Genotyping of Sea Anemone during Early Development

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 g....

Protocol

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....

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 fem.......

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 .......

Materials

List of materials used in this article

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’