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 animals before placing them into the incubator to enhance spawning.</li>
	<li>Leave the animals in the incubator for 1 h at 16 &#176;C.</li>
	<li>Remove the animals from the incubator and leave them on a benchtop with light at room temperature (RT) to allow spawning. Spawning usually occurs within the next 1.5&#8211;2 h.</li>
	<li>If males and females are in separate containers, place egg packages from the female container into a sperm-containing male container by using a transfer pipette whose tip is cut to enlarge the opening so that the eggs are not damaged by mechanical stress during transfer. Allow eggs to be fertilized by leaving them in the male container for at least 15 min.</li>
	<li>De-jelly the egg packages in seawater containing 3% cysteine (pH 7.4) on a Petri dish or in a 15 mL tube. Gently agitate on a shaker for 12 min.</li>
	<li>Use a plastic pipette to break up clumps and continue to agitate for another 2-3 min until completely de-jellied.</li>
	<li>Remove cysteine by replacing the media with fresh seawater for at least 5x.</li>
	<li>Keep the fertilized eggs on a glass Petri dish at 16 &#176;C or RT.</li>
</ol>
2. Surgical removal of an aboral tissue from a gastrula embryo
<ol>
	<li>Prepare a DNA extraction buffer consisting of 10 mM Tris-HCl (pH 8), 50 mM KCl, 1 mM EDTA, 0.3% Tween20, 0.3% NP40, and 1 &#956;g/&#956;L proteinase K. Use 20 &#956;L of the extraction buffer per embryo. Mix well by vortexing and aliquot the buffer into PCR tubes.</li>
	<li>Dissolve 1% agarose in seawater and pour it into a Petri dish to cover the bottom. Cool on a benchtop to make a gel bed. Pour fresh seawater to cover the gel bed in the Petri dish.</li>
	<li>Transfer 24 h post-fertilization (hpf) embryos (early- to mid-gastrula stage) into the Petri dish containing an agarose gel bed.</li>
	<li>Insert a tungsten needle into a needle holder and sterilize by dipping the needle tip in alcohol (70% or higher) and placing it in flame to burn off the alcohol.</li>
	<li>Under a dissecting microscope (at 20x to 40x magnification), use the tungsten needle to make a depression on the agarose bed by removing a piece of surface agarose about the size of an embryo to be manipulated, and place the embryo onto the depression with its lateral side facing down in order to restrict the movement of the embryo for microsurgery.</li>
	<li>Use the tungsten needle to surgically excise a piece of aboral tissue located opposite to the oral blastoporal opening. An aboral one-third to one-quarter of the embryonic tissue along the oral-aboral axis is usually sufficient.</li>
	<li>Use a P20 pipette to transfer the isolated aboral tissue (in &#60;2 &#956;L) to a PCR tube containing 20 &#956;L of DNA extraction buffer.</li>
	<li>Transfer the post-surgery embryo into a well containing at least 500 &#956;L of fresh seawater in a 24- or 96-well plate.</li>
	<li>Repeat steps 2.4&#8211;2.8 for the number of embryos as needed.</li>
	<li>Place the well plate containing post-surgery embryos in an incubator at 16 &#176;C or RT until genotyping is completed.</li>
</ol>
3. Genomic DNA extraction and genotyping PCR
<ol>
	<li>Briefly spin down the PCR tubes containing the DNA extraction buffer and isolated embryonic tissues using a mini-centrifuge (e.g. at 2,680 x g for 10 s).</li>
	<li>To extract genomic DNA from single embryos, incubate the PCR tubes at 55 &#176;C for 3 h. Vortex for 30 s every 30 min to ensure breakup of cell clumps and enhance cell lysis.</li>
	<li>Incubate the PCR tubes at 95 &#176;C for 5 min to inactivate proteinase K.</li>
	<li>Keep gDNA extracts at 4 &#176;C or on ice, and immediately proceed to PCR. 
	NOTE: The protocol can be paused here by placing gDNA extracts in a -20 &#176;C freezer.</li>
	<li>Set up a PCR reaction using extracted gDNA as a template to amplify the genomic locus of interest.
	<ol>
		<li>If different alleles at the locus of interest differ in size so that the size difference can be detected by agarose gel electrophoresis, design a single set of primers to amplify the entire locus.</li>
		<li>Alternatively, use allele-specific primers that generate PCR products only in the presence of the specific allele; for instance, by designing the primer that binds to a region containing insertion/deletion mutations.</li>
		<li>Use a typical 20 &#956;L PCR reaction mix as follows: 5 &#956;L of gDNA extracts, 8 &#956;L of nuclease-free water, 4 &#956;L of PCR buffer, 0.2 &#956;L of 10mM dNTPs, 0.6 &#956;L of DMSO, 1 &#956;L of 10 &#956;M forward primer, 1 &#956;L of 10 &#956;M reverse primer, and 0.2 &#956;L of DNA polymerase (see Table of Materials). 
		NOTE: Multiple primers can be used in a PCR reaction. For instance, one universal forward primer and two allele-specific reverse primers can be combined, as long as the two reverse primers are designed to generate PCR products of distinct sizes so that the presence/absence of the two alleles can be unambiguously determined by gel electrophoresis (see representative results section).</li>
	</ol>
	</li>
	<li>Run agarose gel electrophoresis to determine the size and presence/absence of PCR products. Adjust the condition of agarose gel electrophoresis (e.g., agarose gel percentage, V/cm, and duration) depending on the expected size of PCR products.</li>
	<li>Use the results from PCR regarding the size and presence/absence of PCR products to assign a genotype to each post-surgery embryo. For instance, if different alleles are expected to generate PCR products of different sizes, use the size information to assign the genotype for each embryo. If allele-specific primers are used, the data on the presence/absence of PCR products should be used to assign a genotype to each embryo.</li>
	<li>Sort embryos according to genotype.</li>
</ol>

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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=Sea+anemone+genome+reveals+ancestral+eumetazoan+gene+repertoire+and+genomic+organization.">Sea anemone genome reveals ancestral eumetazoan gene repertoire and genomic organization.</a> Science. 317 (5834), 86-94 (2007).</li><li>Magie, C. R., Daly, M., Martindale, M. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastrulation+in+the+cnidarian+Nematostella+vectensis+occurs+via+invagination+not+ingression.">Gastrulation in the cnidarian Nematostella vectensis occurs via invagination not ingression.</a> Developmental Biology. 305 (2), 483-497 (2007).</li><li>Nakanishi, N., Martindale, M. 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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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The author has nothing to disclose.

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 advantage of the ability of the oral halves of the sea anemone embryo to regulate and develop10,11, and the majority of the embryos (&#62;90%) typically survive the surgery and develop normally under appropriate culture conditions. The tissue amount required for this genotyping assay is less than one-half of an entire gastrula embryo, and negative results due to gDNA extraction failure are rare (&#60;5%). Given that only a small amount of tissue is necessary, it is likely possible to use pre-gastrula stage embryos for this genotyping assay; although, this has yet to be tested. This method can be performed efficiently as long as the animal has not reached a stage of active swimming (i.e., before the free-swimming planula stage). This genotyping method is particularly advantageous in cases requiring the performance of phenotype analyses during development.
One limitation of this protocol is that the number of embryos able to screened may be limited. In particular, surgical removal of an aboral tissue can take one to two minutes per embryo, especially for the uninitiated. An experienced researcher should be able to complete the entire genotyping assay for at least 80 embryos per day, but studies involving hundreds or thousands of embryos are likely too time-consuming to complete in one day.
Researchers also need to be mindful that the phenotype observed in post-surgery mutants may be different from that in intact mutants, for instance, due to the effect of gene knockout on healing and/or embryonic regulation in gastrulae. This possibility should be tested by examining whether the phenotype found in post-surgery mutants is indeed observable in intact mutants.
There are several alternative approaches to the described method of genotyping. First, immunostaining with an antibody against a protein whose expression is lost in knockout mutants can be performed to identify knockout mutant individuals from a genetically heterogeneous population of developing animals. Second, in situ hybridization can be used for this purpose, if the riboprobe can be designed so that it does not hybridize to mutant mRNAs. For instance, if the mutant alleles of a knockout animal carry large deletion mutations in the same region of the gene, the riboprobe can be designed to hybridize to the region of the gene deleted in the knockout mutants. In both cases, knockout mutants are expected to show no labeling, while heterozygous and wild-type individuals should show labeling. However, the animals will need to be sacrificed due to tissue fixation required for the staining. Finally, knockout mutants may be raised to sexual maturity and crossed with each other to generate a progeny, all of which should be knockout mutants, and analyses of developmental phenotype can be performed using this progeny6. This method requires that the knockout animals are viable and capable of reproduction and is thus limited in its application to nonessential genes.
Although the described genotyping protocol is designed for sea anemone embryos, it is possible to use this method with other cnidarians in which both genomic information and embryos are accessible (e.g., corals12 and jellyfish13), as long as the embryos are capable of healing and regulation upon surgical removal. Successful CRISPR-mediated gene modification experiments have been already reported in corals14 as well as hydrozoan jellyfish15,16. Future applications of this genotyping protocol to non-sea anemone cnidarians will be important for studies of the genetic basis of their development. This, in turn, will be key to gaining mechanistic insights into the evolution of remarkably diverse cnidarian development.

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&amp;rsquo;- CATGCGGAGACCAAGCGCAAGGC-3&amp;rsquo;</td></tr><tr><td>Reverse primer specific to &lt;em&gt;glw&lt;sup&gt;-a&lt;/sup&gt;&lt;/em&gt;</td><td></td><td></td><td>5&amp;rsquo;-CCAGATGCCTGGTGATAC-3&amp;rsquo;</td></tr><tr><td>Reverse primer specific to &lt;em&gt;glw&lt;sup&gt;-c &lt;/sup&gt;&lt;/em&gt;</td><td></td><td></td><td>5&amp;rsquo;- CGGCCGGCGCATATATAG-3&amp;rsquo;</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 female carrying a wild-type allele and different knockout allele glw-a at the same locus (genotype: +/glw-a) to generate a progeny. There are four possible genotypes in the progeny: glw-a/glw-c, +/glw-c, glw-a/+, and +/+. Out of all the progeny, eight embryos were randomly selected for this representative genotyping assay. For genotyping PCR, one universal forward primer and two allele-specific reverse primers were designed5. The reverse primer specific to glw-a binds to a region containing insertion mutations, and the expected size of the PCR product is 151 bp. The reverse primer specific to glw-c binds to a region containing both insertion and deletion mutations, and the expected size of the PCR product is 389 bp. Neither reverse primer can bind to the wild-type sequence, and thus no PCR products will be generated from wild-type embryos. Figure 1 shows a representative result of the PCR assay. Embryos 1 and 2 show a single PCR band consistent with the expected size of glw-a. Embryos 3 and 6 show two PCR bands that correspond to the expected sizes for alleles glw-a and glw-c. Embryos 4, 7, and 8 show a single PCR band consistent with the expected size of glw-c. Embryo 5 shows no bands, suggesting the lack of primer binding.
To rule out the possibility of gDNA extraction failure, another PCR was run using a reverse primer that can bind to the wild-type sequence, which showed a PCR product of an expected size (1290 bp; Figure 2). It should be noted that in Figure 2, one of the samples (indicated by *) showed no PCR products, suggesting a failure in gDNA extraction.
Based on the above results, the genotype of each embryo is interpreted to be as follows: embryo 1: +/glw-a, embryo 2: +/glw-a, embryo 3: glw-a/glw-c, embryo 4: +/glw-c, embryo 5: +/+, embryo 6: glw-a/glw-c, embryo 7: +/glw-c, and embryo 8: +/glw-c.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59541/59541fig1.jpg" /> 
Figure 1: Representative results of a genotyping PCR assay. 1-8 represent genotyping PCR results from randomly sampled embryos among the progeny of an F1 heterozygous mutant cross between one +/glw-a female and +/glw-c males. gDNA was extracted from an embryonic tissue fragment and used as a PCR template. The GLWamide locus was targeted for PCR amplification, and one universal forward primer and two allele-specific reverse primers were used (Table of Materials). The reverse primer specific to glw-a generates a 151 bp PCR band (1, 2, 3, 6), while the reverse primer specific to glw-c generates a 389 bp PCR band (3, 4, 6, 7, 8). Neither reverse primer can bind to the wild-type sequence, and thus no PCR products will be generated from wild-type embryos (5). The 1.5% agarose gel was run at 128 V for 25 min. A 100 bp DNA ladder was used. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59541/59541fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59541/59541fig2.jpg" /> 
Figure 2: Representative results of locus-specific PCR to confirm the presence of gDNA.&#160;Ten gDNA extracts that failed to generate PCR products in glw mutant allele-specific PCR experiments, including embryo 5 from Figure 1 (&#8216;5&#8217;), were used as PCR templates for the PCR experiment shown. A universal forward and reverse GLWamide-locus-specific primers were used to generate a 1290 bp PCR product from a wild-type allele. Nine out of ten DNA extracts (except for the one indicated*) showed a PCR band of expected size, including embryo 5 from Figure 1 (&#8216;5&#8217;). This suggests that the failure to generate PCR products from the embryo 5 was not due to the lack of a sufficient gDNA template. The 1.5% agarose gel was run at 128 V for 25 min. 1 kb DNA ladder was used. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59541/59541fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

genotyping-of-sea-anemone-during-early-development

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

5.5K Views.  University of Arkansas. Dit protocol stelt een onderzoeker in staat om gemuteerde zee-anemonen te identificeren tijdens vroege embryogenese, zodat post-embryonale ontwikkelingsfenotype kan worden geanalyseerd. Het belangrijkste voordeel van deze techniek is dat het kan worden gebruikt om genotype individuele zee anemonen vroeg in ontogeny zonder het leven van het dier op te offeren. Het aantonen van deze procedure zal Miguel Silva zijn, een afgestudeerde student van mijn laboratorium.Plaats op de dag voor het...

Video: Genotyping of Sea Anemone during Early Development

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the cephalochordate amphioxus, Branchiostoma lanceolatum. We use constructs for membrane and nuclear targeted mCherry and eGFP that have been modified to accommodate amphioxus codon usage and Kozak consensus sequences. We describe the type of injection needles to be used, the immobilization protocol for the unfertilized oocytes, and the overall injection set-up. This technique generates fluorescently labeled embryos, in which the dynamics of cell behaviors during early development can be analyzed using the latest in vivo imaging strategies. The development of a microinjection technique in this amphioxus species will allow live imaging analyses of cell behaviors in the embryo as well as gene-specific manipulations, including gene overexpression and knockdown. Altogether, this protocol will further consolidate the basal chordate amphioxus as an animal model for addressing questions related to the mechanisms of embryonic development and, more importantly, to their evolution.

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here the robust and efficient expression of fluorescent proteins after mRNA injection into unfertilized oocytes of Branchiostoma lanceolatum. The development of the microinjection technique in this basal chordate will pave the way for far-reaching technical innovations in this emerging model system, including in vivo imaging and gene-specific manipulations.

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the ...

Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis

Cnidarians, and specifically Hydra, were the first animals shown to regenerate damaged or severed structures, and indeed such studies arguably launched modern biological inquiry through the work of Trembley more than 250 years ago. Presently the study of regeneration has seen a resurgence using both &#34;classic&#34; regenerative organisms, such as the Hydra, planaria and Urodeles, as well as a widening spectrum of species spanning the range of metazoa, from sponges through mammals. Besides its intrinsic interest as a biological phenomenon, understanding how regeneration works in a variety of species will inform us about whether regenerative processes share common features and/or species or context-specific cellular and molecular mechanisms. The starlet sea anemone, Nematostella vectensis, is an emerging model organism for regeneration. Like Hydra, Nematostella is a member of the ancient phylum, cnidaria, but within the class anthozoa, a sister clade to the hydrozoa that is evolutionarily more basal. Thus aspects of regeneration in Nematostella will be interesting to compare and contrast with those of Hydra and other cnidarians. In this article, we present a method to bisect, observe and classify regeneration of the aboral end of the Nematostella adult, which is called the physa. The physa naturally undergoes fission as a means of asexual reproduction, and either natural fission or manual amputation of the physa triggers re-growth and reformation of complex morphologies. Here we have codified these simple morphological changes in a Nematostella Regeneration Staging System (the NRSS). We use the NRSS to test the effects of chloroquine, an inhibitor of lysosomal function that blocks autophagy. The results show that the regeneration of polyp structures, particularly the mesenteries, is abnormal when autophagy is inhibited.

Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis

Here we demonstrate how to induce and monitor regeneration in the Starlet Sea Anemone Nematostella vectensis, a model cnidarian anthozoan. We demonstrate how to amputate and categorize regeneration using a morphological staging system, and we use this system to reveal a requirement for autophagy in regenerating polyp structures.

Cnidarians, and specifically Hydra, were the first animals shown to regenerate damaged or severed structures, and indeed such studies arguably launched ...

The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions

Remarkably few cell-to-cell signal transduction pathways are necessary during embryonic development to generate the large variety of cell types and tissues in the adult body form. Yet, each year more components of individual signaling pathways are discovered, and studies indicate that depending on the context there is significant cross-talk among most of these pathways. This complexity makes studying cell-to-cell signaling in any in vivo developmental model system a difficult task. In addition, efficient functional analyses are required to characterize molecules associated with signaling pathways identified from the large data sets generated by next generation differential screens. Here, we illustrate a straightforward method to efficiently identify components of signal transduction pathways governing cell fate and axis specification in sea urchin embryos. The genomic and morphological simplicity of embryos similar to those of the sea urchin make them powerful in vivo developmental models for understanding complex signaling interactions. The methodology described here can be used as a template for identifying novel signal transduction molecules in individual pathways as well as the interactions among the molecules in the various pathways in many other organisms.

The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions

This video article details a straightforward in vivo methodology that can be used to systematically and efficiently characterize components of complex signaling pathways and regulatory networks in many invertebrate embryos.

Remarkably few cell-to-cell signal transduction pathways are necessary during embryonic development to generate the large variety of cell types and ...

In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

Corals are important ocean invertebrates that are critical for overall ocean health as well as human health. However, due to human impacts such as rising ocean temperatures and ocean acidification, corals are increasingly under threat. To tackle these challenges, advances in cell and molecular biology have proven to be crucial for diagnosing the health of corals. Modifying some of the techniques commonly used in human medicine could greatly improve researchers&#39; ability to treat and save corals. To address this, a protocol for in situ hybridization used primarily in human medicine and evolutionary developmental biology has been adapted for use in adult corals under stress.
The purpose of this method is to visualize the spatial expression of an RNA probe in adult coral tissue that has been embedded in paraffin and sectioned onto glass slides. This method focuses on removal of the paraffin and rehydration of the sample, pretreatment of the sample to ensure permeability of the sample, pre-hybridization incubation, hybridization of the RNA probe, and visualization of the RNA probe. This is a powerful method when using non-model organisms to discover where specific genes are expressed, and the protocol can be easily adapted for other non-model organisms. However, the method is limited in that it is primarily qualitative, because expression intensity can vary depending on the amount of time used during the visualization step and the concentration of the probe. Furthermore, patience is required, as this protocol can take up to 5 days (and in many cases, longer) depending on the probe being used. Finally, non-specific background staining is common, but this limitation can be overcome.

In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

The goal of this protocol is to perform in situ hybridization on adult coral samples that have been embedded in paraffin and sectioned onto glass slides. This is a qualitative method used to visualize the spatial expression of an RNA anti-sense probe in paraffin-embedded tissues.

Corals are important ocean invertebrates that are critical for overall ocean health as well as human health. However, due to human impacts such as rising ...

Environment

Chromatin Immunoprecipitation in the Cnidarian Model System Exaiptasia diaphana

Histone post-translational modifications (PTMs) and other epigenetic modifications regulate the chromatin accessibility of genes to the transcriptional machinery, thus affecting an organism&#39;s capacity to respond to environmental stimuli. Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) has been widely utilized to identify and map protein-DNA interactions in the fields of epigenetics and gene regulation. However, the field of cnidarian epigenetics is hampered by a lack of applicable protocols, partly due to the unique features of model organisms such as the symbiotic sea anemone Exaiptasia diaphana, whose high water content and mucus amounts obstruct molecular methods. Here, a specialized ChIP procedure is presented, which facilitates the investigation of protein-DNA interactions in E. diaphana gene regulation. The cross-linking and chromatin extraction steps were optimized for efficient immunoprecipitation and then validated by performing ChIP using an antibody against the histone mark&#160;H3K4me3. Subsequently, the specificity and effectiveness of the ChIP assay were confirmed by measuring the relative occupancy of H3K4me3 around several constitutively activated gene loci using quantitative PCR and by next-generation sequencing for genome-wide scale analysis. This optimized ChIP protocol for the symbiotic sea anemone E. diaphana facilitates the investigation of the protein-DNA interactions involved in organismal responses to environmental changes that affect symbiotic cnidarians, such as corals.

Chromatin Immunoprecipitation in the Cnidarian Model System Exaiptasia diaphana

A chromatin immunoprecipitation (X-ChIP) assay against the histone mark&#160;H3K4me3 for the model organism Exaiptasia diaphana is presented. The specificity and effectiveness of the assay are confirmed by quantitative PCR and next-generation sequencing. This protocol enables the increased investigation of protein-DNA interactions in the sea anemone E. diaphana.

Histone post-translational modifications (PTMs) and other epigenetic modifications regulate the chromatin accessibility of genes to the transcriptional ...

Genetics

Wholemount In Situ Hybridization for Astyanax Embryos

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of genes. Prior research into this emerging model system capitalized on comprehensive genome-wide investigations that have identified numerous quantitative trait loci (QTL) associated with various cave-associated phenotypes. However, the ability to connect genes of interest to the heritable basis for phenotypic change remains a significant challenge. One technique that can facilitate deeper understanding of the role of development in troglomorphic evolution is whole-mount in situ hybridization. This technique can be implemented to directly compare gene expression between cave- and surface-dwelling forms, nominate candidate genes underlying established QTL, identify genes of interest from next-generation sequencing studies, or develop other discovery-based approaches. In this report, we present a simple protocol, supported by a flexible checklist, that can be widely adapted for use well beyond the presented study system. It is hoped that this protocol can serve as a broad resource for the Astyanax community and beyond.

Wholemount In Situ Hybridization for Astyanax Embryos

This protocol enables visualization of gene expression in embryonic Astyanax cavefish. This approach has been developed with the goal of maximizing gene expression signal, while minimizing non-specific background staining.

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of ...

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One challenge is that embryogenesis dynamically unfolds over a period of about 13 h; this half day-long timescale has constrained the scope of experiments by limiting the number of embryos that can be imaged. Here, we describe a semi-high-throughput protocol that allows for the simultaneous 3D time-lapse imaging of development in 80&#8211;100 embryos at moderate time resolution, from up to 14 different conditions, in a single overnight run. The protocol is straightforward and can be implemented by any laboratory with access to a microscope with point visiting capacity. The utility of this protocol is demonstrated by using it to image two custom-built strains expressing fluorescent markers optimized to visualize key aspects of germ-layer specification and morphogenesis. To analyze the data, a custom program that crops individual embryos out of a broader field of view in all channels, z-steps, and timepoints and saves the sequences for each embryo into a separate tiff stack was built. The program, which includes a user-friendly graphical user interface (GUI), streamlines data processing by isolating, pre-processing, and uniformly orienting individual embryos in preparation for visualization or automated analysis. Also supplied is an ImageJ macro that compiles individual embryo data into a multi-panel file that displays maximum intensity fluorescence projection and brightfield images for each embryo at each time point. The protocols and tools described herein were validated by using them to characterize embryonic development following knock-down of 40 previously described developmental genes; this analysis visualized previously annotated developmental phenotypes and revealed new ones. In summary, this work details a semi-high-throughput imaging method coupled with a cropping program and ImageJ visualization tool that, when combined with strains expressing informative fluorescent markers, greatly accelerates experiments to analyze embryonic development.

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

This work describes a semi-high-throughput protocol that allows simultaneous 3D time-lapse imaging of embryogenesis in 80&#8211;100 C. elegans embryos in a single overnight run. Additionally, image processing and visualization tools are included to streamline data analysis. The combination of these methods with custom reporter strains enables detailed monitoring of embryogenesis.

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One ...

Identification of Hemolytic and Phospholipase Activity in Crude Extracts from Sea Anemones by Straightforward Bioassays

Sea anemone venom composition includes polypeptide and non-proteins molecules. Cytolytic components have a high biotechnological and biomedical potential for designing new molecular tools. Sea anemone venom locates in glandular cells from ectoderm and sub-cellular structures called nematocysts, both of which are distributed throughout the sea anemone body. This characteristic implies challenges because the cells and nematocyst must be lysed to release the venom components with other non-toxic molecules. Therefore, first, the venom is derived from a crude extract (mixture of different and diverse molecules and tissue debris). The next step is to detect polypeptides with specific bioactivities. Here, we describe an efficient strategy to obtain the sea anemone crude extract and bioassay to identify the presence of cytolysins. The first step involves inexpensive and straightforward techniques (stirred and freeze-thaw cycle) to release cytolysins. We obtained the highest cytolytic activity and protein (~500 mg of protein from 20 g of dry weight). Next, the polypeptide complexity of the extract was analyzed by SDS-PAGE gel detecting proteins with molecular weights between 10 kDa and 250 kDa. In the hemolytic assay, we used sheep red blood cells and determined HU50 (11.1 &plusmn; 0.3 &#181;g/mL). In contrast, the presence of phospholipases in the crude extract was determined using egg yolk as a substrate in a solid medium with agarose. Overall, this study uses an efficient and inexpensive protocol to prepare the crude extract and applies replicable bioassays to identify cytolysins, molecules with biotechnological and biomedical interests.

Here, we describe a protocol to obtain crude venom extract from sea anemone and detect its hemolytic and phospholipase activity.

Sea anemone venom composition includes polypeptide and non-proteins molecules. Cytolytic components have a high biotechnological and biomedical potential ...

Biochemistry

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...

Biology

High Throughput Microinjections of Sea Urchin Zygotes

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of treatment solution is loaded into a microinjection needle that is used to physically inject individual immobilized cells or embryos. Despite the need for initial training to perform this procedure for high-throughput delivery, microinjection offers maximum efficiency and reproducible delivery of a wide variety of treatment solutions (including complex mixtures of samples) into cells, eggs or embryos. Applications to microinjections include delivery of DNA constructs, mRNAs, recombinant proteins, gain of function, and loss of function reagents. Fluorescent or colorimetric dye is added to the injected solution to enable instant visualization of efficient delivery as well as a tool for reliable normalization of the amount of the delivered solution. The described method enables microinjection of 100-400 sea urchin zygotes within 10-15 min.

Microinjection is a common technique used to deliver DNA constructs, mRNAs, morpholino antisense oligonucleotides or other treatments into eggs, embryos, and cells of various species.

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of ...

Genotyping of Sea Anemone during Early Development

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