Summary
The goal of this protocol is to genotype the sea anemone Nematostella vectensis during gastrulation without sacrificing the embryo.
Abstract
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 4–5 h. This method can be used to identify knockout mutants from a genetically heterogeneous population of embryos and enables analyses of phenotypes during development.
Introduction
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.
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Protocol
1. Induction of spawning, in vitro fertilization, and de-jellying
- Maintain Nematostella vectensis in seawater with a salinity of 12 parts per thousand (ppt) in darkness at 16 °C, feeding Artemia daily.
- 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 °C. Optional: Feed a small piece (<1 mm3) of oyster to individual animals before placing them into the incubator to enhance spawning.
- Leave the animals in the incubator for 1 h at 16 °C.
- 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 1.5–2 h.
- 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.
- 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.
- Use a plastic pipette to break up clumps and continue to agitate for another 2-3 min until completely de-jellied.
- Remove cysteine by replacing the media with fresh seawater for at least 5x.
- Keep the fertilized eggs on a glass Petri dish at 16 °C or RT.
2. Surgical removal of an aboral tissue from a gastrula embryo
- 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 μg/μL proteinase K. Use 20 μL of the extraction buffer per embryo. Mix well by vortexing and aliquot the buffer into PCR tubes.
- 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.
- Transfer 24 h post-fertilization (hpf) embryos (early- to mid-gastrula stage) into the Petri dish containing an agarose gel bed.
- 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.
- 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.
- 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.
- Use a P20 pipette to transfer the isolated aboral tissue (in <2 μL) to a PCR tube containing 20 μL of DNA extraction buffer.
- Transfer the post-surgery embryo into a well containing at least 500 μL of fresh seawater in a 24- or 96-well plate.
- Repeat steps 2.4–2.8 for the number of embryos as needed.
- Place the well plate containing post-surgery embryos in an incubator at 16 °C or RT until genotyping is completed.
3. Genomic DNA extraction and genotyping PCR
- 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).
- To extract genomic DNA from single embryos, incubate the PCR tubes at 55 °C for 3 h. Vortex for 30 s every 30 min to ensure breakup of cell clumps and enhance cell lysis.
- Incubate the PCR tubes at 95 °C for 5 min to inactivate proteinase K.
- Keep gDNA extracts at 4 °C or on ice, and immediately proceed to PCR.
NOTE: The protocol can be paused here by placing gDNA extracts in a -20 °C freezer. - Set up a PCR reaction using extracted gDNA as a template to amplify the genomic locus of interest.
- 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.
- 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.
- Use a typical 20 μL PCR reaction mix as follows: 5 μL of gDNA extracts, 8 μL of nuclease-free water, 4 μL of PCR buffer, 0.2 μL of 10mM dNTPs, 0.6 μL of DMSO, 1 μL of 10 μM forward primer, 1 μL of 10 μM reverse primer, and 0.2 μL 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).
- 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.
- 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.
- Sort embryos according to genotype.
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Representative Results
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.
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. Please click here to view a larger version of this figure.
Figure 2: Representative results of locus-specific PCR to confirm the presence of gDNA. Ten gDNA extracts that failed to generate PCR products in glw mutant allele-specific PCR experiments, including embryo 5 from Figure 1 (‘5’), 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 (‘5’). 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. Please click here to view a larger version of this figure.
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Discussion
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 (>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 (<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.
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Disclosures
The author has nothing to disclose.
Acknowledgments
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.
Materials
| Name | Company | Catalog Number | Comments |
| Drosophila Peltier Refrigerated Incubator | Shellab | SRI6PF | Used for spawning induction |
| Instant ocean sea salt | Instant ocean | 138510 | |
| Brine shrimp cysts | Aquatic Eco-Systems, Inc. | BS90 | |
| L-Cysteine Hydrochloride | Sigma Aldrich | C7352 | |
| Standard Orbital Shaker, Model 3500 | VWR | 89032-092 | |
| TRIS-HCl, 1M, pH8.0 | QUALITY BIOLOGICAL | 351-007-01 | |
| Potassium chloride | VWR | BDH9258 | |
| EDTA, 0.5M pH8 | VWR | BDH7830-1 | |
| Tween 20 | Sigma Aldrich | P9416 | |
| Nonidet-P40 Substitute | US Biological | N3500 | |
| Proteinase K solution (20 mg/mL), RNA grade | ThermoFisher | 25530049 | |
| Agarose | VWR | 710 | |
| Micro Dissecting needle holder | Roboz | RS-6060 | |
| Tungsten dissecting needle | Roboz | RS-6063 | |
| PCR Eppendorf Mastercycler Thermal Cyclers | Eppendorf | E6336000024 | |
| Phusion High-Fidelity DNA polymerase | New England BioLabs | M0530L | |
| dNTP mix | New England BioLabs | N0447L | |
| GLWamide universal forward primer | 5’- CATGCGGAGACCAAGCGCAAGGC-3’ | ||
| Reverse primer specific to glw-a | 5’-CCAGATGCCTGGTGATAC-3’ | ||
| Reverse primer specific to glw-c | 5’- CGGCCGGCGCATATATAG-3’ |
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