Name: genotyping of sea anemone during early development
Uploaded: 2019-05-13T14:30:00
Description: Watch this Scientific Journal Video about Genotyping of Sea Anemone during Early Development at JoVE.com

We thank anonymous reviewers for comments on the earlier version of the manuscript, which improved the manuscript. This work was supported by funds from the University of Arkansas.

1. Induction of spawning, in vitro fertilization, and de-jellying
<ol>
	<li>Maintain Nematostella vectensis in seawater with a salinity of 12 parts per thousand (ppt) in darkness at 16 &#176;C, feeding Artemia daily.</li>
	<li>On the day before spawning induction, place animals in a temperature- and light-controlled incubator. Program the incubator so that the animals are exposed to 8 h of light at 25 &#176;C. Optional: Feed a small piece (&#60;1 mm3) of oyster to individual animal...

<ol>
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Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR+knockouts+reveal+an+endogenous+role+for+ancient+neuropeptides+in+regulating+developmental+timing+in+a+sea+anemone.">CRISPR knockouts reveal an endogenous role for ancient neuropeptides in regulating developmental timing in a sea anemone.</a> eLife. 7, e39742 (2018).</li><li>He, S. N., 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+axial+Hox+code+controls+tissue+segmentation+and+body+patterning+in+Nematostella+vectensis.">An axial Hox code controls tissue segmentation and body patterning in Nematostella vectensis.</a> Science. 361 (6409), 1377 (2018).</li><li>Ikmi, A., McKinney, S. A., Delventhal, K. M., Gibson, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TALEN+and+CRISPR/Cas9-mediated+genome+editing+in+the+early-branching+metazoan+Nematostella+vectensis.">TALEN and CRISPR/Cas9-mediated genome editing in the early-branching metazoan Nematostella vectensis.</a> Nature Communications. 5, 5486 (2014).</li><li>Servetnick, M. D., 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=Cas9-mediated+excision+of+Nematostella+brachyury+disrupts+endoderm+development,+pharynx+formation+and+oral-aboral+patterning.">Cas9-mediated excision of Nematostella brachyury disrupts endoderm development, pharynx formation and oral-aboral patterning.</a> Development. 144 (16), 2951-2960 (2017).</li><li>Kraus, Y., Aman, A., Technau, U., Genikhovich, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-bilaterian+origin+of+the+blastoporal+axial+organizer.">Pre-bilaterian origin of the blastoporal axial organizer.</a> Nature Communications. 7, 11694 (2016).</li><li>Fritzenwanker, J. H., Genikhovich, G., Kraus, Y., Technau, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+development+and+axis+specification+in+the+sea+anemone+Nematostella+vectensis.">Early development and axis specification in the sea anemone Nematostella vectensis.</a> Developmental Biology. 310 (2), 264-279 (2007).</li><li>Lee, P. N., Kumburegama, S., Marlow, H. Q., Martindale, M. Q., Wikramanayake, A. 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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas9-mediated+genome+editing+in+a+reef-building+coral.">CRISPR/Cas9-mediated genome editing in a reef-building coral.</a> Proceedings of the National Academy of Sciences of the United States of America. 115 (20), 5235-5240 (2018).</li><li>Momose, 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=High+doses+of+CRISPR/Cas9+ribonucleoprotein+efficiently+induce+gene+knockout+with+low+mosaicism+in+the+hydrozoan+Clytia+hemisphaerica+through+microhomology-mediated+deletion.">High doses of CRISPR/Cas9 ribonucleoprotein efficiently induce gene knockout with low mosaicism in the hydrozoan Clytia hemisphaerica through microhomology-mediated deletion.</a> Scientific Reports. 8, 11734 (2018).</li><li>Artigas, G. 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Described here a PCR-based protocol to genotype a single sea anemone embryo without sacrificing the animal. Following spawning and de-jellying, the fertilized eggs are allowed to develop into gastrulae. The aboral region of each gastrula embryo is surgically removed, and the isolated aboral tissue is used for subsequent genomic DNA extraction, while the remaining post-surgery embryos heal and continue development. The gDNA extracts are then used for a PCR assay to determine the genotype of each embryo. This method takes ...

Cnidarians represent a diverse group of animals that include jellyfish, corals, and sea anemones. They are diploblasts, composed of ectoderm and endoderm that are separated by an extracellular matrix (mesoglea). Cnidaria is a sister group to speciose Bilateria, to which traditional animal models such as Drosophila and Mus belong1. Additionally, the Cnidaria-Bilateria divergence is thought to have occurred in the pre-Cambrian period2. As such, comparative studies of cnidarians and bilaterians are essential for gaining insights into the biology of their most recent common ancestor. Recently, comparative genomics has revealed that cnidarians and bilaterians share many developmental toolkit genes such as notch and bHLH, implying that their common ancestor already had these genes3. However, the role of these developmental toolkit genes in the last common ancestor of Cnidaria and Bilateria is comparably less well understood. To address this problem, it is critical to study how these deeply conserved genes function in cnidarians.
One of the emerging cnidarian genetic models is the anthozoan Nematostella vectensis. Its genome has been sequenced3, and a variety of genetic tools, including morpholino-mediated gene knockdown, meganuclease-mediated transgenesis, and CRISPR-Cas9-mediated gene knockins and knockouts, are now available for use in this animal. In addition, Nematostella development is relatively well understood. During embryogenesis, gastrulation occurs by invagination4, and the embryo develops into a free-swimming planula larva. The planula subsequently transforms into a sessile polyp with a mouth and circumoral tentacles. The polyp then grows and reaches sexual maturity.
CRISPR-Cas9-mediated targeted mutagenesis is now routinely used to study gene function in Nematostella vectensis5,6,7,8,9. To generate knockout mutants in Nematostella, a cocktail containing locus-specific single-guide RNAs and the endonuclease Cas9 protein is first injected into unfertilized or fertilized eggs to produce F0 founder animals that typically show mosaicism. F0 animals are subsequently raised to sexual maturity and crossed with each other to produce an F1 population, a subset of which may be knockout mutants6. Alternatively, sexually mature F0 animals can be crossed with wild-type animals to generate F1 heterozygous animals, and F1 heterozygotes that carry a knockout allele in the locus of interest can then be crossed with each other to produce F2 offspring, one-quarter of which are expected to be knockout mutants5. Both approaches require a method to identify knockout mutants from a genetically heterogeneous population. Polyp tentacles can be used to extract genomic DNA for genotyping6,7. However, in cases where the developmental function of the gene of interest is being investigated and mutant embryos do not reach the polyp stage (i.e., due to larval lethality associated with the mutation), knockout mutants need to be identified early in ontogeny. Described here is a PCR-based protocol to genotype individual animals at the gastrula stage without sacrificing the animal, which enables identification of knockout mutants from a genetically heterogeneous population of embryos. The duration of the entire genotyping process depends on the number of embryos to be screened, but it minimally requires 4-5 h.

<table>
	<tbody>
		<tr>
			<td>Drosophila Peltier Refrigerated Incubator</td>
			<td>Shellab</td>
			<td>SRI6PF</td>
			<td>Used for spawning induction</td>
		</tr>
		<tr>
			<td>Instant ocean sea salt</td>
			<td>Instant ocean</td>
			<td>138510</td>
			<td></td>
		</tr>
		<tr>
			<td>Brine shrimp cysts</td>
			<td>Aquatic Eco-Systems, Inc.</td>
			<td>BS90</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine Hydrochloride</td>
			<td>Sigma Aldrich</td>
			<td>C7352</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard Orbital Shaker, Model 3500</td>
			<td>VWR</td>
			<td>89032-092</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIS-HCl, 1M, pH8.0</td>
			<td>QUALITY BIOLOGICAL</td>
			<td>351-007-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>VWR</td>
			<td>BDH9258</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA, 0.5M pH8</td>
			<td>VWR</td>
			<td>BDH7830-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma Aldrich</td>
			<td>P9416</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonidet-P40 Substitute</td>
			<td>US Biological</td>
			<td>N3500</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K solution (20 mg/mL), RNA grade</td>
			<td>ThermoFisher</td>
			<td>25530049</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>VWR</td>
			<td>710</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Dissecting needle holder</td>
			<td>Roboz</td>
			<td>RS-6060</td>
			<td></td>
		</tr>
		<tr>
			<td>Tungsten dissecting needle</td>
			<td>Roboz</td>
			<td>RS-6063</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR Eppendorf Mastercycler Thermal Cyclers</td>
			<td>Eppendorf</td>
			<td>E6336000024</td>
			<td></td>
		</tr>
		<tr>
			<td>Phusion High-Fidelity DNA polymerase</td>
			<td>New England BioLabs</td>
			<td>M0530L</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTP mix</td>
			<td>New England BioLabs</td>
			<td>N0447L</td>
			<td></td>
		</tr>
		<tr>
			<td>GLWamide universal forward primer</td>
			<td></td>
			<td></td>
			<td>5&#8217;- CATGCGGAGACCAAGCGCAAGGC-3&#8217;</td>
		</tr>
		<tr>
			<td>Reverse primer specific to glw-a</td>
			<td></td>
			<td></td>
			<td>5&#8217;-CCAGATGCCTGGTGATAC-3&#8217;</td>
		</tr>
		<tr>
			<td>Reverse primer specific to glw-c </td>
			<td></td>
			<td></td>
			<td>5&#8217;- CGGCCGGCGCATATATAG-3&#8217;</td>
		</tr>
	</tbody>
</table>

genotyping of sea anemone during early development

Described here is a PCR-based protocol to genotype the gastrula stage embryo of the anthozoan cnidarian Nematostella vectensis without sacrificing the life of the animal. Following in vitro fertilization and de-jellying, zygotes are allowed to develop for 24 h at room temperature to reach the early- to mid-gastrula stage. The gastrula embryos are then placed on an agarose gel bed in a Petri dish containing seawater. Under the dissecting microscope, a tungsten needle is used to surgically separate an aboral tissue fragment from each embryo. Post-surgery embryos are then allowed to heal and continue development. Genomic DNA is extracted from the isolated tissue fragment and used as a template for locus-specific PCR. The genotype can be determined based on the size of PCR products or presence/absence of allele-specific PCR products. Post-surgery embryos are then sorted according to the genotype. The duration of the entire genotyping process depends on the number of embryos to be screened, but it minimally requires 4&#8211;5 h. This method can be used to identify knockout mutants from a genetically heterogeneous population of embryos and enables analyses of phenotypes during development.

The Nematostella genome has a single locus that encodes a precursor protein for the neuropeptide GLWamide. Three knockout mutant alleles at this locus (glw-a, glw-b, and glw-c) have been previously reported5. Four heterozygous males carrying a wild-type allele (+) and knockout allele glw-c at the GLWamide locus (genotype: +/glw-c) were crossed with a heterozygous fem...

Watch this Scientific Journal Video about Genotyping of Sea Anemone during Early Development at JoVE.com

Genotyping of Sea Anemone during Early Development

The goal of this protocol is to genotype the sea anemone Nematostella vectensis during gastrulation without sacrificing the embryo.

Described here is a PCR-based protocol to genotype the gastrula stage embryo of the anthozoan cnidarian Nematostella vectensis without sacrificing the ...

This protocol enables a researcher to identify mutant sea anemones during early embryogenesis so that post-embryonic developmental phenotype can be analyzed. The main advantage of this technique is that it can be used to genotype individual sea anemones early in ontogeny without sacrificing the life of the animal. Demonstrating this procedure will be Miguel Silva, a graduate student from my laboratory.On the day before spawning induction, place the anemone nematostella vectensis in a temperature-and light-controlled incubator, programming the incubator so that the animals are exposed to eight hours of light at 25 degrees Celsius. On the following day, remove the animals from the incubator and leave them on a bench top at room temperature with the light on to allow spawning, which will occur within the following one and a half to two hours. Use a transfer pipette who's tip is cut to enlarge the opening to place egg packages from the female container into a sperm-containing male container and leave them in the male container for at least 15 minutes to allow for fertilization.After fertilization, to dejelly the egg packages, place them in seawater containing 3%cysteine on a glass Petri dish and gently agitate on a shaker for 12 minutes. Then break up clumps with a plastic pipette and continue to agitate for another two to three minutes until completely dejellied. Keep the fertilized eggs on the same dish at 16 degrees Celsius or at room temperature.On the next day, prepare DNA extraction buffer as described in the manuscript, mix well by vortexing, and aliquot it into PCR tubes by adding 20 microliters to each tube. Dissolve 1%agarose in seawater, pour it into a Petri dish to cover the bottom, and cool on a bench top to make a gel bed. Cover the gel bed with fresh seawater.Transfer 24-hour post-fertilization embryos using a pipette into the agarose gel bed-containing Petri dish. Insert a tungsten needle into a need holder and sterilize by dipping its tip in alcohol and placing it in flame to burn off the alcohol. Under a dissecting microscope, make a depression on the agarose by using the tungsten needle to remove a piece of surface agarose about size of an embryo to be manipulated.Then, using the tungsten needle, place the embryo into the depression with its lateral side facing down in order to restrict the movement of the embryo for microsurgery. In order to perform successful surgery, it is critical to restrict the movement of the embryo. With the tungsten needle, surgically remove a piece of aboral tissue along the oral aboral axis, located opposite to the oral blastoporal opening.With a P20 pipette, transfer the isolated aboral tissue to a PCR tube containing 20 microliters of DNA extraction buffer. Transfer the post-surgery embryo into a well of a 24-well plate containing at least 500 microliters of fresh seawater. And place the plate containing post-surgery embryos in an incubator at 16 degrees Celsius until genotyping is completed.To extract genomic DNA from single embryos, first briefly spin down the PCR tubes containing the DNA extraction buffer and isolated embryonic tissues using a mini centrifuge. Then incubate the tubes at 55 degrees Celsius for three hours while vortexing for 30 second every 30 minutes to ensure breakup of cell clumps and enhance cell lysis. After inactivating proteinase K at 95 degrees Celsius for four minutes, set up a PCR reaction using extracted genomic DNA as a template to amplify the locus of interest.Design desired primers as described in the manuscript. Set up a 20 microliter PCR reaction. After completed PCR, run agarose gel electrophoresis to determine the size and presence or absence of PCR products, making sure to adjust the condition of agarose gel electrophoresis depending on the expected size of PCR products.Use PCR results regarding the size and presence or absence or PCR products to assign a genotype to each post-surgical embryo and then sort embryos according to their genotype. The nematostella genome has a single locus that encodes a precursor protein for the neuropeptide, GLWamide. Three knockout mutant alleles at the locus have been previously reported.PCR results from randomly sampled embryos among the progeny of an F1 cross between one heterozygous female carrying an A knockout allele and heterozygous males carrying a C knockout allele show different genotypes. Embryos one and two show a single PCR band corresponding the expected size of A knockout allele. Embryos three and six show two PCR bands with the expected sizes for knockout alleles A and C.Embryos four, seven, and eight show a single PCR band corresponding to the expected size of C knockout allele.Embryo 5 shows no bands, suggesting the lack of primer binding. To rule out the possibility of genomic DNA extraction failure, another PCR was run using a reverse primer that can bind to the wild type sequence. Where a PCR product of an expected size was detected, genomic DNA extraction was successful.Whereas no product suggested a failure in extraction. Although this protocol is designed for sea anemone embryos, it sure be possible to apply this method to other cnidarians, such as corals and jellyfish, where genomic information and embryos are accessible. Following this procedure, the identified mutants are used to assess developmental phenotype.For instance, histological techniques such as immunostaining can be used to assess defects in morphology, while in situ hybridization, realtime quantitative RT-PCR, and RNA-seq can be used to assess defects in gene expression. Also, live imaging can be used to assess defects in behavior during post-embryonic development, such as in larvae or in early polyps.

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