A Method for Microinjection of Patiria minata Zygotes

Summary

Methods that produce morphant embryos are essential to study developmental mechanisms and gene regulatory networks. The sea star Patiria miniata is an emerging model system for these studies. Here we present a protocol for obtaining gametes, producing cultures of embryos, and rapid microinjection of zygotes from this species.

Abstract

Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and evolution of developmental processes. The sea star, Patiria miniata, is gaining prevalence as a model system for these types of studies which were previously performed almost exclusively in the sea urchins, Strongylocentrotus purpuratus and Lytechinus variegatus. An advantage of these model systems is the ease of producing modified embryos in which a particular gene is up or downregulated, labeling a group of cells, or introducing a reporter gene. A single microinjection method is capable of creating a wide variety of such modified embryos. Here, we present a method for obtaining gametes from P. miniata, producing zygotes, and introducing perturbing reagents via microinjection. Healthy morphant embryos are subsequently isolated for quantitative and qualitative studies of gene function. The availability of genome and transcriptome data for this organism has increased the types of studies that are performed and the ease of executing them.

Introduction

The sea star, Patiria miniata, (commonly known as the bat star) is emerging as an interesting and versatile model system for a variety of cellular^1-3, developmental^4,5, evolutionary^6-8, and ecological studies^9-11. Adult P. miniata are distributed along the pacific coast from Sitka, Alaska to Baja, California¹² and are readily maintained in marine aquaria. Oocytes are obtainable year round and each female can shed tens of thousands of eggs. Oocytes are easily matured and fertilized externally¹³. The resulting embryos are transparent allowing for easy obs....

Protocol

Keep all sea water or artificial seawater (SW), adult animals, and cultures at 15 °C as much as is practical. Ensure eggs and zygotes are kept immersed in SW.

Commercially prepared sea salts reconstituted with distilled or reverse osmosis water serves well as a source of SW. Check salinity using a hydrometer and adjust salts or water to achieve optimum levels. Keep specific gravity levels between 1.020 to 1.025. Keep all glassware and plasticware separate from all other labware to avoid any contamination with chemicals. Clean embryo grade labware by rinsing with deionized water or occasional soaking in dilute sodium hypochlorite follow....

Results

The goal of this protocol is to introduce reagents into embryos. We demonstrate the effectiveness of the protocol by injecting a DNA reporter construct that drives the expression of green fluorescent protein (GFP). Injected embryos express GFP in clonal patches (Figure 4A-B) as the DNA incorporates during early cleavage. Many reagents that are desirable to introduce into embryos are toxic in high quantities and to suboptimal batches of embryos. Toxicity manifests by delaying development, arresting develo.......

Discussion

There are two critical steps that are difficult for novice users of this technique but are essential for successfully creating morphant embryos. The first is selecting healthy oocytes that will mature and fertilize properly. The percentage of normal development in a culture depends on the season, the health of the animal, and the number of times that oocytes have been harvested from a single individual. Oocytes tend to be of better quality from April through October. It is important to look carefully at the oocytes befor.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1-Methyladenine	Acros Organics (Fisher Scientific)	AC20131-1000
190 micron nitex nylon filter	Small Parts (originally Sefar)	CMN-0185-C/5PK-05
100 micron nitex nylon filter	Small Parts (originally Sefar)	CMN-0105-C/5PK-05
Polystyrene Petri dishes, 60 mm x 15 mm	Fisher Scientific	FB0875713A
Capillary tubing	FHC, Inc	30-30-0	For pulling microinjection needles
Model P-97 Needle Puller	Sutter Instruments	P-97
Dextran, Rhodamine Green	Life Technologies	D7163	If injecting a GFP expression reporter, it is helpful to substitute Texas Red dextran as an injection tracer
Instant Ocean Sea Salt	Doctors Foster and Smith	CD-116528	Also available in many pet stores
Microloader tips	Eppendorf	5242 956.003