Name: shifting zebrafish lethal skeletal mutant penetrance by progeny testing
Uploaded: 2017-09-01T15:00:00
Description: Watch this Scientific Journal Video about Shifting Zebrafish Lethal Skeletal Mutant Penetrance by Progeny Testing at JoVE.com

We would like to thank Chuck Kimmel for guidance, John Dowd for help in developing this breeding strategy, Macie Walker for her work in perfecting the skeletal stain, and Charline Walker and Bonnie Ullmann for helpful zebrafish advice. This work was supported by K99/R00 DE024190 to JTN.

All experiments described in this protocol were completed in accordance and compliance with the University of Colorado and the University of Oregon Institutional Animal Care and Use Committees (IACUC).
1. Preparing the Unselected Starting Stock
<ol>
	<li>Identify heterozygous carriers of the mutant allele of interest by fin clip11 and genotyping a stock of full-sibling animals by a method of choice, such as KASP12,1...

<ol>
	<li>Nichols, J. T., 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=Ligament+versus+bone+cell+identity+in+the+zebrafish+hyoid+skeleton+is+regulated+by+mef2ca.">Ligament versus bone cell identity in the zebrafish hyoid skeleton is regulated by mef2ca.</a> Development. 143 (23), 4430-4440 (2016).</li><li>Sheehan-Rooney, K., Swartz, M. E., Zhao, F., Liu, D., Eberhart, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ahsa1+and+Hsp90+activity+confers+more+severe+craniofacial+phenotypes+in+a+zebrafish+model+of+hypoparathyroidism,+sensorineural+deafness+and+renal+dysplasia+(HDR).">Ahsa1 and Hsp90 activity confers more severe craniofacial phenotypes in a zebrafish model of hypoparathyroidism, sensorineural deafness and renal dysplasia (HDR).</a> Dis Model Mech. 6 (5), 1285-1291 (2013).</li><li>Cox, S. G., 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=An+essential+role+of+variant+histone+H3.3+for+ectomesenchyme+potential+of+the+cranial+neural+crest.">An essential role of variant histone H3.3 for ectomesenchyme potential of the cranial neural crest.</a> PLoS Genet. 8 (9), e1002938 (2012).</li><li>DeLaurier, A., 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=Role+of+mef2ca+in+developmental+buffering+of+the+zebrafish+larval+hyoid+dermal+skeleton.">Role of mef2ca in developmental buffering of the zebrafish larval hyoid dermal skeleton.</a> Dev Biol. 385 (2), 189-199 (2014).</li><li>McGurk, P. D., Ben Lovely, C., Eberhart, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+Craniofacial+Morphogenesis+in+Zebrafish+Using+4D+Confocal+Microscopy.">Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy.</a> J Vis Exp. (83), e51190 (2014).</li><li>Kemp, H. A., Carmany-Rampey, A., Moens, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generating+chimeric+zebrafish+embryos+by+transplantation.">Generating chimeric zebrafish embryos by transplantation.</a> J Vis Exp. (29), e1394 (2009).</li><li>Lush, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progeny+test+and+individual+performance+as+indicators+of+an+animal's+breeding+value.">Progeny test and individual performance as indicators of an animal's breeding value.</a> J Dairy Science. 18 (1), 1-19 (1935).</li><li>Lerner, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Population Genetics and Animal Improvement. , (1950).</li><li>Shinya, M., Sakai, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+Highly+Homogeneous+Strains+of+Zebrafish+Through+Full+Sib-Pair+Mating.">Generation of Highly Homogeneous Strains of Zebrafish Through Full Sib-Pair Mating.</a> G3. 1 (5), 377-386 (2011).</li><li>Neff, M. M., Neff, J. D., Chory, J., Pepper, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=dCAPS,+a+simple+technique+for+the+genetic+analysis+of+single+nucleotide+polymorphisms:+experimental+applications+in+Arabidopsis+thaliana+genetics.">dCAPS, a simple technique for the genetic analysis of single nucleotide polymorphisms: experimental applications in Arabidopsis thaliana genetics.</a> Plant J. 14 (3), 387-392 (1998).</li><li>Xing, L. Y., Quist, T. S., Stevenson, T. J., Dahlem, T. J., Bonkowsky, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+Efficient+Zebrafish+Genotyping+Using+PCR+with+High-resolution+Melt+Analysis.">Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis.</a> Jove-Journal of Visualized Experiments. (84), e51138 (2014).</li><li>He, C., Holme, J., Anthony, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SNP+genotyping:+the+KASP+assay.">SNP genotyping: the KASP assay.</a> Methods Mol Biol. 1145, 75-86 (2014).</li><li>Semagn, K., Babu, R., Hearne, S., Olsen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+nucleotide+polymorphism+genotyping+using+Kompetitive+Allele+Specific+PCR+(KASP):+overview+of+the+technology+and+its+application+in+crop+improvement.">Single nucleotide polymorphism genotyping using Kompetitive Allele Specific PCR (KASP): overview of the technology and its application in crop improvement.</a> Molecular Breeding. 33 (1), 1-14 (2014).</li><li>Yuan, J., Wen, Z., Gu, C., Wang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+of+high+throughput+and+cost+effective+SNP+genotyping+platforms+in+soybean.">Introduction of high throughput and cost effective SNP genotyping platforms in soybean.</a> Plant Genet Genomics Biotech. 2 (1), 90-94 (2014).</li><li>Walker, M. B., Kimmel, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+two-color+acid-free+cartilage+and+bone+stain+for+zebrafish+larvae.">A two-color acid-free cartilage and bone stain for zebrafish larvae.</a> Biotech Histochem. 82 (1), 23-28 (2007).</li><li>Nasiadka, A., Clark, M. 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+breeding+in+the+laboratory+environment.">Zebrafish breeding in the laboratory environment.</a> ILAR J. 53 (2), 161-168 (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>Schilling, T. F., 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=Jaw+and+branchial+arch+mutants+in+zebrafish+I:+branchial+arches.">Jaw and branchial arch mutants in zebrafish I: branchial arches.</a> Development. 123, 329-344 (1996).</li><li>McCune, A. R., Carlson, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Twenty+ways+to+lose+your+bladder:+common+natural+mutants+in+zebrafish+and+widespread+convergence+of+swim+bladder+loss+among+teleost+fishes.">Twenty ways to lose your bladder: common natural mutants in zebrafish and widespread convergence of swim bladder loss among teleost fishes.</a> Evol Dev. 6 (4), 246-259 (2004).</li><li>Mrakov&#269;i&#269;, M., Haley, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inbreeding+depression+in+the+Zebra+fish+Brachydanio+rerio+(Hamilton+Buchanan).">Inbreeding depression in the Zebra fish Brachydanio rerio (Hamilton Buchanan).</a> J Fish Biol. 15 (3), 323-327 (1979).</li><li>Charlesworth, D., Willis, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+inbreeding+depression.">The genetics of inbreeding depression.</a> Nat Rev Genet. 10 (11), 783-796 (2009).</li><li>McCune, A. R., 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+low+genomic+number+of+recessive+lethals+in+natural+populations+of+bluefin+killifish+and+zebrafish.">A low genomic number of recessive lethals in natural populations of bluefin killifish and zebrafish.</a> Science. 296 (5577), 2398-2401 (2002).</li><li>Streisinger, G., Walker, C., Dower, N., Knauber, D., Singer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+clones+of+homozygous+diploid+zebra+fish+(Brachydanio+rerio).">Production of clones of homozygous diploid zebra fish (Brachydanio rerio).</a> Nature. 291 (5813), 293-296 (1981).</li><li>Dreosti, E., Lopes, G., Kampff, A. R., Wilson, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+social+behavior+in+young+zebrafish.">Development of social behavior in young zebrafish.</a> Front Neural Circuits. 9, 39 (2015).</li><li>Eames, B. F., 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=FishFace:+interactive+atlas+of+zebrafish+craniofacial+development+at+cellular+resolution.">FishFace: interactive atlas of zebrafish craniofacial development at cellular resolution.</a> BMC Dev Bio. 13 (1), 23 (2013).</li><li>Nichols, J. T., Pan, L., Moens, C. B., Kimmel, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=barx1+represses+joints+and+promotes+cartilage+in+the+craniofacial+skeleton.">barx1 represses joints and promotes cartilage in the craniofacial skeleton.</a> Development. 140 (13), 2765-2775 (2013).</li><li>Sasaki, M. M., Nichols, J. T., Kimmel, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=edn1+and+hand2+Interact+in+early+regulation+of+pharyngeal+arch+outgrowth+during+zebrafish+development.">edn1 and hand2 Interact in early regulation of pharyngeal arch outgrowth during zebrafish development.</a> PLoS One. 8 (6), e67522 (2013).</li></ol>

Selective Breeding Unveils Subtleties of Gene Function
Shifting mutant phenotypes to be either more or less severe by selective breeding is a straightforward way to gain new insights into gene function. When compared with standard methods of unselected breeding, the protocol presented here can yield a much more complete understanding of mutant phenotypes. Specifically, by generating strains that are severe, the full breadth of mutant phenotypes may be revealed, including some ...

The zebrafish is a powerful model system for understanding skeletal development. With mutant zebrafish strains, biologists can decipher gene function during skeletogenesis. However, zebrafish skeletal mutant phenotypes can present with variable penetrance1,2,3,4 which can hinder developmental and genetic analyses. The purpose of this method is threefold. First, generating zebrafish mutant lines which consistently produce severe phenotypes enables downstream developmental studies like time-lapse recording5 and transplantation6. These sorts of studies can be crippled by attempting to study phenotypes that only manifest inconsistently. Second, inbreeding zebrafish strains can decrease genetic background variation, thus promoting experimental consistency and reproducibility. For example, performing all in situ hybridization analyses on one selectively inbred strain can reduce confounding variability and strengthen conclusions. Third, generating severe and mild strains will reveal the entire phenotypic series that can result from a particular mutation.
At first glance, selective breeding of lethal mutants seems impossible. How can one breed for penetrance when the animals that are scored for selection are dead? Fortunately, methods for selective breeding by family selection, specifically progeny testing, have demonstrated effectiveness in livestock breeding programs for many years7,8. These programs are mainly used for selective breeding for traits that are only present in one sex, like milk production in cows or egg production in hens. The males of these species cannot be scored directly, but their progeny are scored and a value is then assigned to the parents. Borrowing from this strategy, the protocol presented here involves scoring the fixed and stained mutant offspring from a pair of zebrafish that are heterozygous for a mutant gene of interest. The penetrance of a phenotype in the homozygous lethal mutant offspring is assigned to the parents when deciding which individuals will produce the next generation in the line. We find that this method is an effective means of shifting penetrance in zebrafish lethal skeletal mutants1.
Similar to other studies, this selective breeding protocol takes under consideration criteria like clutch size, survival of offspring, normal development of embryos, and sex ratio9. However, these factors are all considered in the context of a mutant background with the objective of shifting the mutant penetrance. Therefore, this protocol extends previous selective breeding paradigms by offering a method to strengthen developmental mutant analyses as well as increase background homogeneity.
This protocol requires extensive genotyping, so it is important to develop a reliable, rapid genotyping protocol in advance. There are many genotyping protocols available10,11, however we find the KASP genotyping12,13,14 is faster, more cost efficient, and more reliable than methods based on restriction enzyme cleavage of amplified sequences10. Therefore, we include a KASP protocol in this work. Additionally, we focus on skeletal mutant phenotypes in this protocol and include a procedure for Alcian Blue/Alizarin Red staining modified from previous protocols15.
The method described here is a straightforward strategy for shifting lethal mutant penetrance upward or downward. While this protocol focuses on skeletal mutant phenotypes, we believe it will be a useful strategy for husbandry of all mutant zebrafish lines. In fact, the utility of this breeding strategy likely extends beyond zebrafish. We predict that this protocol can be modified to shift penetrance in a broad range of organisms. Shifting lethal penetrance by progeny testing can help push forward the progress of any developmental geneticist.

<table border="0" cellpadding="0" cellspacing="0" width="627">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Paraformaldehyde, pelleted, solid</td>
			<td style="width:125px;">Ted Pella Co.</td>
			<td style="width:119px;">18501</td>
			<td style="width:167px;">Pelleted PFA is a safer alternative to powdered PFA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnesium Chloride, solid</td>
			<td style="width:125px;">Acros Organics</td>
			<td>223210010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10x PBS, Aqueous</td>
			<td style="width:125px;">Fisher</td>
			<td style="width:119px;">BP3994</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">190 proof Ethanol</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Alcian Blue, solid</td>
			<td style="width:125px;">Anatech Ltd.</td>
			<td style="width:119px;">867</td>
			<td>Must be from Anatech</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Alizarin Red, solid</td>
			<td style="width:125px;">Sigma</td>
			<td style="width:119px;">A5533-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycerol, liquid</td>
			<td style="width:125px;">Fisher</td>
			<td style="width:119px;">BP229 1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hydrogen peroxide, liquid</td>
			<td style="width:125px;">Fisher</td>
			<td style="width:119px;">BP263500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Potassium hydroxide,&#160; solid</td>
			<td style="width:125px;">Fisher</td>
			<td style="width:119px;">P250 500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">StepOnePlus Real-time PCR Machine</td>
			<td style="width:125px;">Applied Biosystems</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">MicroAmp Fast Optical 96-well Reaction Plate with Barcode (0.1mL)</td>
			<td style="width:125px;">Applied Biosystems</td>
			<td style="width:119px;">4346906</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microseal &#39;B&#39; seal</td>
			<td style="width:125px;">BioRad</td>
			<td style="width:119px;">MSB1001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">KASP Master Mix, High ROX</td>
			<td style="width:125px;">LGC</td>
			<td>KBS-1016-022</td>
			<td>https://www.lgcgroup.com/products/kasp-genotyping-chemistry/#.WOPX41UrKUk</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">KASP By Design Primer Mix</td>
			<td style="width:125px;">LGC</td>
			<td style="width:119px;">KBS-2100-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris HCl, solid</td>
			<td style="width:125px;">Fisher</td>
			<td style="width:119px;">BP153 500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">potassium chloride, solid</td>
			<td style="width:125px;">Fisher</td>
			<td style="width:119px;">BP366 500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tween-20, liquid</td>
			<td style="width:125px;">Fisher</td>
			<td style="width:119px;">BP337 100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nonidet P40</td>
			<td style="width:125px;">ThermoFisher</td>
			<td>28324</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tricaine-S</td>
			<td style="width:125px;">Western Chemicals</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Proteinase K</td>
			<td style="width:125px;">Fisher</td>
			<td style="width:119px;">BP1700 100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">T100 Thermal Cycler</td>
			<td style="width:125px;">BioRad</td>
			<td>1861096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Controlled Drop Pasteur Pipets</td>
			<td style="width:125px;">Fisher</td>
			<td>13-678-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nanodrop</td>
			<td style="width:125px;">ThermoFisher</td>
			<td style="width:119px;"></td>
			<td>for DNA quantitation</td>
		</tr>
	</tbody>
</table>

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.

This protocol is a long-term husbandry technique useful for understanding zebrafish skeletal mutants (Figure 1). Selective breeding by progeny testing should yield a shift in overall penetrance both downward and upward in a few generations (Figure 2). In our previous work, two rounds of selective breeding drove the average penetrance downward from 17% to 3%1. Similarly, in our upward line, we shifted the a...

Watch this Scientific Journal Video about Shifting Zebrafish Lethal Skeletal Mutant Penetrance by Progeny Testing at JoVE.com

Shifting Zebrafish Lethal Skeletal Mutant Penetrance by Progeny Testing

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 ...

The overall goal of this breeding strategy is to manipulate mutant penetrance upward or downward. This selective breeding method enables a more complete understanding of mutant phenotypes. By generating high and low penetrance mutant strains, the entire phenotypic series of a mutation can be understood.Most zebrafish scale where mutants are recessive lethal, so homozygous mutants die before they can be directly bred. Therefore, we employ the classical selective breeding strategy of progeny testing, which we demonstrate here. We first had the item for this method when attempting to time lapse record ectopic bone cell development.Frustratingly, extra bone cells appeared in it frequently, so we set out to alter mutant penetrants by selective breeding. Although we perform this method on a zebrafish scale with a mutant line, it can also be applied to other zebrafish mutant phenotypes and likely, other organisms. After preparing unselected starting zebrafish stock according to the text protocol, carry out KASP genotyping as follows.Place the zebrafish tail tissue in 50 microliters of lysis buffer in the wells of a 96 well plate. Add 10 microliters of 10 milligrams per milliliter proteinase k per well and digest the tissue samples in a thermocycler at 55 degree celsius for two to five hours. Followed by a 20 minute 94 degree celsius inactivation step.Refrigerate the lysis product after digestion. Use a spectrophotometer to quantify the genomic DNA. Then, using molecular biology grade water, dilute the genomic template so that each reaction contains approximately 20 to 30 nanograms.Add 0.14 microliters of the KASP primer mix and five microliters of the KASP master mix per sample on ice, into a 1.5 milliliter microcentrifuge tube and mix well. Then, briefly centrifuge to collect the contents at the bottom of the tube. Pipette five microliters of primer master mix solution into the wells of a 96 well optical PCR plate suitable for the real time PCR machine being used.Then, pipette five microliters of diluted DNA template samples into each well and aspirate with a pipette to mix. Place an optically clear film seal over the plate, ensuring that the film is fully sealed on each well. Briefly centrifuge the plate at 600 x G to collect the contents at the bottom and to remove bubbles from the mix.After performing the main KASP reaction, view the resulting scatter plot produced by the analysis computer. House the identified heterozygous animals together on the main water system. After the animal recover from the fin clip, set up pairwise intercrosses, use dividers to ensure embryos are synchronized.Collect the embryos, and keep the parental pairs isolated in breeding cages. Monitor isolated adults, so that aggressive fish can be separated or provided with cover. To rear the embryos on day five or six, when the swim bladders is inflated in 75%of a clutch, use a glass pipette to collect phenotypically wild type animals.Place phenotypic wild types in the nursery, recording which parents yielded them. After anesthetizing larvae that do not inflate their swim bladders according to the text protocol, use a wide bore glass pasteur pipette to collect the animals into 1.5 milliliters microcentrifuge tubes. Remove the embryo water from each tube and replace it with one milliliter of 2%paraformaldehyde in 1x PBS.Rock the tubes for one hour to fix the animals. Track the tubes regularly during this and all subsequent rocking steps to ensure no larvae have become stuck to the side or clumped in the bottom of the tube. Use a glass pipette to remove the fixative from the tubes.Then add one milliliter of 50%ethanol. Rock the samples for an additional 10 minutes. Prepare fresh staining solution by adding 20 microliters of 0.5%alizarin red per one milliliter of Alcian premix.Remove the 50%ethanol, and add one milliliter of staining solution to each tube. Then, rock the tubes overnight. Within a few days after bleaching the embryos according to the text protocol, score stained mutant offspring for penetrants.In this example, we score for any occurrence of a topic bone. For the next generation, choose two high penetrants and two low penetrants families. Give each parental pair a sub stock identifier, for example, family 4.01, 02, et cetera.It's also helpful to indicate the or inbred generation number on all labels. House parental pairs on the main system with several mixed sex companion fish. Companion fish can be any zebrafish line with permanent obvious distinguishing features, such as altered pigmentation or fin structure.Label the tanks containing the wild type sibling larvae from selected families with the penetrants from the mutant siblings and raise the fish to adulthood as normal. Selective breeding by progeny testing, should yield a shift in overall penetrants both downward and upward in a few generations. In these examples, apply to the mef2ca b1086 mutant, high or low penetrants of a topic bone between the opercal and the branchiostegal ray in lethal homozygous mutants was selected for.In a successful cast procedure, type groupings of samples corresponding to gene type should be recognized by the computer program and the NTCs should be located near the origin of the plot after sufficient cycles have been performed. As demonstrated here, successful Alcian blue, alizarin red stain will have vibrantly stained bone and cartilage elements that are not transparent or faint. The boundaries of the cartilage elements should appear crisp and individual contracytes will be discernible.Alcian blue that is too concentrated were not clear from other tissues making analysis impossible. Implementing this technique from zebrafish line maintenance will likely alter penetrants in a few generations. Following this procedure, other methods like NC2 hybridization and timelapse imaging studies may yield more consistent results.We use this technique to explore the heritability of phenotype variation. After watching this video, you should have a good understanding of how to selectively breed your mutant strains to alter penetrance, even if the mutation is lethal.

shifting-zebrafish-lethal-skeletal-mutant-penetrance-progeny

Research

Education

JoVE Journal

JoVE Core

Molecular Biology

Developmental Biology

Unit 2

Mendelian Genetics

SCIENTISTS IN ACTION

Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is the nephron. Mammals are able to repair existing nephrons after injury, but lose the ability to form new nephrons soon after birth. In contrast to mammals, adult fish produce new nephrons (neonephrogenesis) throughout their lives in response to growth requirements or injury. Recently, lhx1a has been shown to mark nephron progenitor cells in the adult zebrafish kidney, however mechanisms controlling the formation of new nephrons after injury remain unknown. Here we show our method for robust and reproducible injury in the adult zebrafish kidney by intraperitoneal (i.p.) injection of gentamicin, which uses a noninvasive visual screening process to select for fish with strong but nonlethal injury. Using this method, we can determine optimal gentamicin dosages for injury and go on to demonstrate the effect of higher temperatures on kidney regeneration in zebrafish.

Here we present a reliable method to study adult kidney regeneration by inducing acute kidney injury by gentamicin injection. We show that injury is dependent on gentamicin dosage and environmental temperature using in situ hybridization to label lhx1a+ developing new nephrons.

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for candidate genes of interest, migratory behaviors of cells, and secreted signals in distinct genetic settings. Because parabiotic animals share a common circulation, any blood or blood-borne factor from one animal will be exchanged with its partner and vice versa. Thus, cells and molecular factors derived from one genetic background can be studied in the context of a second genetic background. Parabiosis of adult mice has been &#160;used extensively to research aging, cancer, diabetes, obesity, and brain development. More recently, parabiosis of zebrafish embryos has been used to study the developmental biology of hematopoiesis. In contrast to mice, the transparent nature of zebrafish embryos permits the direct visualization of cells in the parabiotic context, making it a uniquely powerful method for investigating fundamental cellular and molecular mechanisms. The utility of this technique, however, is limited by a steep learning curve for generating the parabiotic zebrafish embryos. This protocol provides a step-by-step method on how to surgically fuse the blastulae of two zebrafish embryos of different genetic backgrounds to investigate the role of candidate genes of interest. In addition, the parabiotic zebrafish embryos are tolerant to heat shock, making temporal control of gene expression possible. This method does not require a sophisticated set-up and has broad applications for studying cell migration, fate specification, and differentiation in vivo during embryonic development.

This protocol provides step-by-step instruction on how to generate parabiotic zebrafish embryos of different genetic backgrounds. When combined with the unparalleled imaging capabilities of the zebrafish embryo, this method provides a uniquely powerful means to investigate cell-autonomous versus non-cell-autonomous functions for candidate genes of interest.

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for ...

Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced skeletal muscle regeneration is a widely used, powerful model system to study the events involved in muscle regeneration as well as the mechanisms and different players. Indeed, a detailed knowledge of this process is essential for a better understanding of the pathological conditions that lead to skeletal muscle degeneration, and it aids in identifying new targeted therapeutic strategies. The present work describes a detailed and reproducible protocol to induce acute skeletal muscle regeneration in mice through a single intramuscular injection of cardiotoxin (CTX). CTX belongs to the family of snake venom toxins and causes myolysis of myofibers, which eventually triggers the regeneration events. The dynamics of skeletal muscle regeneration is evaluated by histological analysis of muscle sections. The protocol also illustrates the experimental procedures for dissecting, freezing, and cutting the Tibialis Anterior muscle, as well as the routine Hematoxylin &#38; Eosin staining that is widely used for subsequent morphological and morphometric analysis.

This manuscript describes a detailed protocol to induce acute skeletal muscle regeneration in adult mice and subsequent manipulations of the muscles, such as dissection, freezing, cutting, routine staining, and myofiber cross-sectional area analysis.

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced ...

Dissection of Larval Zebrafish Gonadal Tissue

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex determination system, where several genes distributed throughout the genome collectively determine the sex identities of individual fish. Currently, the genes involved in regulating gonad development and how they work remain elusive. Normally, isolating gonadal tissue is the first step to examine the sex developmental processes. Here, we present a procedure to isolate gonadal tissue from 17 dpf (days post fertilization) and 25 dpf zebrafish larvae. The isolated gonadal tissue may be subsequently examined by morphology and gene expression profiling.

Here, we present a protocol for isolating gonadal tissue of larval zebrafish, which will facilitate investigations of zebrafish sex differentiation and maintenance.

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex ...

Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where they act as front line sentinels of infection and tissue damage. While other immune cells are continuously renewed from hematopoietic stem and progenitor cells (HSPC) located in the bone marrow, a lineage of macrophages, known as resident macrophages, have been shown to be self-maintained in tissues without input from bone marrow HSPCs. This lineage is exemplified by microglia in the brain, Kupffer cells in the liver and Langerhans cells in the epidermis among others. The intestinal and colon lamina propria are the only adult tissues devoid of HSPC-independent resident macrophages. Recent investigations have identified that resident macrophages originate from the extra-embryonic yolk sac hematopoiesis from progenitor(s) distinct from fetal hematopoietic stem cells (HSC). Among yolk sac definitive hematopoiesis, erythromyeloid progenitors (EMP) give rise both to erythroid and myeloid cells, in particular resident macrophages. EMP are only generated within the yolk sac between E8.5 and E10.5 days of development and they migrate to the fetal liver as early as circulation is connected, where they expand and differentiate until at least E16.5. Their progeny includes erythrocytes, macrophages, neutrophils and mast cells but only EMP-derived macrophages persist until adulthood in tissues. The transient nature of EMP emergence and the temporal overlap with HSC generation renders the analysis of these progenitors difficult. We have established a tamoxifen-inducible fate mapping protocol based on expression of the macrophage cytokine receptor Csf1r promoter to characterize EMP and EMP-derived cells in vivo by flow cytometry.

While infiltrating macrophages are continuously recruited to adult tissues from circulating precursors, resident macrophages seed their tissue during development, where they are maintained without further input from progenitors. The progenitors for resident macrophages were recently identified. Here, we present methods for the genetic fate mapping of the resident macrophage progenitors.

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where ...

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The M&#252;ller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both M&#252;ller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina, i.e. they migrate between the basal Inner Nuclear Layer (INL) and the Outer Nuclear Layer (ONL), respectively, in a process described as Interkinetic Nuclear Migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled M&#252;ller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP]mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

Zebrafish retinal regeneration has mostly been studied using fixed retinas. However, dynamic processes such as interkinetic nuclear migration occur during the regenerative response and require live-cell imaging to investigate the underlying mechanisms. Here, we describe culture and imaging conditions to monitor Interkinetic Nuclear Migration (INM) in real-time using multiphoton microscopy.

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The ...

Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles

Pericytes are perivascular multipotent cells that show heterogeneity in different organs or even within the same tissue. In skeletal muscles, there are at least two pericyte subpopulations (called type I and type II), which express different molecular markers and have distinct differentiation capabilities. Using NG2-DsRed and Nestin-GFP double-transgenic mice, type I (NG2-DsRed+Nestin-GFP-) and type II (NG2-DsRed+Nestin-GFP+) pericytes have been successfully isolated. However, the availability of these double-transgenic mice prevents the widespread use of this purification method. This work describes an alternative protocol that allows for the easy and simultaneous isolation of type I and type II pericytes from skeletal muscles. This protocol utilizes the fluorescence-activated cell sorting (FACS) technique and targets PDGFR&#946;, rather than NG2, together with the Nestin-GFP signal. Following isolation, type I and type II pericytes show distinct morphologies. In addition, type I and type II pericytes isolated with this new method, like those isolated from the double-transgenic mice, are adipogenic and myogenic, respectively. These results suggest that this protocol can be used to isolate pericyte subpopulations from skeletal muscles and possibly from other tissues.

This work describes a FACS-based protocol that allows for easy and simultaneous isolation of type I and type II pericytes from skeletal muscles.

Pericytes are perivascular multipotent cells that show heterogeneity in different organs or even within the same tissue. In skeletal muscles, there are at ...

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, antibodies for specific cell markers are applied on tissue sections. In adult skeletal muscle, satellite cells (SCs) are stem cells that contribute to muscle repair and regeneration. Therefore, it is important to visualize and trace the satellite cell population under different physiological conditions. In resting skeletal muscle, SCs reside between the basal lamina and myofiber plasma membrane. A commonly used marker for identifying SCs on the myofibers or in cell culture is the paired box protein Pax7. In this article, an optimized Pax7 immunofluorescence protocol on skeletal muscle sections is presented that minimizes non-specific staining and background. Another antibody that recognizes a protein (laminin) of the basal lamina was also added to help identify SCs. Similar protocols can also be used to perform double or triple labeling with Pax7 and antibodies for additional proteins of interest.

The precise identification of satellite cells is essential for studying their functions under various physiological and pathological conditions. This article presents a protocol to identify satellite cells on adult skeletal muscle sections by immunofluorescence-based staining.

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, ...

Performing Human Skeletal Muscle Xenografts in Immunodeficient Mice

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this failure is the use of inadequate laboratory models. It is challenging to model complex human diseases in traditional laboratory organisms, but this issue can be circumvented through the study of human xenografts. The surgical method we describe here allows for the creation of human skeletal muscle xenografts, which can be used to model muscle disease and to carry out preclinical therapeutic testing. Under an Institutional Review Board (IRB)-approved protocol, skeletal muscle specimens are acquired from patients and then transplanted into NOD-Rag1null IL2r&gamma;null (NRG) host mice. These mice are ideal hosts for transplantation studies due to their inability to make mature lymphocytes and are thus unable to develop cell-mediated and humoral adaptive immune responses. Host mice are anaesthetized with isoflurane, and the mouse tibialis anterior and extensor digitorum longus muscles are removed. A piece of human muscle is then placed in the empty tibial compartment and sutured to the proximal and distal tendons of the peroneus longus muscle. The xenografted muscle is spontaneously vascularized and innervated by the mouse host, resulting in robustly regenerated human muscle that can serve as a model for preclinical studies.

Complex human diseases can be challenging to model in traditional laboratory model systems. Here, we describe a surgical approach to model human muscle disease through the transplantation of human skeletal muscle biopsies into immunodeficient mice.

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this ...