This work was supported by the USDA National Institute of Food and Agriculture, Hatch project 1016879 and Maryland Agricultural Experiment Station via MAES Grant No. 2956952.

1. Preparation of dosage curve for effective mutagenesis
<ol>
	<li>Soak 100 seeds with the genotype of interest in six 250 mL glass flasks (100 in each flask) containing 50 mL of distilled water. Shake at 100 rpm for 8 h at room temperature (RT) for imbibition by the seeds.</li>
	<li>In a fume hood, prepare 50 mL of 0.4%, 0.6%, 0.8%, 1.0%, and 1.2% (w/v) ethyl methanesulfonate (EMS) solution by dissolving 0.167, 0.249, 0.331, 0.415, and 0.498 mL of EMS in distilled water, respectively. 
	NOTE: EMS is liquid at RT with a density of 1.206 g/mL. 
	CAUTION: Use appropriate personal protective equipment (PPE) while handling EMS.</li>
	<li>Decant the water out of five flasks and add 50 mL of EMS solution in each flask containing imbibed seed so that there are six different treatments with 0.0%, 0.4%, 0.6%, 0.8%, 1.0%, and 1.2% EMS solution. Shake flasks for 16 h at 75 rpm and RT.</li>
	<li>Decant the EMS solution and collect the treated seeds separately for each treatment using cheese cloth. Inactivate the used EMS solution by adding one volume of EMS-inactivating solution (0.1 M NaOH, 20% w/v Na2S2O3) for 24 h. Also treat the contaminated flasks and pipette tips with the EMS-inactivating solution for 24 h.</li>
	<li>Wash the EMS-treated seeds under running tap water for 2 h. Transplant each seed individually into root trainers containing potting soil.</li>
	<li>Grow plants at 20&#8211;25 &#176;C under a 16 h light period.</li>
	<li>Record data on plant survival after 15 days of transplantation. Use the following equation to calculate the survival rate for each treatment: 
	<img alt="Equation 1" src="/files/ftp_upload/59743/59743eq1.jpg" /> 
	NOTE: If the germination rate is lower than 100% in controls, accurate survival rates in all the treatments should be calculated after subtracting the number of seeds that failed to germinate in the controls. A survival rate of 40%&#8211;60% is desirable for effective mutagenesis. It may be required to perform a second round of dosage optimization with a modified concentration according to the survival of the treated seeds until achieving the desirable lethality rate of 40%&#8211;60%.</li>
</ol>
2. Mutagenesis and maintenance of mutant population
<ol>
	<li>Soak the final batch of at least 3,000 seeds dividing equally (600 seeds each) in five 1,000 mL flasks containing 300 mL distilled water. Shake for 8 h at 100 rpm under RT for imbibition. 
	NOTE: The final size of the desirable population will depend on the mutation frequency and ploidy level of the genotype, but it is advisable to use at least 3,000 seeds for hexaploids, 4,000 seeds for tetraploids, and more than 7,000 seeds for diploids.</li>
	<li>In a fume hood, prepare 1,500 mL of the optimized EMS concentration solution in distilled water.</li>
	<li>Decant the water out of the flasks and add 300 ml of the optimal concentration of EMS solution in each flask containing 600 imbibed seeds. Shake the flasks for 16 h at 75 rpm and RT.</li>
	<li>Decant the EMS and collect the treated seeds in cheese cloth. Inactivate the EMS solution and treatment containers with EMS-inactivating solution as done in step 1.4.</li>
	<li>Wash the EMS-treated seeds under running tap water for 2 h. Transplant each EMS-treated M1 seed individually into root trainers.</li>
	<li>Grow M1 plants (derived from M0 seeds) at 20&#8211;25 &#176;C under a 16 h light period. 
	NOTE: It may be required to vernalize the seedlings at the two leaf stage for 6 weeks at 4 &#176;C, if the genotype of interest has a winter-type growth habit.</li>
	<li>Allow the M1 plants to self-pollinate, and harvest the M2 seeds separately for each fertile M1 plant. 
	NOTE: To avoid chances of potential outcrossing, cover the spikes of M1 plants with pollination bags before anthesis.</li>
	<li>Plant a single M2 seed from each M1 plant to avoid genetic redundancy.</li>
	<li>Collect tissue from M2 plants at the two-leaf stage in 1.1 mL of racked 96 well microtubes. Collect around 80 mm of leaf tissue from each plant and record the ID of each sample in a tissue collection plan.</li>
	<li>Freeze-dry the leaf tissue using a lyophilizer and store at -80 &#176;C.</li>
	<li>Maintain the M2 plants at 20&#8211;25 &#176;C under a 16 h light period.</li>
	<li>Record data on mutant phenotypes of the M2 plants at regular intervals. The expected phenotypes are albino, chlorina, grassy shoot, variegated, partially fertile, sterile, etc.</li>
	<li>Allow the M2 plants to self-fertilize and mature. Separately harvest and save the M3 seeds of the M2 plants (Figure 1). 
	NOTE: M seeds are used for validating the phenotype in reverse genetics studies. Therefore, M should be carefully catalogued and stored under cool and dry conditions. Alternatively, if the seed must be increased in field, head rows can be planted for each M plant, and M seeds from each row can be harvested and saved separately as the TILLING population resource.</li>
</ol>
3. Cel-1 assay for genetic characterization of mutants
<ol>
	<li>Extract DNA from the leaf tissue of M2 using a plant DNA extraction kit with a DNA purification system (see Table of Materials) following the manufacturer&#8217;s recommendations.</li>
	<li>Quantify DNA using a spectrophotometer and normalize the DNA concentrations to 25 ng/&#181;L with nuclease-free water in 96 well blocks. 
	NOTE: It is important to check the quality of the DNA by running it on a gel, as low-quality DNA (smeared DNA) may result in false negatives in pooled samples.</li>
	<li>Create 4x DNA pools by combining DNA from four 96 well blocks into one plate, while maintaining the row and column identity of each sample. Add 50 &#181;L of DNA from each individual sample into the pool plate so that each pool plate well contains a total 200 &#181;L of DNA from four different 96 well blocks.</li>
	<li>Catalogue the identity of pooled DNA in the format of Pool Plate-Row-Column (e.g., Pool 1 A1 = Box1A1 + Box2A1 + Box3A1 + Box4A1).</li>
	<li>Design genome-specific primers [A1] for the gene of interest using the Genome Specific Primers (GSP) page &#60;https://probes.pw.usda.gov/GSP&#62; with default settings for polyploid species. For diploids, use Primer 3 &#60;http://primer3.ut.ee/&#62; for designing primers using default settings. Design multiple primers, if needed, to cover the entire coding region of the gene of interest. 
	NOTE: The latest IWGSC assembly can be used to obtain sequences for the gene of interest in wheat using the URGI BLAST tool &#60;https://wheat-urgi.versailles.inra.fr/Seq-Repository/BLAST&#62;. The optimal amplicon length is in the range of 800&#8211;1,500 bp. Table 1 shows examples of primers for waxy genes in hexaploid wheat.</li>
	<li>Run a PCR for gene-specific primers on pooled DNA as follows:
	<ol>
		<li>Add 5 &#181;L of PCR buffer, 2 &#181;L (each) of 4 &#181;M forward and reverse primers, 0.1 &#181;L of DNA polymerase (see Table of Materials), 5 &#181;L of pooled DNA template, then increase the volume to 25 &#181;L using nuclease-free water.</li>
		<li>Use a touch down profile to run the PCR reaction on a thermal cycler as follows: 95 &#176;C for 1 min, seven cycles of 95 &#176;C for 1 min, 67 &#176;C to 60 &#176;C for 1 min with temperature decreases of 1 &#176;C per cycle and 72 &#176;C for 2 min, followed by 30 cycles of 95 &#176;C for 1 min, 60 &#176;C for 1 min, 72 &#176;C for 2 min, and final extension at 72 &#176;C for 7 min. 
		NOTE: Above PCR profile works on wheat DNA template for most of the primers designed using primer 3 default settings. In case of non-specific amplification, PCR profile should be made stringent before moving to the next steps.</li>
	</ol>
	</li>
	<li>Generate heteroduplexes between mismatched DNA by incubating PCR products in thermal cycler using profile as follows: 95 &#176;C for 2 min, five cycles of 95 &#176;C for 1 s, 95 &#176;C to 85 &#176;C for 1 min with temperature decreases of 2 &#176;C per cycle, and 60 cycles of 85 &#176;C to 25 &#176;C with decreases of 1 &#176;C per cycle.</li>
	<li>Add 2.5 &#181;L of homemade Cel-1 endonuclease to the heteroduplexed PCR products and incubate for 45 min at 45 &#176;C. Terminate the Cel-1 reaction by adding 2.5 &#181;L of 0.5 M EDTA (pH 8.0). 
	NOTE: Cel-1 endonuclease can be extracted from fresh celery stalks using the protocol performed by Till et al.19 It is very important to test the activity and optimum amount of Cel-1 endonuclease, which can be tested using previously characterized mutants or a commercially available mutation detection kit.</li>
	<li>Run the Cel-1 treated products on a 3.0% agarose gel at 100 V for 2.5 h. The wells containing smaller and unique cleaved band(s), in addition to full-length uncleaved bands, will contain the mutant DNA sample.</li>
	<li>Denconvolute mutant pools.
	<ol>
		<li>Follow steps 3.6, 3.7, 3.8, and 3.9 for individual M2 DNA samples constituting the mutant pools identified in step 3.9.</li>
		<li>To determine the zygosity of mutants, run two PCR (as described in step 3.6) for individual M2 DNA, in which the first reaction contains 2.5 &#181;L of M2 DNA and 2.5 &#181;L of wild-type DNA and the second reaction contains only 5 &#181;L of M2 DNA. If the mutation is heterozygous, an additional cleaved band will be present in both reactions. On the other hand, additional cleaved bands will be found only in the first reaction if the mutant is homozygous.</li>
	</ol>
	</li>
	<li>To identify the nature of the mutation, sequence the PCR products of the confirmed mutants using a Sanger sequencing platform following the manufacturer&#8217;s instructions.</li>
</ol>
4. Calculation of mutation frequency
NOTE: Mutation frequency of a TILLING population refers to the average physical distance in which one mutation occurs in the individuals of that population. For example, a mutation frequency of 1/35 kb in a TILLING population means that an average individual of that population possesses 1 mutation per every 35 kb in the genome.
<ol>
	<li>To determine the mutation frequency of a TILLING population, calculate the total number of bases screened.</li>
	<li>To calculate the total number of bases screened, multiply the PCR product size by the total number of individuals screened.</li>
	<li>Divide the total number of bases screened by the number of unique mutations observed using the following equation, which will yield the physical region possessing 1 mutation in the given TILLING population: 
	<img alt="Equation 2" src="/files/ftp_upload/59743/59743eq2.jpg" /> 
	NOTE: To account for the limitation in resolution of 50 bp at both the ends based on an agarose gel-based platform, subtract 100 bp from the product size in the calculation.</li>
</ol>

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V., 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=Uncovering+hidden+variation+in+polyploid+wheat.">Uncovering hidden variation in polyploid wheat.</a> Proceedings of the National Academy of Sciences. , 201619268 (2017).</li><li>Dong, C., Dalton-Morgan, J., Vincent, K., Sharp, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Modified+TILLING+Method+for+Wheat+Breeding.">A Modified TILLING Method for Wheat Breeding.</a> The Plant Genome. 2 (1), 39-47 (2009).</li><li>Till, B. J., Zerr, T., Comai, L., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+TILLING+and+Ecotilling+in+plants+and+animals.">A protocol for TILLING and Ecotilling in plants and animals.</a> Nature Protocols. 1 (5), 2465-2477 (2006).</li><li>Wu, J. -. 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=Chemical-+and+Irradiation-induced+Mutants+of+Indica+Rice+IR64+for+Forward+and+Reverse+Genetics.">Chemical- and Irradiation-induced Mutants of Indica Rice IR64 for Forward and Reverse Genetics.</a> Plant Molecular Biology. 59 (1), 85-97 (2005).</li><li>Feldman, M., Levy, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+Evolution+Due+to+Allopolyploidization+in+Wheat.">Genome Evolution Due to Allopolyploidization in Wheat.</a> Genetics. 192 (3), 763-774 (2012).</li><li>Comai, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+advantages+and+disadvantages+of+being+polyploid.">The advantages and disadvantages of being polyploid.</a> Nature Reviews Genetics. 6 (11), 836-846 (2005).</li><li>Guo, H., 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=Development+of+a+High-Efficient+Mutation+Resource+with+Phenotypic+Variation+in+Hexaploid+Winter+Wheat+and+Identification+of+Novel+Alleles+in+the+TaAGP.L-B1+Gene.">Development of a High-Efficient Mutation Resource with Phenotypic Variation in Hexaploid Winter Wheat and Identification of Novel Alleles in the TaAGP.L-B1 Gene.</a> Frontiers in Plant Science. 8, (2017).</li><li>Rakszegi, M., 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=Diversity+of+agronomic+and+morphological+traits+in+a+mutant+population+of+bread+wheat+studied+in+the+Healthgrain+program.">Diversity of agronomic and morphological traits in a mutant population of bread wheat studied in the Healthgrain program.</a> Euphytica. 174 (3), 409-421 (2010).</li><li>Tsai, H., Ngo, K., Lieberman, M., Missirian, V., Comai, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tilling+by+Sequencing.">Tilling by Sequencing.</a> Plant Functional Genomics: Methods and Protocols. , 359-380 (2015).</li></ol>

Authors declare no competing financial interests.

TILLING is a highly valuable reverse genetics tool for gene validation, especially for small grains where transformation-based approaches have serious bottlenecks11. Developing a mutagenized population with a high mutation frequency is one of the critical steps in conducting functional genomics studies. The most important step in developing a robust TILLING population is to determine the optimal concentration of EMS. The 40%-60% survival rate in the M1 has been found to be a good indica...

Point mutations in genomes can serve many useful purposes for researchers. Depending on their nature and location, these mutations can be used to assign functions to genes or even distinct domains of proteins of interest. On the other hand, as a source of novel genetic variation, useful mutations can be selected for desired traits using phenotyping screens and further used in crop improvement. TILLING is a powerful reverse genetics tool that includes chemical mutagenesis and detection of sequence variation in the target gene. First developed in Arabidopsis1 and Drosophilia melanogaster2, TILLING populations have been developed and utilized in many small grain crops such as hexaploid bread wheat (Triticum aestivum)3, barley (Hordeum vulgare)4, tetraploid durum wheat (T. dicoccoides durum)5, diploid wheat (T. monococcum)6 and the &#34;D&#34; genome progenitor of wheat Aegilops tauschii7. These resources have been used to validate the roles of genes in regulating abiotic and biotic stress tolerance8, regulating flowering time9, and developing nutritionally superior crop varieties5.
TILLING, along with the use of alkylating mutagenic agents such as ethyl methanesulfonate (EMS), sodium azide, N-methyl-N-nitrosourea (MNU), and methyl methanesulfonate (MMS), has advantages over other reverse genetics tools for several reasons. First, mutagenesis can be conducted on practically any species or variety of plant10 and is independent of the transformation bottleneck, which is particularly challenging in the case of small grains11. Second, in addition to generating knockout mutations that can be obtained by other gene validation approaches, a range of missense and splicing mutations can be induced, which can discern functions of individual domains of the proteins of interest12. Moreover, TILLING generates an immortal collection of mutations throughout the genome; thus, a single population can be used for functional validation of multiple genes. In contrast, other reverse genetics tools generate resources specific to only the gene under study13. Useful mutations identified through TILLING can be deployed for breeding purposes and are not subject to regulation, unlike gene editing, whose non-transgenic classification is still uncertain in many countries. This becomes especially relevant to small grains that are internationally traded14.
TILLING is a simple and efficient gene validation strategy and requires mutagenized populations to be developed for investigating genes of interest. Developing an effective mutagenized population is key to determining the efficiency of a TILLING-based gene validation study. A TILLING population with a low overall mutation frequency indicates that an impractically large population must be screened for desired mutations, whereas a high mutagen concentration leads to high mortality in the population and an insufficient number of mutagenized individuals. Once a good population is developed, there are multiple ways to detect mutations in the genes of interest, and the choice of platform depends on the experimental scale and availability of resources. Whole genome sequencing and exome sequencing has been used to characterize all mutations in TILLING populations in plants with small genomes15,16. Exome sequencing of two TILLING populations has been performed in bread and durum wheat and is available to the public for identifying desirable mutations and ordering mutant lines of interest17. It is a great public resource in terms of availability of desirable mutations; however, in gene validation studies, the wild-type line should possess the candidate gene of interest. Unfortunately, it is still cost-prohibitive to sequence the exome of the entire TILLING population for reverse genetics-based validation of a few candidate genes in another background. Amplicon sequencing and Cel-1-based assays have been used in detecting mutations in targeted populations in wheat, and Cel-1 assays are simpler, requiring no computational knowledge, and are especially suitable for validation of a small number of genes with basic lab equipment6,18.
In the present article, described are methods for the development of a good TILLING population, including preparation of the dosage curve, mutagenesis and maintenance of the mutant population, and screening of the mutant population using the PCR-based Cel-1 assay. This protocol has already been implemented successfully in developing and utilizing mutagenized populations of Triticum aestivum, Triticum monoccocum6, barley, Aegilops tauchii7, and several others. Included are explicit details of these methods along with useful tips that will help researchers develop TILLING populations, using EMS as a mutagen in any small grain plant of choice.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96 well 1.1 ml microtubes in microracks</td>
			<td>National Scientific</td>
			<td>TN0946-08R</td>
			<td>For collecting leaf tissues</td>
		</tr>
		<tr>
			<td>Agarose I biotechnology grade</td>
			<td>VWR</td>
			<td>0710-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosprint 96 DNA Plant Kit</td>
			<td>Qiagen</td>
			<td>941558</td>
			<td>Kit for DNA extraction</td>
		</tr>
		<tr>
			<td>Cel-1 endonuclease</td>
			<td>Extracted as described by Till et al 2006</td>
			<td></td>
			<td>Single strand specific endonuclease</td>
		</tr>
		<tr>
			<td>Centrifuge 5430 R</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl methanesulfonate</td>
			<td>Sigma Aldrich</td>
			<td>M-0880-25G</td>
			<td>EMS, Chemical mutagen</td>
		</tr>
		<tr>
			<td>Freeze Dry/Shell freeze system</td>
			<td>Labconco</td>
			<td></td>
			<td>For lyophilization of leaf tissue</td>
		</tr>
		<tr>
			<td>Kingfisher Flex purification system</td>
			<td>Thermo fisher scientific</td>
			<td>5400610</td>
			<td>High throughput DNA extraction robot</td>
		</tr>
		<tr>
			<td>My Taq DNA Polymerase</td>
			<td>Bioline</td>
			<td>BIO-21107</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease free water</td>
			<td>Sigma aldrich</td>
			<td>W4502-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>NuGenius gel imaging system</td>
			<td>Syngene</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Orbit Environ-shaker</td>
			<td>Lab-line</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SPECTROstar Nano</td>
			<td>BMG LABTECH</td>
			<td></td>
			<td>Nano drop for DNA quantification</td>
		</tr>
		<tr>
			<td>T100 Thermal cycler</td>
			<td>BIO-RAD</td>
			<td>1861096</td>
			<td></td>
		</tr>
	</tbody>
</table>

development of targeting induced local lesions in genomes (tilling) populations in small grain crops by ethyl methanesulfonate mutagenesis

Targeting Induced Local Lesions IN Genomes (TILLING) is a powerful reverse genetics tool that includes chemical mutagenesis and detection of sequence variation in target genes. TILLING is a highly valuable functional genomics tool for gene validation, especially in small grains in which transformation-based approaches hold serious limitations. Developing a robust mutagenized population is key to determining the efficiency of a TILLING-based gene validation study. A TILLING population with a low overall mutation frequency indicates that an impractically large population must be screened to find desired mutations, whereas a high mutagen concentration leads to high mortality in the population, leading to an insufficient number of mutagenized individuals. Once an effective population is developed, there are multiple ways to detect mutations in a gene of interest, and the choice of platform depends upon the experimental scale and availability of resources. The Cel-1 assay and agarose gel-based approach for mutant identification is convenient, reproducible, and a less resource-intensive platform. It is advantageous in that it is simple, requiring no computational knowledge, and it is especially suitable for validation of a small number of genes with basic lab equipment. In the present article, described are the methods for development of a good TILLING population, including preparation of the dosage curve, mutagenesis and maintenance of the mutant population, and screening of the mutant population using the PCR-based Cel-1 assay.

Figure 2 shows the dosage curve of hexaploid bread wheat cultivar Jagger, diploid wheat Triticum monococcum6, and a genome donor of wheat Aegilops tauschii7. The EMS doses for desired 50% survival rates were about 0.25%, 0.6% and 0.7% for T. monococcum, Ae. tauschii, and T. aestivum, respectively. The higher EMS tolerance of hexaploid wheat is due to its genome buffer...

Watch this Scientific Journal Video about Development of Targeting Induced Local Lesions IN Genomes TILLING Populations in Small Grain Crops by Ethyl Methanesulfonate Mutagenesis at JoVE.com

Development of Targeting Induced Local Lesions IN Genomes TILLING Populations in Small Grain Crops by Ethyl Methanesulfonate Mutagenesis

Described is a protocol for developing a Targeting Induced Local Lesions IN Genomes (TILLING) population in small grain crops with use of ethyl methanesulfonate (EMS) as a mutagen. Also provided is a protocol for mutation detection using the Cel-1 assay.

Development of Targeting Induced Local Lesions IN Genomes (TILLING) Populations in Small Grain Crops by Ethyl Methanesulfonate Mutagenesis

Targeting Induced Local Lesions IN Genomes (TILLING) is a powerful reverse genetics tool that includes chemical mutagenesis and detection of sequence ...

development-targeting-induced-local-lesions-genomes-tilling

Research

JoVE Journal

Developmental Biology

11.5K Views.  University of Maryland, College Park. Described is a protocol for developing a Targeting Induced Local Lesions IN Genomes (TILLING) population in small grain crops with use of ethyl methanesulfonate (EMS) as a mutagen. Also provided is a protocol for mutation detection using the Cel-1 assay.

Video: Development of Targeting Induced Local Lesions IN Genomes TILLING Populations in Small Grain Crops by Ethyl Methanesulfonate Mutagenesis

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 ...

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development and organogenesis are two areas of research that have benefitted greatly from these advances, due to the high quality of data that can be obtained directly from imaging pre-implantation embryos or ex vivo organs. While pre-implantation embryos have yielded data with especially high spatial resolution, later stages have been less amenable to three-dimensional reconstruction. Obtaining high-quality 3D or volumetric data for known embryonic structures in combination with fate mapping or genetic lineage tracing will allow for a more comprehensive analysis of the morphogenetic events taking place during embryogenesis.
This protocol describes a whole-mount immunofluorescence approach that allows for the labeling, visualization, and quantification of progenitor cell populations within the developing cardiac crescent, a key structure formed during heart development. The approach is designed in such a way that both cell- and tissue-level information can be obtained. Using confocal microscopy and image processing, this protocol allows for three-dimensional spatial reconstruction of the cardiac crescent, thereby providing the ability to analyze the localization and organization of specific progenitor populations during this critical phase of heart development. Importantly, the use of reference antibodies allows for successive masking of the cardiac crescent and subsequent quantitative measurements of areas within the crescent. This protocol will not only enable a detailed examination of early heart development, but with adaptations should be applicable to most organ systems in the gastrula to early somite stage mouse embryo.

Here, we describe a protocol for whole mount immunofluorescence and image-based quantitative volumetric analysis of early stage mouse embryos. We present this technique as a powerful approach to qualitatively and quantitatively assess cardiac structures during development, and propose that it may be widely adaptable to other organ systems.

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the Notch receptor with ligands from neighboring cells induces activation of the signaling pathway (trans-activation), while interaction with ligands from the same cell inhibits signaling (cis-inhibition). Proper balance between trans-activation and cis-inhibition helps establish optimal levels of Notch signaling in some contexts during animal development. Because of the overlapping expression domains of Notch and its ligands in many cell types and the existence of feedback mechanisms, studying the effects of a given post-translational modification on trans- versus cis-interactions of Notch and its ligands in vivo is difficult. Here, we describe a protocol for using Drosophila S2 cells in cell-aggregation assays to assess the effects of knocking down a Notch pathway modifier on the binding of Notch to each ligand in trans and in cis. S2 cells stably or transiently transfected with a Notch-expressing vector are mixed with cells expressing each Notch ligand (S2-Delta or S2-Serrate). Trans-binding between the receptor and ligands results in the formation of heterotypic cell aggregates and is measured in terms of the number of aggregates per mL composed of &#62;6 cells. To examine the inhibitory effect of cis-ligands, S2 cells co-expressing Notch and each ligand are mixed with S2-Delta or S2-Serrate cells and the number of aggregates is quantified as described above. The relative decrease in the number of aggregates due to the presence of cis-ligands provides a measure of cis-ligand-mediated inhibition of trans-binding. These straightforward assays can provide semi-quantitative data on the effects of genetic or pharmacological manipulations on the binding of Notch to its ligands, and can help deciphering the molecular mechanisms underlying the in vivo effects of such manipulations on Notch signaling.

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Complexity of in vivo systems makes it difficult to distinguish between the activation and inhibition of Notch receptor by trans- and cis-ligands, respectively. Here, we present a protocol based on in vitro cell-aggregation assays for qualitative and semi-quantitative evaluation of the binding of Drosophila Notch to trans-ligands vs cis-ligands.

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical insult to secondary injury processes, including apoptosis and inflammation. Animal modeling has been valuable in the search to unravel injury mechanisms and evaluate potential neuroprotective therapies. This protocol describes the controlled cortical impact (CCI) model of focal, open-head TBI. Specifically, parameters for producing a mild unilateral cortical injury are described. Behavioral consequences of CCI are analyzed using the adhesive tape removal test of bilateral sensorimotor integration. Regarding experimental therapy for TBI pathology, this protocol also illustrates a process for transplanting cultured cells into the brain. Neural cell cultures derived from human induced pluripotent stem cells (hiPSCs) were chosen for their potential to show superior functional restoration in human TBI patients. Chronic survival of hiPSCs in the host mouse brain tissue is detected using a modified DAB immunohistochemical process.

This protocol demonstrates methodologies for a mouse model of open-skull traumatic brain injury and transplantation of cultured human induced pluripotent stem cell-derived cells into the injury site. Behavioral and histologic tests of outcomes from these procedures are also described in brief.

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical ...

Murine Neural Plate Targeting by In Utero Nano-Injection (NEPTUNE) at Embryonic Day 7.5

Manipulating gene expression in the developing mouse brain in utero holds great potential for functional genetics studies. However, it has previously largely been restricted to the manipulation of embryonic stages post-neurulation. A protocol was developed to inject the amniotic cavity at embryonic day (E)7.5 and deliver lentivirus, encoding cDNA or shRNA, targeting &gt;95% of the neural plate and neural crest cells, contributing to the future brain, spinal cord, and peripheral nervous system. This protocol describes the steps necessary to achieve successful transduction, including grinding of the glass capillary needles, pregnancy verification, developmental staging using ultrasound imaging, and optimal injection volumes matched to embryonic stages. 
Following this protocol, it is possible to achieve transduction of &gt;95% of the developing brain with high-titer lentivirus and thus perform whole-brain genetic manipulation. In contrast, it is possible to achieve mosaic transduction using lower viral titers, allowing for genetic screening or lineage tracing. Injection at E7.5 also targets ectoderm and neural crest contributing to distinct compartments of the eye, tongue, and peripheral nervous system. This technique thus offers the possibility to manipulate gene expression in mouse neural-plate- and ectoderm-derived tissues from preneurulation stages, with the benefit of reducing the number of mice used in experiments.

Murine Neural Plate Targeting by In Utero Nano-Injection NEPTUNE at Embryonic Day 7.5

In this protocol, we describe how to inject the mouse amniotic cavity at E7.5 with lentivirus, leading to uniform transduction of the entire neural plate, with minimal detrimental effects on survival or embryonic development.

Manipulating gene expression in the developing mouse brain in utero holds great potential for functional genetics studies. However, it has previously ...

Isolation of Rat Adipose Tissue Mesenchymal Stem Cells for Differentiation into Insulin-producing Cells

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of stem cells that does not pose any ethical concerns. However, current methods to isolate Ad-MSCs are not standardized and employ complicated protocols that require special equipment. We isolated Ad-MSCs from the epididymal fat of Sprague-Dawley rats using a simple, reproducible method. The isolated Ad-MSCs usually appear within 3 days post isolation, as adherent cells display fibroblastic morphology. Those cells reach 80% confluency within 1 week of isolation. Afterward, at passage 3-5 (P3-5), a full characterization was carried out for the isolated Ad-MSCs by immunophenotyping for characteristic MSC cluster of differentiation (CD) surface markers such as CD90, CD73, and CD105, as well as inducing differentiation of these cells down the osteogenic, adipogenic, and chondrogenic lineages. This, in turn, implies the multipotency of the isolated cells. Furthermore, we induced the differentiation of the isolated Ad-MSCs toward the insulin-producing cells (IPCs) lineage via a simple, relatively short protocol by incorporating high glucose Dulbecco&#39;s modified Eagle medium (HG-DMEM), &#946;-mercaptoethanol, nicotinamide, and exendin-4. IPCs differentiation was genetically assessed, firstly, via measuring the expression levels of specific &#946;-cell markers such as MafA, NKX6.1, Pdx-1, and Ins1, as well as dithizone staining for the generated IPCs. Secondly, the assessment was also carried out functionally by a glucose-stimulated insulin secretion (GSIS) assay. In conclusion, Ad-MSCs can be easily isolated, exhibiting all MSC characterization criteria, and they can indeed provide an abundant, renewable source of IPCs in the lab for diabetes research.

Adipose tissue-derived mesenchymal stem cells (Ad-MSCs) can be a potential source of MSCs that differentiate into insulin-producing cells (IPCs). In this protocol, we provide detailed steps for the isolation and characterization of rat epididymal Ad-MSCs, followed by a simple, short protocol for the generation of IPCs from the same rat Ad-MSCs.

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of ...

Development of Targeting Induced Local Lesions IN Genomes (TILLING) Populations in Small Grain Crops by Ethyl Methanesulfonate Mutagenesis

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Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

Technique to Target Microinjection to the Developing Xenopus Kidney

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells

Murine Neural Plate Targeting by In Utero Nano-Injection (NEPTUNE) at Embryonic Day 7.5

Isolation of Rat Adipose Tissue Mesenchymal Stem Cells for Differentiation into Insulin-producing Cells