Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

Summary

We report here the robust and efficient expression of fluorescent proteins after mRNA injection into unfertilized oocytes of Branchiostoma lanceolatum. The development of the microinjection technique in this basal chordate will pave the way for far-reaching technical innovations in this emerging model system, including in vivo imaging and gene-specific manipulations.

Abstract

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the cephalochordate amphioxus, Branchiostoma lanceolatum. We use constructs for membrane and nuclear targeted mCherry and eGFP that have been modified to accommodate amphioxus codon usage and Kozak consensus sequences. We describe the type of injection needles to be used, the immobilization protocol for the unfertilized oocytes, and the overall injection set-up. This technique generates fluorescently labeled embryos, in which the dynamics of cell behaviors during early development can be analyzed using the latest in vivo imaging strategies. The development of a microinjection technique in this amphioxus species will allow live imaging analyses of cell behaviors in the embryo as well as gene-specific manipulations, including gene overexpression and knockdown. Altogether, this protocol will further consolidate the basal chordate amphioxus as an animal model for addressing questions related to the mechanisms of embryonic development and, more importantly, to their evolution.

Introduction

During development, a single cell gives rise to an entire organism in a highly complex process that involves both cell divisions and movements. To better understand the biological principles underlying the dynamics of cell behavior, developmental biologists have started to use fluorescence-based in vivo imaging techniques. Specific compartments of cells, such as cell membranes, can either be labeled by treatments with fluorescent dyes, an approach hampered by a lack of specificity and of tissue penetration¹, or by the specific introduction into the embryo of exogenous mRNAs encoding fluorescent proteins². Different techniques can be used for the efficient delivery of exogenous compounds, such as mRNAs. These include, but are not limited to, microinjection, electroporation, bombardment with microparticles, lipofection and transduction^3,4. Although all of these approaches can be used to introduce exogenous compounds into a developing embryo, only microinjection allows the application of predefined and precise quantities into each cell³. Microinjection techniques have been described for all major developmental model systems⁴ (e.g., fruit flies, nematode worms, zebrafish, frogs, mice) as well as for some alternative models⁴, including those used for comparative studies aimed at understanding the evolution of developmental mechanisms (e.g., sea anemones, annelid worms, sea urchins, ascidian tunicates, the cephalochordate amphioxus).

Cephalochordates, which together with tunicates and vertebrates establish the chordate phylum, are particularly well-suited models to study the evolution of chordates and the diversification of vertebrates from an invertebrate ancestor^5-8. The cephalochordate lineage diverged very early during chordate evolution; and extant cephalochordates, which are subdivided into three genera (Branchiostoma, Asymmetron and Epigonichthys), resemble vertebrates both in terms of overall anatomy and genome architecture^5-8. Of the about 30 species of cephalochordates that have been described so far, five are available for embryological and developmental studies^6,9: Asymmetron lucayanum (the Bahama lancelet), Branchiostoma floridae (the Florida amphioxus), Branchiostoma lanceolatum (the European amphioxus), Branchiostoma belcheri (the Chinese amphioxus) and Branchiostoma japonicum (the Japanese amphioxus). Ripe adults of three of these species (B. lanceolatum, B. belcheri and B. japonicum) can be induced to spawn on-demand during the breeding season^10,11. In addition, at least for B. lanceolatum, efficient spawning can also be induced in artificial sea water¹², thereby making this particular cephalochordate species accessible for laboratories that do not have access to natural seawater. The combination, in B. lanceolatum, of a convenient and reliable access to embryos with an efficient delivery method, such as microinjection, so far the only delivery technique developed in amphioxus (in both B. floridae and B. belcheri)^13-15, will enable the development of a novel suite of manipulative techniques, including lineage tracing- and dynamic cell behavior-based approaches.

A protocol for the efficient microinjection of mRNAs to express fluorescent proteins in the B. lanceolatum embryo was hence developed. Furthermore, to provide a basic toolkit for live imaging of B. lanceolatum embryos, vector systems were developed that allow membrane-associated and nuclear expression of fluorescent proteins. For membrane targeting, enhanced green fluorescent protein (eGFP) was fused to the human HRAS CAAX box and nuclear localization of mCherry and eGFP was obtained by fusion to the zebrafish histone 2B (H2B) exon (Figure 1, Supplementary File 1). Furthermore, with the goal to optimize protein translation, the Kozak sequences and codons of the constructs have been modified and adapted to usage in B. lanceolatum. Taken together, the injection method and expression vectors presented here will serve as a basis for the generation of new experimental approaches for cephalochordates, notably analyses using the latest fluorescence-based in vivo imaging techniques.

Protocol

1. Preparation of Instruments and Reagents

1. Transfer Pasteur pipettes
   1. Generate a series of transfer Pasteur pipettes with different tip diameters by pulling 230 mm long Pasteur pipettes above a flame at different speeds. Ensure that the taper is as long as possible for smooth and fine control of aspiration.
   2. With a diamond scribe, scratch the pipette along a line perpendicular to the length of the pipette. With both hands, pull the pipette parallel to its length to generate a blunt cut. Swiftly flame-polish the pipette without sealing the tip.
   3. Vary the diameter of the tip with the stage of the oocytes/embryos to be pipetted: 300-400 µm for unfertilized oocytes (around 150 µm in diameter), 600 µm for fertilized eggs (around 500 µm in diameter), 200-400 µm for hatched neurulae. Use pipettes with a mouth-monitored aspiration tube to transfer oocytes/embryos from one dish to another.
2. Injection needles
   1. Manipulate the capillaries with gloves to ensure RNase-free conditions. Use borosilicate glass with filament capillaries with dimensions of OD 1.20 mm, ID 0.94 mm, length 10 mm.
   2. If capillaries are pulled on the type of heating-filament needle puller described in the Materials List, use the following settings: Heat 600, Pull 50, Velocity 80, Time 60, Pressure 200 or 300. Otherwise, ensure that the shape, which is crucial for successful injections, is as shown in Figure 2 with the following properties: 4-8 µm outer tip diameter, 2 cm taper length.
      NOTE: Needles can be pulled before the spawning season and used throughout the season.
3. 5% Phenol Red stock solution (4x)
   1. Prepare fresh before each spawning season.
   2. In a 0.22 µm-filtration tube, weigh out 25 mg of Phenol Red powder.
   3. Add 0.5 ml of DNase- and RNase-free water to the tube containing the powder.
   4. Spin for 3-5 min at 18,000 x g at room temperature to filter-sterilize the solution and remove crystals that could clog the injection needle.
   5. Store at 4 °C or store 250 µl aliquots at -20 °C.
4. 0.25 mg/ml poly-lysine solution
   1. Prepare fresh before each spawning season.
   2. Dissolve 5 mg of poly-L-lysine in 20 ml distilled water. Store 5 ml aliquots at -20 °C.
   3. Use a defrosted aliquot immediately and only once to ensure reproducible and robust adhesion of the oocytes to the poly-lysine-coated dish.
5. mRNA synthesis
   1. Prepare fresh before each spawning season.
   2. Linearize 5 µg of DNA of interest with the adequate enzyme (usually for 2 hr, at 37 °C). To check the completeness of the digestion, run 2% in volume of the digestion mix on a 1% agarose-TBE gel in TBE buffer at 150 W for 20 min.
   3. Extract the linearized DNA with 25:24:1 phenol (pH 8.0):chloroform:isoamyl alcohol. Vortex for 20 sec, centrifuge 10 min at 18,000 x g and collect the aqueous (upper) phase.
   4. Extract the aqueous phase again with 24:1 chloroform:isoamyl alcohol. Vortex for 20 sec, centrifuge 10 min at 18,000 x g and collect the aqueous phase.
   5. Precipitate the linearized DNA with 100:10:300 linearized DNA:3 M sodium acetate (pH 5.2):100% ethanol overnight at -20 °C.
   6. Centrifuge 20 min at 18,000 x g at 4 °C. Rinse in 70% ethanol.
   7. Centrifuge 10 min at 18,000 x g at 4 °C. Let dry and resuspend in RNase-free water at a final concentration of 0.5 µg/µl.
   8. Transcribe 1 µg of linearized DNA using an mRNA synthesis kit with the appropriate polymerase, according to manufacturer’s instructions.
   9. Extract the mRNA with 5:1 phenol (pH 4.7):chloroform and ammonium acetate stop solution provided with the kit.
   10. Vortex for 20 sec, centrifuge 10 min at 18,000 x g and collect the aqueous phase.
   11. Extract the aqueous phase again with 24:1 chloroform:isoamyl alcohol. Vortex for 20 sec, centrifuge 10 min at 18,000 x g and collect the aqueous phase.
   12. Precipitate the mRNA with 100% isopropanol overnight at -20 °C.
   13. Centrifuge 20 min at 18,000 x g at 4 °C. Rinse in 80% ethanol.
   14. Centrifuge 10 min at 18,000 x g at 4 °C. Let the pellet dry at room temperature for no longer than 5-10 min as it will then be difficult to resuspend. Resuspend in DNase- and RNase-free water to a final concentration of at least 2 µg/µl to ensure a decent final mRNA concentration in the injection mix.
   15. To check the quality and size of the transcription product, run 0.5 µl of the mRNA on a RNase-free 1% agarose-TBE gel in TBE buffer at 150 W for 20 min. Store 2 µl aliquots at -80 °C.
6. Poly-lysine-coated dishes
   NOTE: Poly-lysine coated dishes are used to immobilize the oocytes during injection.
   1. For each 35 mm cell-culture Petri dish (5 in total), cover the bottom of the Petri dish with 1 ml of the thawed 0.25 mg/ml poly-lysine solution. Incubate at room temperature for 5 min.
   2. For each 35 mm cell-culture Petri dish (5 in total), transfer the 0.25 mg/ml poly-lysine solution into another 35 mm cell-culture Petri dish. Incubate at room temperature for 5 min.
   3. Discard the 0.25 mg/ml poly-lysine solution.
   4. Let the Petri dishes dry, upside-down at room temperature for 2 hr.
   5. Store the poly-lysine-coated dishes wrapped in a plastic wrap at 4 °C to avoid contamination for one week maximum.
7. Agarose-coated dishes
   NOTE: They are used to culture injected embryos. The agarose provides a cushion for the injected embryos and prevents them from sticking to the bottom of the dish.
   1. Make artificial seawater (ASW) using 37-38 g/L commercial salts + 0.25 mM NaHCO₃ in reverse osmosis water.
   2. Dissolve agarose to a 1% concentration in 0.22 µm-filtered ASW by heating the solution in a microwave.
   3. Swiftly pour the warm agarose solution from one 35 mm Petri dish into another one in order to ensure a very thin agarose coating of the dish.
   4. Store the agarose-coated dishes wrapped in Saran wrap at 4 °C to avoid contamination for one week maximum.
8. Injection mix and loading of the injection needles
   1. About 2 hr before starting the injections, make a 2 µl injection mix in RNase- and DNase-free water with final concentrations of 1-1.8 µg/µl of mRNA, 15% glycerol, 1.25% Phenol Red.
      NOTE: The Phenol Red colors the solution, which allows monitoring of the injection efficiency and the identification of successfully injected embryos. Glycerol favors mRNA diffusion within the oocyte.
   2. Centrifuge 4 min at 18,000 x g to pellet crystals. Keep on ice until use.
   3. With a 10 µl pipette, collect 0.5 µl of the injection mix, avoiding the bottom of the tube, where the crystals have been pelleted.
   4. Backfill at least two injection needles (in case one breaks during the injection) by pipetting the 0.5 µl drop of injection mix at the large opening of the needle.
   5. Install the needles in a storage jar with liquid at the bottom at 4 °C to prevent evaporation of the injection mix. Let the injection mix slowly travel to the tip of the injection needle for at least 1 hr to minimize the creation of bubbles.
   6. Store additional injection mix at -80 °C for maximum of three additional uses, after which the mRNA quality deteriorates (data not shown).

2. Collection of Biological Material, Microinjection and Embryo Culture

1. Oocyte and sperm collection
   NOTE: See Theodosiou et al.¹² for a detailed protocol for inducing spawning and for gamete collection.
   1. Shock males and females in ASW at 23 °C for 24 hr.
   2. One to two hours before sunset, transfer the adults into individual cups in ASW at 19 °C because most adults will spawn 1-2 hr after sunset.
   3. Rinse 35 mm Petri dishes in filtered ASW and let them dry upside-down to prevent the oocytes from sticking to the bottom of the dish.
   4. Upon spawning, immediately collect sperm and oocytes with a 1,000 µl pipette.
      1. Keep sperm and oocytes separate from adult amphioxus because the contact is detrimental for gamete health (data not shown).
      2. Furthermore, avoid startling the adults, which leads to movements that dissipate, and hence dilute, both sperm and oocytes. To keep the sperm active as long as possible and to optimize fertilization rate, collect the sperm as concentrated as possible.
   5. Keep sperm on ice in a 1.5 ml tube.
   6. Transfer oocytes in filtered ASW into the pre-rinsed 35 mm Petri dishes.
   7. Transfer 100-500 oocytes with a previously pulled 300-400 µm transfer Pasteur pipette to another 35 mm Petri dish to perform the injections.
   8. Fertilize the remainder of the clutch as a control for sperm and oocyte quality or for other experiments.
2. Oocyte injection
   1. Install the injection needle on the micromanipulator at a 50° angle relative to the horizontal plane.
      NOTE: Angles of less than 50° will push the oocytes around on the dish, while angles of more than 50° will not allow an appropriate monitoring of the needle position relative to the oocyte.
   2. Under a fluorescent dissecting scope with 25X oculars, transfer 30 oocytes with the 300-400 µm transfer Pasteur pipette on a poly-lysine-coated dish containing filtered ASW.
   3. Deposit the oocytes along a line to carry out injections in an ordered way and to distinguish injected from non-injected oocytes. Inject small numbers (30 oocytes) to minimize the exposure time of oocytes to poly-lysine, which tends to deform developing embryos (data not shown).
   4. Use the dark field illumination to render the oocytes as translucent as possible.
   5. With the coarse movement knob of the micromanipulator, bring the injection needle close to an oocyte.
   6. With fine forceps, cut open the needle at the level where the tip starts to be curved. By pulsing with the injector, verify that red injection mix is actually flowing out of the needle.
   7. At 200X magnification and with the fine movement knob of the micromanipulator, gently move the injection needle inside the core of the oocyte.
      NOTE: If inserted too superficially, the injected solution will not remain inside the oocyte. If inserted too far, the oocyte will be destroyed.
   8. Inject with 1-3 pulses of 120 msec duration and 1-10 psi pressure. If the needle is fine enough, inject with continuous flow at constant pressure. Ensure that the injection volume corresponds to 1/5 to 1/3 of the volume of a single oocyte.
   9. Following injection, pull the needle out swiftly to avoid leakage of the oocyte.
   10. Verify that the injected solution remains within the oocyte and that after a few sec, the injected solution spreads throughout the oocyte.
   11. Move on to the next oocyte in line.
   12. Keep some uninjected embryos of each series as negative control to estimate the background fluorescence when scanning for injected embryos.
3. Fertilization, selection of injected embryos and embryo culture
   1. Fertilize the oocytes as soon as a series has been injected. As oocyte quality declines with time, inject and fertilize oocytes within 1 hr after spawning¹².
   2. Depending on the sperm concentration, add 1-5 drops of sperm to the oocytes and swirl the dish.
      NOTE: The fertilization envelope should become apparent on the embryos after about 1 min.
   3. Allow the embryos to detach from the poly-lysine-coated dish, while injecting another series of oocytes.
   4. Transfer the embryos with the 600 µm transfer Pasteur pipette into an agarose-coated Petri dish. Remove the embryos from the poly-lysine-coated dish as soon as possible, if at all possible before the 2-cell stage.
      NOTE: In case of prolonged exposure to poly-lysine, the embryos tend to become densely-packed blastulae, flattened on the side touching the bottom of the dish.
   5. At the 2-cell to 4-cell stage, select with a fluorescent dissecting scope with DSR filter the successfully-injected embryos, i.e., those with a normal morphology that exhibit a Phenol Red-derived red fluorescent signal.
   6. Keep the embryos in culture in filtered ASW in agarose-coated Petri dishes at 19 °C until the desired stage for in vivo imaging.

Results

The protocol detailed above provides the basis for the microinjection of B. lanceolatum oocytes and hence for the introduction into developing B. lanceolatum embryos of mRNA encoding fluorescent proteins for in vivo imaging. Although the technique is certainly robust and reliable, the rate of successful injections using this protocol remains variable (Table 1). The very likely explanation for this intriguing fact is the extreme variability of oocyte clutches: different egg batc...

Discussion

In this article, we present, for the first time, a detailed and reproducible protocol for the injection of B. lanceolatum oocytes, which, after B. floridae^13,14 and B. belcheri¹⁵, is thus the third amphioxus species, for which such a technique has been described. Importantly, the protocol described here also includes the description of vector systems suited for the production of fluorescent proteins in B. lanceolatum from injected mRNA produced in vitro (...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Consumables
35 mm Petri dishes	Falcon	353001	Culture-treated
Filtration unit (Stericup 1 L)	Fisher	W21719	0.22 μm
Spin-X tubes	Costar	8160	0.22 μm
Needle storage jar for 1.2 mm diameter capillaries	WPI	E212
Pasteur pipettes			230 mm long
Aspiration tube	Dutscher	75056
Capillaries for injection needles	Sutter	BF 120-94-10	Borosilicate glass with filament, OD 1.20 mm, ID 0.94 mm, length 10 mm
Reagents
Low-melting agarose	Sigma	A9414
Phenol Red	Sigma	114537
Glycerol	Sigma	G2025
Poly-L-lysine hydrobromide	Sigma	P9155
H2O, DNase/RNase-free	Gibco	10977-035
mMessage mMachine SP6 Transcription kit	Ambion	AM1340	mRNA synthesis kit
Phenol pH 8	Sigma	P4557
24:1 chloroform:isoamylic alcohol	Sigma	C0549
5:1 phenol pH 4.7:chloroform	Sigma	P1944
Reef Crystal salts (200 kg)	Europrix		Commercial salts
NaHCO3	Sigma	S6014
Equipment
Fluorescent dissecting scope with 200X magnification	Leica	MZ16F	25X oculars, DSR and GFP2 filters
Micromanipulator	Marzhauzer	M-33
Injector	Picospritzer	model II or III
Needle puller	Sutter	P97	Heating-filament needle puller
Fine forceps	Fine Science Tools GmbH	11252-30	Dumont #5