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.
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.
The sea star, Patiria miniata, (commonly known as the bat star) is emerging as an interesting and versatile model system for a variety of cellular1-3, developmental4,5, evolutionary6-8, and ecological studies9-11. 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 observation; they develop synchronously, and require only sea water for development. Whole genome assembly and multiple transcriptomes are also available for P. miniata (Echinobase.org). Such advantages make them ideal for a range of research and teaching purposes.
In recent years, P. miniata has become a model system for developmental gene regulatory network analyses14-16. The aim of such studies is to identify the entire compliment of regulatory genes and determine the network of their interactions. Much of this work entails perturbing gene expression through introduction of antisense oligonucleotides or in vitro synthesized mRNAs. Additionally, cis regulatory analyses are used to characterize the function of regulatory DNA15. These analyses require introduction of perturbation reagents and/or DNA reporter constructs into embryos. Furthermore, to characterize the downstream effects of these perturbations, one must assay many embryos for changes in gene expression of potential targets. Techniques for microinjection of many hundreds of zygotes are central for this work.
Echinoderms, including P. miniata, require many months to reach sexual maturity. Because of this, it is generally not practical to develop and maintain transgenic lines of these animals for experimentation. Therefore, breeding of transgenic adults cannot efficiently create modified embryos. Instead, perturbation must occur de novo through microinjection. Microinjection offers an opportunity to modify embryos with reagents that are not cell-permeable. The following protocol describes a method to introduce DNA, mRNA, cell tracers, and morpholino antisense oligonucleotides into hundreds of fertilized eggs in one 2-3 hr sitting through microinjection. This produces sufficient material for a variety of downstream experiments including, but not limited to, qPCR, in situ hybridization, RNA-Seq, and western blotting.
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 followed by rinsing several times in water.
1. Obtaining and Maturing Gametes from Patiria miniata Adults
2. Fertilization of Mature Oocytes
3. De-jellying and Rowing Fertilized Eggs
4. Injection of Zygotes
5. Collecting Injected Embryos for Downstream Analysis
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 development in early cleavage or at the fertilized egg stage, or by imposing aberrant development (Figure 5A-C).
Figure 1. Obtaining gametes from P. miniata adults. A) To excise gonads make an incision on the side of an arm proximal to the middle of the animal. B) The dark brown material pulled from this incision is gut tissue. Avoid pulling gut tissue as it will injure the animal. C) Ovary tissue is typically orange in color as shown here, but may also be yellow or light brown. D) Testes are white or beige in color, as shown. All scale bars denote 1 cm.
Figure 2. Maturation and fertilization of oocytes. A) Healthy oocytes that are ready for maturation are large and clear with a visible germinal vesicle. B) Oocytes that are not ready for maturation are smaller, brown, and grainy in appearance. C) An Oocyte that has been matured with 1-methyladenine. The germinal vesicle has broken down. D) A zygote surrounded by a fertilization envelope. E) Avoid cultures with large oocytes that cannot be matured because these oocytes will not be removed by filtration. Such oocytes are slightly smaller than the one in A. and are darker brown and grainy textured. The scale bar in A denotes 50 μm and represents the scale for images A-E.
Figure 3. Apparatuses for assembly. A-A’) A mesh filter fastened to a 50 ml conical tube. The tapered-end of the tube and the middle of the lid are cut out to allow cultures to be poured through the tube and filter. B) An injection dish constructed from the lid of a 60 x 15 mm polystyrene culture dish. A circle drawn around the center outlines the field of view of the injection microscope and serves as a guide for rowing embryos. A line scored into the dish is useful for breaking injection needles. C-C’) Mouth pipette set-up. Fit a mouthpiece into one end of a several foot long rubber tube. Fit the other end to a Pasteur pipette, which has been shaped by briefly melting and pulling the narrow end of the glass. D) Schematic demonstrating an ideal rowing Pasteur pipette shape and width to promote sticking without damaging embryos. When breaking the end off of the pulled pipette, it is helpful if the end is tapered to prevent emerging zygotes from floating away from the dish. All scale bars denote 3 cm.
Figure 4. Reagents Successfully Introduced by Microinjection. A) DIC of a blastula stage embryo injected with a GFP reporter plasmid. The embryo has normal morphology. B) GFP expression in the embryo shown in A. C) DIC image of two morphologically normal blastula stage embryos. D) One embryo from C emits a red fluorescence from the introduction of Texas Red dextran. The other embryo is un-injected and exhibits no fluorescence. Scale bars denote 50 μm. Scale bar in A represents the scale for A and B. Scale bar in C represents the scale for C and D.
Figure 5. Overinjection and reagent toxicity result in arrested or aberrant development. All images are of embryos 24 hr post-injection from the same injection batch. A-A’) An overinjected zygote arrested at the one-cell stage. B-B’) An embryo arrested in early cleavage. A healthy embryo will divide symmetrically, while this embryo arrested due to abnormal cell divisions. C-C’) An embryo with wildly aberrant development due to toxicity. While this embryo is shaped roughly like a blastula embryo (D), it is asymmetrical and abnormally thickened. D-D’) A properly developing injected blastula stage embryo. Scale bar in A denotes 50 μm and represents the scale for all panels.
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 before proceeding to ensure that the majority look like Figure 2A rather than Figure 2B. It is permissible to have a small number of oocytes that will not mature, particularly if they are small enough to filter out as the oocytes are processed. Occasionally, oocytes that are undeveloped are almost as large as fully developed oocytes that are ready for maturation. They are distinguishable by their grainy, brown coloring (Figure 2E). Do not use batches of oocytes of this type because filtration will not isolate fully developed oocytes from undeveloped oocytes and they often result in abnormal embryo cultures that are more likely to exhibit the defects seen in Figure 5.
The other critical step is injection bolus size. Introduce an amount of perturbing reagent that is sufficiently large to exert an effect, but not so large as to cause non-specific effects. The first time a perturbing reagent is used, it is helpful to perform a titration in which several concentrations of reagent are tried. Perform subsequent microinjections with the highest concentration that does not cause developmental abnormalities that are unrelated to the expected phenotype, or delay development (Figure 5A). Avoid injecting a bolus that is greater than 30% the diameter of the embryo as this may disrupt normal development. Overly small bolus sizes, by contrast, are difficult to visually inspect for consistent sizing which can lead to an excessive range of perturbation phenotypes across replicates.
Perturbing reagents may cause developmental delays or non-specific phenotypes at effective concentration ranges. It is very important, therefore, to inject sibling controls with a benign reagent such as a standard control MASO, standard DNA constructs, or rhodamine tracer. Assay morphant embryos only if these controls show normal development. Injected embryos, including controls, sometimes exhibit a wrinkled blastula phenotype but they will frequently proceed through the rest of development normally. Additionally, if a particular reagent consistently causes developmental delays in the resulting morphant embryos, compare these embryos to control embryos to determine how many hours of delay they are experiencing.
There are a few attributes of this method that may limit its usefulness in specific experimental scenarios. One such issue is the fact that one can only introduce reagents at fertilized egg or early cleavage stages. This is particularly problematic if the gene or pathway of interest is especially crucial in early development and resultant morphant embryos are inviable or too developmentally abnormal for meaningful study. Sometimes this issue is resolved by injecting fewer copies of the perturbing reagent, resulting in a gentler, incomplete knockdown. If an appropriate cell-permeable drug for the target gene or pathway exists, it is helpful to compare the phenotype of embryos injected with reagents at the fertilized egg stage with embryos treated with the drug at a later, more appropriate stage. Because injection of MASOs or mRNAs offers greater specificity, it is more powerful to use this method in conjunction with a drug-treatment study as opposed to abandoning microinjection in favor of drug treatment.
Finally, although this microinjection method will produce a sufficient amount of embryos for a wide variety of downstream applications, it will at best produce several hundred to one thousand healthy, successfully injected embryos. Some desired experiments may require considerably more material than this. Other methods successfully used in the sea urchin system may be modified to create even larger numbers of morphant sea star embryos, such as pantropic retroviruses20, although this method is novel and has not yet gained widespread use.
The authors have nothing to disclose.
This work was supported by the National Science Foundation IOS 0844948 and IOS 1024811
Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
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, 60mm x 15mm | 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 |