Genotyping of Sea Anemone during Early Development

Podsumowanie

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

Streszczenie

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.

Wprowadzenie

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 vectensis^5,6,7,8,9. 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 genotyping^6,7. 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.

Protokół

1. Induction of spawning, in vitro fertilization, and de-jellying

1. Maintain Nematostella vectensis in seawater with a salinity of 12 parts per thousand (ppt) in darkness at 16 °C, feeding Artemia daily.
2. 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.
3. Leave the animals in the incubator for 1 h at 16 °C.
4. 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.
5. 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.
6. 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.
7. Use a plastic pipette to break up clumps and continue to agitate for another 2-3 min until completely de-jellied.
8. Remove cysteine by replacing the media with fresh seawater for at least 5x.
9. 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

1. 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.
2. 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.
3. Transfer 24 h post-fertilization (hpf) embryos (early- to mid-gastrula stage) into the Petri dish containing an agarose gel bed.
4. 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.
5. 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.
6. 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.
7. Use a P20 pipette to transfer the isolated aboral tissue (in <2 μL) to a PCR tube containing 20 μL of DNA extraction buffer.
8. Transfer the post-surgery embryo into a well containing at least 500 μL of fresh seawater in a 24- or 96-well plate.
9. Repeat steps 2.4–2.8 for the number of embryos as needed.
10. 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

1. 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).
2. 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.
3. Incubate the PCR tubes at 95 °C for 5 min to inactivate proteinase K.
4. 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.
5. Set up a PCR reaction using extracted gDNA as a template to amplify the genomic locus of interest.
   1. 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.
   2. 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.
   3. 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).
6. 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.
7. 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.
8. Sort embryos according to genotype.

Wyniki

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...

Dyskusje

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 ...

Materiały

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’