailed information). Alternatively, pull the glass injection needle with a needle puller.</li>
	<li>Turn on the air compressor and adjust the pressure setting to 50 psi. Turn on the dissecting microscope light source and Pico micro-injection pump and adjust the settings as follows: hold pressure 20 psi, eject pressure 10 psi, period value of 2.5 and 100 &#956;sec range of gating.</li>
	<li>Load the glass pre-pulled needle with 5 &#956;l of the injection solution and place the needle in a vertical position with the tip pointing down. Wait until all the solution reaches the needle tip with no visible air bubbles. Place the needle holder in an appropriate position next to the microscope and insert the glass needle into the holder. Adjust the injection angle to 45 degrees.</li>
	<li>Bring the needle tip into view under the microscope, high off the stage, and focus on the thinnest region of the tip. Break off the needle tip with fine point tweezers.</li>
	<li>Place a ruler and a capillary tube with inner diameter of 0.15 mm side by side under the microscope; inject the solution into one end of the capillary tube to make a liquid column. Adjust the eject time to reach every 17 drops per 1 mm length liquid column, then the volume of every drop is 1 nl (drop volume = &#960; x radius2 x length (of liquid column) / count (of drops). 
	NOTE: A drop with a diameter of 0.1 mm gives a injection volume of about 500 pl (drop volume = 4/3 x &#960; x radius3).</li>
</ol>
3. Preparation of Fertilized Zebrafish Embryos and Injection with Morpholinos
<ol>
	<li>Set up Tg(wt1b:GFP) transgenic zebrafish breeding pairs according to published protocols for standard zebrafish husbandry and maintenance. Collect embryos after natural spawn, and keep them in a 10 cm Petri dish filled with E3 water (5mM NaCl, 0.17mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4). Start MO injection right away 13. Monitor embryo morphology under the microscope and make sure MO injection is at the one-cell stage, or no later than the four-cell stage.
	<ol>
		<li>Transfer the embryos to a 10 cm petri dish with wedge-shaped agar troughs. Aspirate the extra E3 water and gently press the embryos into the troughs.</li>
		<li>Manipulate the embryos with the micropipette to visualize 1-cell stage embryos under the dissecting microscope. Penetrate the chorion and then the yolk to inject one or two drops of MO into the yolk (1 drop = 500 pl). Transfer the injected embryos to a 10 cm petri dish with E3 water and incubate at 28.5 &#176;C.</li>
	</ol>
	</li>
	<li>After the injections, remove the dead embryos and record the number of injected embryos. Replace the E3 water in the dish every 24 to 48 hr.</li>
</ol>
4. Phenotype Rescue Experiments
<ol>
	<li>Select an orthologue from another species that has a different primary base pair structure (usually 3-7 base pair mismatches) and is, therefore, resistant to the MO, for rescue. For example, in this experiment, we chose mouse because the mouse Wnt5a mRNA sequence is different from the zebrafish sequence.</li>
	<li>Design a primer set with the forward primer starting at the translational site and the reverse primer at close to the end of the mRNA coding region. Add a T7 promoter sequence at the 5&#8217;- end of the forward primer sequence. In this experiment, we used the following the primer sequences; forward: 5&#8217;- taa tac gac tca cta tag gga cta tga tgc tgc tga agc tga a- 3&#8217; and reverse: 5&#8217;- tca ctt gca gac gta ctg gtc- 3&#8217;.</li>
	<li>Perform a 50 &#956;l Polymerase Chain Reaction (PCR) with the primer set (0.5 &#956;M of each primer) designed above and 0.1 &#956;g template DNA containing the mouse Wnt5a cDNA sequence with the following PCR program: 1 cycle at 95 &#176;C for 5 min; 35 cycles of 95 &#176;C (30 sec), 58.5 &#176;C (30 sec), 72 &#176;C (1 min); and final extension step at 72 &#176;C for 7 min.</li>
	<li>After finishing the cycle, run 2 &#956;l of the PCR product on 1% agarose gel to make sure the band is the right size. Purify the PCR product with a PCR purification kit according to manufacturer&#8217;s instructions. Check DNA concentration with a spectrophotometer. Store the purified PCR product at -20 &#176;C or directly use in capped RNA transcription in the next step.</li>
	<li>Synthesize in vitro capped mRNA with a capped RNA synthesis kit according to the manufacturer&#8217;s instructions. Dilute the mRNA in 20 &#956;l RNase free water and determine the concentration. Aliquot the RNA and store at -80 &#176;C.</li>
	<li>On the day of injection, prepare a working solution of the mRNA with RNase free sterile water. Note that 40 pg is generally the highest RNA dose at which nonspecific or toxic effects of the RNA do not occur. Keep the working solution on ice. Inject the embryo with MO and then the rescue mRNA, or co-inject both together. For example, if the concentration of the mRNA prepared in step 4.5 is 0.6 &#956;g/&#956;l, add 0.67 &#956;l of mRNA (0.4 &#956;g) to 4.33 &#956;l of RNase free water to make a final concentration of 0.08 &#956;g/&#956;l, then one drop (500 pl) of each injection contains 40 pg of mRNA. Prepare MO as described in step 3.</li>
</ol>
5. Preparation of Injected Embryos for Fluorescent Imaging
<ol>
	<li>After incubation at 28.5 &#176;C O/N, add 0.003% N-Phenylthiourea (PTU) to the E3 water to prevent melanization, which affects observation of pronephros structure using fluorescence microscopy. However, do not add PTU before gastrulation, because it affects early embryonic development.</li>
	<li>At 48 hpf, manually dechorionate the embryos with fine tweezers. Take images of 48 and 72 hpf embryos under a light dissection microscope. 
	NOTE: These pictures can be taken without anesthesia.</li>
	<li>Approximately 10 min prior to imaging under the fluorescence microscope, anesthetize the embryos by placing them into a 10 cm petri dish containing 160 &#956;g/ml buffered Tricaine. Wait for 5-10 min, and then lightly touch the zebrafish to confirm that they do not move and therefore the anesthesia is working.</li>
	<li>After the embryos are anesthetized, mount them in 3% methyl cellulose and orient them in a prone position under a dissecting microscope (please refer to Table 1).</li>
	<li>Observe the pronephros structure under a fluorescence microscope (please refer to Table 1) and take pictures at different magnifications (10X and 20X).</li>
</ol>

<ol>
	<li>Choi, S. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cdc42+deficiency+causes+ciliary+abnormalities+and+cystic+kidneys.">Cdc42 deficiency causes ciliary abnormalities and cystic kidneys.</a> J Am Soc Nephrol. 24 (9), 1435-1450 (2013).</li><li>Hostetter, C. L., Sullivan-Brown, J. L., Burdine, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+pronephros:+a+model+for+understanding+cystic+kidney+disease.">Zebrafish pronephros: a model for understanding cystic kidney disease.</a> Dev Dyn. 228 (3), 514-522 (2003).</li><li>Nyengaard, J. R., Bendtsen, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glomerular+number+and+size+in+relation+to+age,+kidney+weight,+and+body+surface+in+normal+man.">Glomerular number and size in relation to age, kidney weight, and body surface in normal man.</a> Anat Rec. 232 (2), 514-201 (1992).</li><li>Keller, G., Zimmer, G., Mall, G., Ritz, E., Amann, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nephron+number+in+patients+with+primary+hypertension.">Nephron number in patients with primary hypertension.</a> N Engl J Med. 348 (2), 101-108 (2003).</li><li>Cullen-McEwen, L. A., Kett, M. M., Dowling, J., Anderson, W. P., Bertram, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nephron+number,+renal+function,+and+arterial+pressure+in+aged+GDNF+heterozygous+mice.">Nephron number, renal function, and arterial pressure in aged GDNF heterozygous mice.</a> Hypertension. 41 (2), 335-340 (2003).</li><li>Gerlach, G. F., Wingert, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kidney+organogenesis+in+the+zebrafish:+insights+into+vertebrate+nephrogenesis+and+regeneration.">Kidney organogenesis in the zebrafish: insights into vertebrate nephrogenesis and regeneration.</a> Wiley Interdiscip Rev Dev Biol. 2 (5), 559-585 (2013).</li><li>Igarashi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview:+nonmammalian+organisms+for+studies+of+kidney+development+and+disease.">Overview: nonmammalian organisms for studies of kidney development and disease.</a> J Am Soc Nephrol. 16 (2), 296-298 (2005).</li><li>Zhou, W., Boucher, R. C., Bollig, F., Englert, C., Hildebrandt, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+mesonephric+development+and+regeneration+using+transgenic+zebrafish.">Characterization of mesonephric development and regeneration using transgenic zebrafish.</a> Am J Physiol Renal Physiol. 299 (5), 1040-1047 (2010).</li><li>Liu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+defect+in+a+novel+Nek-family+kinase+causes+cystic+kidney+disease+in+the+mouse+and+in+zebrafish.">A defect in a novel Nek-family kinase causes cystic kidney disease in the mouse and in zebrafish.</a> Development. 129 (24), 5839-5846 (2002).</li><li>Slanchev, K., Putz, M., Schmitt, A., Kramer-Zucker, A., Walz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nephrocystin-4+is+required+for+pronephric+duct-dependent+cloaca+formation+in+zebrafish.">Nephrocystin-4 is required for pronephric duct-dependent cloaca formation in zebrafish.</a> Hum Mol Genet. 20 (16), 3119-3128 (2011).</li><li>Huang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+Wnt5a+in+prostate+gland+development.">The role of Wnt5a in prostate gland development.</a> Dev Biol. 328 (2), 188-199 (2009).</li><li>Andre, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Wnt+coreceptor+Ryk+regulates+Wnt/planar+cell+polarity+by+modulating+the+degradation+of+the+core+planar+cell+polarity+component+Vangl2.">The Wnt coreceptor Ryk regulates Wnt/planar cell polarity by modulating the degradation of the core planar cell polarity component Vangl2.</a> J Biol Chem. 287 (53), 44518-44525 (2012).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev Dyn. 203 (3), 253-310 (1995).</li><li>Torres, V. E., Harris, P. C., Pirson, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autosomal+dominant+polycystic+kidney+disease.">Autosomal dominant polycystic kidney disease.</a> Lancet. 369 (9569), 1287-1301 (2007).</li></ol>

The authors have nothing to disclose.

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 development of renal cysts, and so far there is no ideal tool to screen these genes. We describe a fast and easy way to observe kidney cyst formation after gene knockdown in zebrafish. This method can likely be used to screen other candidate PKD genes.
One of the strengths of this technique is its simplicity. In Tg(wt1b:GFP) zebrafish, the expression of GFP is in glomeruli, tubules and ducts, which makes it a particularly suitable model to study the entire pronephric kidney structure. The Tg(wt1b:GFP) zebrafish allows direct visualization of pronephric structure and kidney cyst formation after gene knock down. It was suspected that Wnt5a knockdown could cause PCP pathway defects and kidney cyst formation in mice. However, global knockout of Wnt5a is postnatal lethal in mice and could not be used to observe kidney cyst formation after birth. By using the method described here, in a short period of time it was determined that wnt5a knockdown in zebrafish resulted in kidney cyst formation. With this result, the next step will be to generate kidney specific Wnt5a knockout mice.
This technique also has a few limitations. It is difficult to estimate the efficacy of MOs without a good antibody, and to rule out the possibility that the MO might inhibit the function of an irrelevant gene to cause the phenotype. Extensive control experiments are required to overcome these problems. Two distinct MOs, the AUG-MO and the splice- MO were used to evaluate wnt5a knockdown. Both morphants phenocopied each other, and 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 &#8220;off-target&#8221; effects of the MO.
Our model demonstrates that wnt5a knockdown causes kidney cyst formation in zebrafish embryos. This methodology can be employed to test many other genes that are suspected to cause renal cyst formation, as it is simple and less time consuming. Once it is demonstrated that the gene mutation results in kidney cyst formation in zebrafish, further experiments can be conducted to investigate the detailed mechanisms of kidney cyst formation. In this experiment, since the Wnt5a global knockout mice are postnatal lethal and cannot be used to observe kidney cyst formation after birth, the next step will be to generate kidney-specific Wnt5a knockout mice using the Cre-lox system.

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 nephrons3,4 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&#8217;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.

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

a possible zebrafish model of polycystic kidney disease: knockdown of wnt5a causes cysts in zebrafish kidneys

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 &#8220;off-target&#8221; effects of the morpholino. This work supports the validity of using a zebrafish model to study wnt5a function in the kidney.

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&#160;1A). The AUG- and splice- morphants phenocopied each other with multiple defects, including curly tail down body axis and pericardial edema (Figure&#160;1B). Mouse Wnt5a mRNA partially rescues the morphant phenotype (Figure&#160;4B), demonstrating that the phenotype is not due to off-target effects. The Tg(wt1b:GFP) transgenic zebrafish, which expresses GFP driven by the wt1b promoter so that the GFP recapitulates endogenous expression of wt1b, was used to outline the pronephric structure. The Tg(wt1b:GFP) fish showed glomerular cyst formation after wnt5a knockdown at 48 hpf, whereas control embryos injected with phenol-red alone displayed normal pronephric development, forming a fused glomerulus in a &#8220;U&#8221; shape with tubules that extend laterally (Figure&#160;2). At 72 hpf, glomerular structure further developed with easily visualized vascular loops, with both AUG- and splice-MOs developed cysts in the glomeruli and proximal tubules (Figure&#160;3). H&#38;E staining of the transverse histological sections of 72 hpf morphant embryos revealed cyst formation, disrupted glomerular structures and dilated renal tubules (Figure&#160;4A).
<img alt="Figure 1" src="/files/ftp_upload/52156/52156fig1highres.jpg" /> 
Figure 1: Morpholino design and light microscopic appearance of zebrafish embryos at 72 hr post fertilization (hpf) following injection with phenol-red control, AUG-MO, or splice-MO. (A)&#160;Diagram showing the zebrafish wnt5a gene, the coding region, and the regions targeted by the AUG- and splice- MO. The wnt5a AUG-MO was designed to target the ATG start codon to block protein translation; the splice-MO was designed to target the border of exons 3 and 4. Splice-MO oligo directed against any splice junction is expected to generate either a complete or partial single exon deletion or a complete or partial single intron insertion. We chose to knock down wnt5a with both an AUG-MO, which affects maternal and zygotic message, and a splice-MO, which affects only the zygotic transcript, in order to enhance specificity. (B)&#160;The embryo injected with phenol-red (injection control) showed normal body curvature, length and pericardium. The embryo injected with the AUG-MO showed downward curly tail structure and pericardial edema, and the embryo injected with the splice-MO phenocopied the appearance of the embryo injected with AUG-MO. Bar = 2mm.
<img alt="Figure 2" src="/files/ftp_upload/52156/52156fig2highres.jpg" /> 
Figure 2: Fluorescence microscopic appearance of Tg (wt1b:GFP) zebrafish embryos at 48 hpf after injection with phenol-red control, wnt5a AUG-MO or wnt5a Splice-MO. In the phenol-red injection control, pronephros development was complete at 48 hpf. Two fused glomeruli and the proximal tubules can be seen. Cyst formation can be observed in glomeruli in both the AUG- and splice-MO embryos at 48 hpf by the end of zebrafish pronephros development (bar = 100 &#956;m).
<img alt="Figure 3" src="/files/ftp_upload/52156/52156fig3highres.jpg" /> 
Figure 3: Fluorescence microscopic appearance of Tg(wt1b:GFP) zebrafish embryos at 72 hpf after injection with phenol-red control, wnt5a AUG-MO or wnt5a splice-MO. At 72 hpf, glomerular structure can be clearly visualized. In the phenol-red injection control, two fused glomeruli and vascular loops can be observed. Wnt5a knockdown resulted in cyst formation in all the nephron structures that could be visualized, including the glomeruli and proximal tubular region (bar = 100 &#956;m).
<img alt="Figure 4" src="/files/ftp_upload/52156/52156fig4highres.jpg" /> 
Figure 4: (A)&#160;H&#38;E staining of the transverse histological sections of 72&#160;hpf morphant embryos revealed cyst formation, disrupted glomerular structures and dilated renal tubules. Arrow: pronephric glomeruli; arrowhead: dilated renal tubule. Bar = 20 &#956;m. (B)&#160;The AUG-MO phenotype could be partially rescued with mouse Wnt5a, resistant to the MO due to a difference in primary base pair structure, thereby demonstrating that the phenotype was not due to off-target effects. The difference was statistically significant at p &#60;0.05 (* P &#60;0.05 vs Phenol-red group; # P &#60;0.05 vs AUG-MO 15 ng group).

Watch this Scientific Journal Video about A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys at JoVE.com

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

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.

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

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 ...

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Nanyang Technological University

Research

JoVE Journal

Medicine

11.1K Views.  Eastern Virginia Medical School. 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. 

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

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method described here allows time-lapse analysis of normal and impaired kidney development in zebrafish embryos by using a fluorescence dissecting microscope equipped for structured illumination and z-stack acquisition. To visualize nephrogenesis, transgenic zebrafish (Tg(wt1b:GFP)) with fluorescently labeled kidney structures were used. Renal defects were triggered by injection of an antisense morpholino oligonucleotide against the Wilms tumor gene wt1a, a factor known to be crucial for kidney development.
The advantage of the experimental setup is the combination of a zoom microscope with simple strategies for re-adjusting movements in x, y or z direction without additional equipment. To circumvent focal drift that is induced by temperature variations and mechanical vibrations, an autofocus strategy was applied instead of utilizing a usually required environmental chamber. In order to re-adjust the positional changes due to a xy-drift, imaging chambers with imprinted relocation grids were employed.
In comparison to more complex setups for time-lapse recording with optical sectioning such as confocal laser scanning&#160;or light sheet microscopes, a zoom microscope is easy to handle. Besides, it offers dissecting microscope-specific benefits such as high depth of field and an extended working distance.
The method to study organogenesis presented here can also be used with fluorescence stereo microscopes not capable of optical sectioning. Although limited for high-throughput, this technique offers an alternative to more complex equipment that is normally used for time-lapse recording of developing tissues and organ dynamics.

The method described here allows time-lapse analysis of organ development in zebrafish embryos by using a fluorescence dissecting microscope capable of performing optical sectioning and simple strategies of readjustment to correct focal and planar drift.

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

Developmental Biology

Precise Cellular Ablation Approach for Modeling Acute Kidney Injury in Developing Zebrafish

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore adequate kidney function after supportive treatment. However, a better understanding of how nephron cell death and repair occur on the cellular level is required to minimize cell death and to enhance the regenerative process. The zebrafish pronephros is a good model system to accomplish this goal because it contains anatomical segments that are similar to the mammalian nephron. Previously, the most common model used to study kidney injury in fish was the pharmacological gentamicin model. However, this model does not allow for precise spatiotemporal control of injury, and hence it is difficult to study cellular and molecular processes involved in kidney repair. To overcome this limitation, this work presents a method through which, in contrast to the gentamicin approach, a specific Green Fuorescent Protein (GFP)-expressing nephron segment can be photoablated using a violet laser light (405 nm). This novel model of AKI provides many advantages that other methods of epithelial injury lack. Its main advantages are the ability to &#34;dial&#34; the level of injury and the precise spatiotemporal control in the robust in vivo animal model. This new method has the potential to significantly advance the level of understanding of kidney injury and repair mechanisms.

This work presents a new in vivo model of segmental kidney injury using kidney GFP transgenic zebrafish. The model allows for the induction of the targeted ablation of kidney epithelial cells to show the cellular mechanisms of nephron injury and repair.

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore ...

Shifting Zebrafish Lethal Skeletal Mutant Penetrance by Progeny Testing

Zebrafish mutant phenotypes are often incompletely penetrant, only manifesting in some mutants. Interesting phenotypes that inconsistently appear can be difficult to study, and can lead to confounding results. The protocol described here is a straightforward breeding paradigm to increase and decrease penetrance in lethal zebrafish skeletal mutants. Because lethal mutants cannot be selectively bred directly, the classic selective breeding strategy of progeny testing is employed. This method also includes protocols for Kompetitive Allele Specific PCR (KASP) genotyping zebrafish and staining larval zebrafish cartilage and bone. Applying the husbandry strategy described here can increase the penetrance of an interesting skeletal phenotype enabling more reproducible results in downstream applications. In addition, decreasing the mutant penetrance through this selective breeding strategy can reveal the developmental processes that most crucially require the function of the mutated gene. While the skeleton is specifically considered here, we propose that this methodology will be useful for all zebrafish mutant lines.

The goal of this protocol is to alter the penetrance of lethal skeletal mutant phenotypes in zebrafish by selective breeding. Lethal mutants cannot be grown to adulthood and bred themselves, therefore this protocol describes a method for tracking and selecting penetrance through multiple generations by progeny testing.

Zebrafish mutant phenotypes are often incompletely penetrant, only manifesting in some mutants. Interesting phenotypes that inconsistently appear can be ...

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and optical transparency. In particular, the zebrafish embryo has become an important organism to study vertebrate kidney organogenesis as well as multiciliated cell (MCC) development. To visualize MCCs in the embryonic zebrafish kidney, we have developed a combined protocol of whole-mount fluorescent in situ hybridization (FISH) and whole mount immunofluorescence (IF) that enables high resolution imaging. This manuscript describes our technique for co-localizing RNA transcripts and protein as a tool to better understand the regulation of developmental programs through the expression of various lineage factors.

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

Cilia development is vital to proper organogenesis. This protocol describes an optimized method to label and visualize ciliated cells of the zebrafish.

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and ...

Behavioral And Physiological Analysis In A Zebrafish Model Of Epilepsy

Epilepsy represents one of the most common neurological disorders, affecting an estimated 50 million people worldwide. Recent advances in genetic research have uncovered a large spectrum of genes implicated in various forms of epilepsy, highlighting the heterogeneous nature of this disorder. Appropriate animal models are essential for investigating the pathological mechanisms triggered by genetic mutations implicated in epilepsy and for developing specialized, targeted therapies. In recent years, zebrafish has emerged as a valuable vertebrate organism for modeling epilepsies, with the use of both genetic manipulation and exposure to known epileptogenic drugs, such as pentylenetetrazole (PTZ), to identify novel anti-epileptic therapeutics. Deleterious mutations in the mTOR regulator DEPDC5 have been associated with various forms of focal epilepsies and knock-down of the zebrafish orthologue causes hyperactivity associated with spontaneous seizure-like episodes, as well as enhanced electrographic activity and characteristic turn wheel swimming. Here, we described the method involved in generating the DEPDC5 loss-of-function model and illustrate the protocol for assessing motor activity at 28 and 48 h post fertilization (hpf), as well as a method for recording field activity in the zebrafish optic tectum. An illustration of the effect of the epileptogenic drug PTZ on neuronal activity over time is also provided.

Here, we present a protocol for the development and the characterization of a zebrafish model of epilepsy resulting from the transient inhibition of the DEPDC5 gene.

Epilepsy represents one of the most common neurological disorders, affecting an estimated 50 million people worldwide. Recent advances in genetic research ...

Neuroscience

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

Biology

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants.

Visualization of experimental data has become a key element in presenting results to the scientific community. Generation of live time-lapse recording of growing embryos contributes to better presentation and understanding of complex developmental processes. This protocol is a step-by-step guide to cell labeling via photoconversion of kaede protein in zebrafish.

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, ...

Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.

The zebrafish adult kidney is an excellent system for renal regeneration and disease studies. An essential aspect of such research is the assessment of nephron structure and function. This protocol describes several methodologies that can be implemented to assess nephron tubule composition and to evaluate renal reabsorption.

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys ...

Acute Kidney Injury Model Induced by Cisplatin in Adult Zebrafish

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney due to its low molecular weight. Such accumulation causes the death of tubular cells and can induce the development of Acute Kidney Injury (AKI), which is characterized by a quick decrease in kidney function, tissue damage, and immune cells infiltration. If administered in specific doses cisplatin can be a useful tool as an AKI inducer in animal models. The zebrafish has appeared as an interesting model to study renal function, kidney regeneration, and injury, as renal structures conserve functional similarities with mammals. Adult zebrafish injected with cisplatin shows decreased survival, kidney cell death, and increased inflammation markers after 24 h post-injection (hpi). In this model, immune cells infiltration and cell death can be assessed by flow cytometry and TUNEL assay. This protocol describes the procedures to induce AKI in adult zebrafish by intraperitoneal cisplatin injection and subsequently demonstrates how to collect the renal tissue for flow cytometry processing and cell death TUNEL assay. These techniques will be useful to understand the effects of cisplatin as a nephrotoxic agent and will contribute to the expansion of AKI models in adult zebrafish. This model can also be used to study kidney regeneration, in the search for compounds that treat or prevent kidney damage and to study inflammation in AKI. Moreover, the methods used in this protocol will improve the characterization of tissue damage and inflammation, which are therapeutic targets in kidney-associated comorbidities.

This protocol describes the procedures to induce Acute Kidney Injury (AKI) in adult zebrafish using cisplatin as a nephrotoxic agent. We detailed the steps to evaluate the reproducibility of the technique and two techniques to analyze inflammation and cell death in the renal tissue, flow cytometry and TUNEL, respectively.

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney ...

Evaluation of Zebrafish Kidney Function Using a Fluorescent Clearance Assay

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish kidney develops and functions in almost the same way as humans. A major difference in the construction of the human kidney is the presence of millions of nephrons compared to the zebrafish that has only two. However, simplifying such a complex system into basic functional units has aided our understanding of how the kidney develops and operates. In zebrafish, the midline located glomerulus is responsible for the initial blood filtration into two pronephric tubules that diverge to run bilaterally down the embryonic axis before fusing to each other at the cloaca. The pronephric tubules are heavily populated by motile cilia that facilitate the movement of filtrate along the segmented tubule, allowing the exchange of various solutes before finally exiting via the cloaca2-4. Many genes responsible for CKD, including those related to ciliogenesis, have been studied in zebrafish5. However, a major draw back has been the difficulty in evaluating zebrafish kidney function after genetic manipulation. Traditional assays to measure kidney dysfunction in humans have proved non translational to zebrafish, mainly due to their aquatic environment and small size. For example, it is not physically possible to extract blood from embryonic staged fish for analysis of urea and creatinine content, as they are too small. In addition, zebrafish do not produce enough urine for testing on a simple proteinuria &#8216;dipstick&#8217;, which is often performed during initial patient examinations. We describe a fluorescent assay that utilizes the optical transparency of the zebrafish to quantitatively monitor the clearance of a fluorescent dye, over time, from the vasculature and out through the kidney, to give a read out of renal function1,6-9.

The zebrafish is a popular tool to model chronic kidney disease (CKD). However, their small size makes it impossible to evaluate renal function using traditional methods. We describe a fluorescent dye kidney clearance assay1 that allows quantitative analysis of zebrafish kidney function in CKD.

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish ...

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

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