The authors would like to acknowledge the support by the &#8220;Animalerie Centrale de Gif-sur-Yvette&#8221; for animal husbandry. This work was supported by funds from ANR (ANR-09-BLAN-0262-02 and ANR-11-JSV2-002-01) to Michael Schubert, by the European Union FP6 grant &#8220;Embryomics&#8221; and by the ANR grant &#8220;ANR-10-BLAN-121801 Dev-Process&#8221; to Jean-Fran&#231;ois Nicolas and Nadine Peyri&#233;ras. Jo&#227;o Emanuel Carvalho is financed by a FCT doctoral fellowship (SFRH/BD/86878/2012).
Requests for the vectors described here can be addressed directly to the authors.

1. Preparation of Instruments and Reagents
<ol style="margin-left: 40px;">
	<li>Transfer Pasteur pipettes
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>Vary the diameter of the tip with the stage of the oocytes/embryos to be pipetted: 300-400 &#181;m for unfertilized oocytes (around 150 &#181;m in diameter), 600 &#181;m for fertilized eggs (around 500 &#181;m in diameter), 200-400 &#181;m for hatched neurulae. Use pipettes with a mouth-monitored aspiration tube to transfer oocytes/embryos from one dish to another.</li>
	</ol>
	</li>
	<li>Injection needles
	<ol>
		<li>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.</li>
		<li>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 &#181;m outer tip diameter, 2 cm taper length. 
		NOTE: Needles can be pulled before the spawning season and used throughout the season.</li>
	</ol>
	</li>
	<li>5% Phenol Red stock solution (4x)
	<ol>
		<li>Prepare fresh before each spawning season.</li>
		<li>In a 0.22 &#181;m-filtration tube, weigh out 25 mg of Phenol Red powder.</li>
		<li>Add 0.5 ml of DNase- and RNase-free water to the tube containing the powder.</li>
		<li>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.</li>
		<li>Store at 4 &#176;C or store 250 &#181;l aliquots at -20 &#176;C.</li>
	</ol>
	</li>
	<li>0.25 mg/ml poly-lysine solution
	<ol>
		<li>Prepare fresh before each spawning season.</li>
		<li>Dissolve 5 mg of poly-L-lysine in 20 ml distilled water. Store 5 ml aliquots at -20 &#176;C.</li>
		<li>Use a defrosted aliquot immediately and only once to ensure reproducible and robust adhesion of the oocytes to the poly-lysine-coated dish.</li>
	</ol>
	</li>
	<li>mRNA synthesis
	<ol>
		<li>Prepare fresh before each spawning season.</li>
		<li>Linearize 5 &#181;g of DNA of interest with the adequate enzyme (usually for 2 hr, at 37 &#176;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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Precipitate the linearized DNA with 100:10:300 linearized DNA:3 M sodium acetate (pH 5.2):100% ethanol overnight at -20 &#176;C.</li>
		<li>Centrifuge 20 min at 18,000 x g at 4 &#176;C. Rinse in 70% ethanol.</li>
		<li>Centrifuge 10 min at 18,000 x g at 4 &#176;C. Let dry and resuspend in RNase-free water at a final concentration of 0.5 &#181;g/&#181;l.</li>
		<li>Transcribe 1 &#181;g of linearized DNA using an mRNA synthesis kit with the appropriate polymerase, according to manufacturer&#8217;s instructions.</li>
		<li>Extract the mRNA with 5:1 phenol (pH 4.7):chloroform and ammonium acetate stop solution provided with the kit.</li>
		<li>Vortex for 20 sec, centrifuge 10 min at 18,000 x g and collect the aqueous phase.</li>
		<li>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.</li>
		<li>Precipitate the mRNA with 100% isopropanol overnight at -20 &#176;C.</li>
		<li>Centrifuge 20 min at 18,000 x g at 4 &#176;C. Rinse in 80% ethanol.</li>
		<li>Centrifuge 10 min at 18,000 x g at 4 &#176;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 &#181;g/&#181;l to ensure a decent final mRNA concentration in the injection mix.</li>
		<li>To check the quality and size of the transcription product, run 0.5 &#181;l of the mRNA on a RNase-free 1% agarose-TBE gel in TBE buffer at 150 W for 20 min. Store 2 &#181;l aliquots at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Poly-lysine-coated dishes 
	NOTE: Poly-lysine coated dishes are used to immobilize the oocytes during injection.
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>Discard the 0.25 mg/ml poly-lysine solution.</li>
		<li>Let the Petri dishes dry, upside-down at room temperature for 2 hr.</li>
		<li>Store the poly-lysine-coated dishes wrapped in a plastic wrap at 4 &#176;C to avoid contamination for one week maximum.</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>Make artificial seawater (ASW) using 37-38 g/L commercial salts + 0.25 mM NaHCO3 in reverse osmosis water.</li>
		<li>Dissolve agarose to a 1% concentration in 0.22 &#181;m-filtered ASW by heating the solution in a microwave.</li>
		<li>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.</li>
		<li>Store the agarose-coated dishes wrapped in Saran wrap at 4 &#176;C to avoid contamination for one week maximum.</li>
	</ol>
	</li>
	<li>Injection mix and loading of the injection needles
	<ol>
		<li>About 2 hr before starting the injections, make a 2 &#181;l injection mix in RNase- and DNase-free water with final concentrations of 1-1.8 &#181;g/&#181;l 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.</li>
		<li>Centrifuge 4 min at 18,000 x g to pellet crystals. Keep on ice until use.</li>
		<li>With a 10 &#181;l pipette, collect 0.5 &#181;l of the injection mix, avoiding the bottom of the tube, where the crystals have been pelleted.</li>
		<li>Backfill at least two injection needles (in case one breaks during the injection) by pipetting the 0.5 &#181;l drop of injection mix at the large opening of the needle.</li>
		<li>Install the needles in a storage jar with liquid at the bottom at 4 &#176;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.</li>
		<li>Store additional injection mix at -80 &#176;C for maximum of three additional uses, after which the mRNA quality deteriorates (data not shown).</li>
	</ol>
	</li>
</ol>
2. Collection of Biological Material, Microinjection and Embryo Culture
<ol style="margin-left: 40px;">
	<li>Oocyte and sperm collection 
	NOTE: See Theodosiou et al.12 for a detailed protocol for inducing spawning and for gamete collection.
	<ol>
		<li>Shock males and females in ASW at 23 &#176;C for 24 hr.</li>
		<li>One to two hours before sunset, transfer the adults into individual cups in ASW at 19 &#176;C because most adults will spawn 1-2 hr after sunset.</li>
		<li>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.</li>
		<li>Upon spawning, immediately collect sperm and oocytes with a 1,000 &#181;l pipette.
		<ol>
			<li>Keep sperm and oocytes separate from adult amphioxus because the contact is detrimental for gamete health (data not shown).</li>
			<li>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.</li>
		</ol>
		</li>
		<li>Keep sperm on ice in a 1.5 ml tube.</li>
		<li>Transfer oocytes in filtered ASW into the pre-rinsed 35 mm Petri dishes.</li>
		<li>Transfer 100-500 oocytes with a previously pulled 300-400 &#181;m transfer Pasteur pipette to another 35 mm Petri dish to perform the injections.</li>
		<li>Fertilize the remainder of the clutch as a control for sperm and oocyte quality or for other experiments.</li>
	</ol>
	</li>
	<li>Oocyte injection
	<ol>
		<li>Install the injection needle on the micromanipulator at a 50&#176; angle relative to the horizontal plane. 
		NOTE: Angles of less than 50&#176; will push the oocytes around on the dish, while angles of more than 50&#176; will not allow an appropriate monitoring of the needle position relative to the oocyte.</li>
		<li>Under a fluorescent dissecting scope with 25X oculars, transfer 30 oocytes with the 300-400 &#181;m transfer Pasteur pipette on a poly-lysine-coated dish containing filtered ASW.</li>
		<li>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).</li>
		<li>Use the dark field illumination to render the oocytes as translucent as possible.</li>
		<li>With the coarse movement knob of the micromanipulator, bring the injection needle close to an oocyte.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Following injection, pull the needle out swiftly to avoid leakage of the oocyte.</li>
		<li>Verify that the injected solution remains within the oocyte and that after a few sec, the injected solution spreads throughout the oocyte.</li>
		<li>Move on to the next oocyte in line.</li>
		<li>Keep some uninjected embryos of each series as negative control to estimate the background fluorescence when scanning for injected embryos.</li>
	</ol>
	</li>
	<li>Fertilization, selection of injected embryos and embryo culture
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>Allow the embryos to detach from the poly-lysine-coated dish, while injecting another series of oocytes.</li>
		<li>Transfer the embryos with the 600 &#181;m 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.</li>
		<li>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.</li>
		<li>Keep the embryos in culture in filtered ASW in agarose-coated Petri dishes at 19 &#176;C until the desired stage for in vivo imaging.</li>
	</ol>
	</li>
</ol>

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Genet. Genomics. 41, 215-219 (2014).</li><li>Zhang, Q. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+culture+of+two+lancelets+and+production+of+the+second+filial+generations+in+the+laboratory.">Continuous culture of two lancelets and production of the second filial generations in the laboratory.</a> J. Exp. Zool. B. 308, 464-472 (2007).</li><li>Yasui, K., Igawa, T., Kaji, T., Henmi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+aquaculture+of+the+Japanese+lancelet+Branchiostoma+japonicum+for+7+years.">Stable aquaculture of the Japanese lancelet Branchiostoma japonicum for 7 years.</a> J. Exp. Zool. B. 320, 538-547 (2013).</li><li>Benito-Guti&#233;rrez, E., Weber, H., Bryant, D. V., Arendt, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+generating+year-round+access+to+amphioxus+in+the+laboratory.">Methods for generating year-round access to amphioxus in the laboratory.</a> PLoS One. 8, e71599 (1371).</li></ol>

The authors have nothing to disclose.

In this article, we present, for the first time, a detailed and reproducible protocol for the injection of B. lanceolatum oocytes, which, after B. floridae13,14 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 (described below). Together, these new tools allow in vivo imaging of the early development of amphioxus and the analysis of dynamic cell behaviors that underlie the earliest morphogenetic events in the embryo, such as cleavage and gastrulation.
In general, the microinjection technique has the benefit of allowing the delivery, into a target cell, of a pre-defined volume of a specific compound. With this being said, one major limitation of this technique is the limited number of cells that can be injected during a given experiment. For example, the protocol described here for B. lanceolatum allows the injection of 100 to 120 eggs per injection session, of which about half will be labeled (Table 3). For B. floridae, the numbers are very similar with 500 or more eggs that can be injected per day, of which more than 50% will survive the injection13,14. While the protocols for injecting B. lanceolatum and B. floridae eggs closely resemble each other, the B. belcheri technique is slightly different: the eggs are placed on poly-lysine-coated coverslips and injections are performed under an inverted microscope using a microinjector of a different brand. This approach apparently allows the injection of between 200 and 300 B. belcheri eggs per injection session with a survival rate, after hatching at the neurula stage, of 89.39% to 95.83%15. Considering the differences in the protocols, it is difficult to know whether the differences in success rates are due to species-related or protocol-related differences. One would have to test the different species in parallel with the different protocols to answer this question. Nonetheless, to further increase the number of oocytes targeted for the delivery of exogenous compounds, other protocols will have to be developed for amphioxus. Candidate techniques include electroporation, bombardment with microparticles, lipofection, and transduction4. Of note, electroporation has already been established as a standard technique for the introduction of exogenous material into fertilized ascidian tunicate eggs, another invertebrate chordate model used for studying the evolution of developmental mechanisms8.
For successful microinjection and subsequent in vivo imaging of fluorescent proteins, suitable expression vectors are a mandatory prerequisite. Towards this end, three plasmids have been developed and experimentally validated (Figure 1, Supplementary File 1): for membrane targeting, the eGFP gene was fused at its 3&#8217; end to the human HRAS CAAX box (resulting in a eGFP:CAAX box construct)16 and, for nuclear targeting, both the eGFP and the mCherry genes were fused at their 5&#8217; ends to the zebrafish histone 2B (H2B) exon (resulting, respectively, in a H2B:eGFP and a H2B:mCherry construct)17. Each one of these constructs has been cloned into the pCS2+ plasmid containing a SV40 late polyadenylation site and a SP6 promoter allowing in vitro capped mRNA synthesis18,19. Furthermore, with the goal to optimize translation of the constructs in B. lanceolatum, both the Kozak sequences and the codons have been adapted using single nucleotide mutagenesis (Tables 2 and 3, Supplementary File 1). Based on the approach by Nakagawa20, a small pool of B. lanceolatum genes was analyzed to identify a preferred Kozak sequence in this species. The closest known naturally-occurring sequence to this theoretical preferred sequence is that of the B. lanceolatum actin gene. The Kozak sequences of the three constructs were hence modified to match this actin gene sequence. Furthermore, the codon usage of B. lanceolatum was analyzed using the codon usage database21 and codons in the constructs with a low usage probability in B. lanceolatum (&#60;10%) were replaced, wherever possible, by equivalent codons with a higher usage probability.
The results of the injections show that the level of protein expression from these vectors is suitable for in vivo imaging analyses. Given that the expression output of the modified vectors has not been compared with that of unmodified constructs, we cannot conclude on the efficiency of the modifications that were introduced in the Kozak consensus and the coding sequences. It would certainly be interesting to test whether these modifications indeed have an impact on protein expression levels. Along these lines, a recent analysis based on microinjections into B. belcheri suggests that other, unmodified vectors can also be used for the synthesis of mRNA for fluorescent protein production in amphioxus15.
Amphioxus contains endogenous green fluorescent proteins22. The embryos therefore exhibit low levels of green as well as red fluorescence (our unpublished observations). However, these endogenous levels are negligible with respect to the fluorescence levels resulting from the injection of our mRNA constructs. Therefore, the endogenous fluorescence of amphioxus embryos does not disturb experimental observations using fluorescent binoculars or multi-laser scanning microscopes. In addition, the injection experiments reveal that the intensity of the exogenous fluorescence generated by mRNA injection depends on the actual amount of injected material, which is directly correlated with the final concentration of mRNA used in the injection mix. B. lanceolatum embryos tend to tolerate a concentration of up to 1.8 &#181;g/&#181;l mRNA, although, independent of the actual mRNA concentration, some mRNA combinations seem to be less tolerated by B. lanceolatum embryos than others. Thus, the combination of nuclear mCherry and membrane eGFP mRNAs appeared to be more toxic than the injection of nuclear eGFP alone.
Texas Red dextran has previously been used as a tracer for successful injections into amphioxus oocytes13-15. Intriguingly, a fluorescent signal derived from the injected mRNA was never obtained after injection of solutions containing Texas Red dextran and this in both amphioxus oocytes (n = 4 experiments) and zebrafish embryos (n = 3 experiments). When an injection mix containing in vitro transcribed mRNA and Texas Red dextran is loaded on a RNase-free agarose gel (Figure 3, lane 2), the band corresponding to the mRNA is absent. In contrast, in vitro transcribed mRNA is detectable on an RNase-free agarose gel in the presence of Phenol Red (Figure 3, lane 1). Given that there is no evidence for mRNA degradation on the gel, these results suggest that Texas Red dextran tends to trap mRNA. When used in an injection solution, Texas Red dextran might thus prevent the translation of the injected mRNA. Following this observation, the interactions of other dyes (FITC dextran, Rhodamine dextran and Oregon Green dextran) with mRNA were tested, both on RNase-free agarose gels and by injection into B. lanceolatum or zebrafish (Figure 3). The analyses indicate that FITC dextran does not trap mRNA on gel (Figure 3, lane 4) and leads to fluorescent protein expression in zebrafish (n = 1 experiment), but not in embryos of B. lanceolatum (n=1 experiment) (data not shown). Rhodamine dextran does not trap mRNA on gel (Figure 3, lane 5) and leads to fluorescent protein expression in zebrafish embryos (n = 1 experiment) (data not shown), but its activity could unfortunately not be tested in B. lanceolatum embryos. Finally, as the Oregon Green dextran migrates at the same level as the mRNA, mRNA trapping could not be assessed by agarose gel (Figure 3, lane 3). This latter dye prevented fluorescent protein expression in amphioxus (n = 1 experiment), but not zebrafish embryos (n=1 experiment) (data not shown). In sum, these data suggest that some dextrans can efficiently inhibit the translation of injected mRNA. Given that dose-response experiments were not carried out, these data are qualitative. Intriguingly, this inhibitory effect seems to be dependent on the animal species (zebrafish versus amphioxus), which highlights the need for further studies to understand the mechanisms underlying this effect. Furthermore, these results call for preparatory analyses, if dextrans are to be used as color tracers for injections.
The development of the microinjection technique for a given model opens the door for gene-specific manipulations. In amphioxus, for example, the first description of microinjections (in B. floridae) was followed by the successful knockdown of a gene, hox1, using morpholino oligonucleotide injections23,24. The number of B. lanceolatum embryos that can successfully be injected every day using the protocol presented here is certainly compatible with this type of study. Furthermore, with these methods and the novel constructs at hand, one can now also design and perform overexpression and knockdown studies by injection of mRNAs of interest (native or dominant-negative forms) or use other strategies for disrupting gene expression (for example microRNA- or shRNA-based strategies). So far, amphioxus injections can only be performed at the oocyte stage, simply because this is the only stage that can efficiently be immobilized. After injection at the oocyte stage, the molecule of interest is ubiquitously expressed in the embryo. Thus, if gene expression needs to be altered or monitored only in a subset of cells, DNA constructs need to be injected, as these are expressed mosaically in the developing amphioxus embryo15,25-27. DNA construct injections can also be used to test the activity of regulatory regions during amphioxus development in transient transgenic assays, with the aforementioned drawback of mosaic expression15,25-27.
Ultimately, the amphioxus microinjection technique also opens the door for stable transgenesis, including the targeted knock-out or knock-in of specific genetic loci. Systems for stable insertion of exogenous DNA already exist, for example with the fish transposon-based Tol2 system that seems to function in both insects and amniote vertebrates28. Furthermore, approaches based on modified zinc-finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs) or RNA-guided genome editing tools (CRISPR/Cas system) could be used to generate point mutations and knock-in modifications in specific regions of the genome29. These efforts will require the year-round breeding of amphioxus in captivity, which has already been set up for B. belcheri11,30 and B. japonicum30,31 and is currently being developed for A. lucayanum, B. floridae and the amphioxus species featured here, B. lanceolatum32.

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 transduction3,4. 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 ancestor5-8. 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 architecture5-8. Of the about 30 species of cephalochordates that have been described so far, five are available for embryological and developmental studies6,9: 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 season10,11. 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.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Consumables&lt;/strong&gt;</td></tr><tr><td>35 mm Petri dishes</td><td>Falcon</td><td>353001</td><td>Culture-treated</td></tr><tr><td>Filtration unit (Stericup 1 L)</td><td>Fisher</td><td>W21719</td><td>0.22 &amp;#956;m</td></tr><tr><td>Spin-X tubes</td><td>Costar</td><td>8160</td><td>0.22 &amp;#956;m</td></tr><tr><td>Needle storage jar for 1.2 mm diameter capillaries</td><td>WPI</td><td>E212</td><td></td></tr><tr><td>Pasteur pipettes</td><td></td><td></td><td>230 mm long</td></tr><tr><td>Aspiration tube</td><td>Dutscher</td><td>75056</td><td></td></tr><tr><td>Capillaries for injection needles</td><td>Sutter</td><td>BF 120-94-10</td><td>Borosilicate glass with filament, OD 1.20 mm, ID 0.94 mm, length 10 mm</td></tr><tr><td colspan="4">&lt;strong&gt;Reagents&lt;/strong&gt;</td></tr><tr><td>Low-melting agarose</td><td>Sigma</td><td>A9414</td><td></td></tr><tr><td>Phenol Red</td><td>Sigma</td><td>114537</td><td></td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G2025</td><td></td></tr><tr><td>Poly-L-lysine hydrobromide</td><td>Sigma</td><td>P9155</td><td></td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O, DNase/RNase-free</td><td>Gibco</td><td>10977-035</td><td></td></tr><tr><td>mMessage mMachine SP6 Transcription kit</td><td>Ambion</td><td>AM1340</td><td>mRNA synthesis kit</td></tr><tr><td>Phenol pH 8</td><td>Sigma</td><td>P4557</td><td></td></tr><tr><td>24:1 chloroform:isoamylic alcohol</td><td>Sigma</td><td>C0549</td><td></td></tr><tr><td>5:1 phenol pH 4.7:chloroform</td><td>Sigma</td><td>P1944</td><td></td></tr><tr><td>Reef Crystal salts (200 kg)</td><td>Europrix&amp;nbsp;</td><td></td><td>Commercial salts</td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>S6014</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt;</td></tr><tr><td>Fluorescent dissecting scope with 200X magnification</td><td>Leica</td><td>MZ16F</td><td>25X oculars, DSR and GFP2 filters</td></tr><tr><td>Micromanipulator</td><td>Marzhauzer</td><td>M-33</td><td></td></tr><tr><td>Injector</td><td>Picospritzer</td><td>model II or III</td><td></td></tr><tr><td>Needle puller</td><td>Sutter</td><td>P97</td><td>Heating-filament needle puller</td></tr><tr><td>Fine forceps</td><td>Fine Science Tools GmbH</td><td>11252-30</td><td>Dumont #5</td></tr></tbody></table>

expression of fluorescent proteins in branchiostoma lanceolatum by mrna injection into unfertilized oocytes

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.

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 batches do indeed behave very differently, when subjected to the injection pressure. Some oocytes are rather elastic and tend to flatten at the bottom of the dish, while others are quite stiff and remain round upon injection. Furthermore, some oocyte batches tend to inflate their chorion membranes upon injection, while others simply lyse (data not shown). No obvious correlation could be established between oocyte behavior upon injection and embryonic development after fertilization. It is thus difficult to anticipate which category of oocytes is best suited for microinjection. However after fertilization, embryos undergoing normal development can be identified as early as cleavage stages: 2-cell to 4-cell stage embryos can be considered normal when the contact surface between blastomeres is small, while a compacted cleavage-stage embryo, where individual cells are difficult to discern, is definitely abnormal.
In our hands, about half of the oocytes do not survive the trauma caused by the injection and about half of the embryos that do survive the injection exhibit a specific fluorescent label. Thus, we estimate that with this injection protocol, between 50 and 120 embryos can be successfully injected on a given spawning day yielding between 15 and 60 labeled embryos.
The red fluorescence of the Phenol Red in 2-cell to 4-cell stage embryos very reliably marks embryos that have been properly injected, as 100% of embryos selected in this manner subsequently produce the fluorescent proteins encoded by the injected mRNA. Additionally, there is a very clear positive correlation between the intensity of Phenol Red fluorescence at the 2-cell to 4-cell stage and the time of onset of the fluorescence produced by the proteins encoded by the injected mRNA. This correlation thus allows the identification of those embryos that have received the highest quantity of mRNA during the injection process.
If the signal of the fluorescent protein encoded by the injected mRNA is not detected after selection of Phenol Red-positive embryos, we recommend to run the injection mix on an RNAse-free gel in order to check for mRNA degradation or trapping (Figure 3). In lane 1, an intact mRNA band is detected. Injection of this mix led to strong fluorescent signal in the embryo. On the contrary, in lane 2 which corresponds to another experiment where Phenol Red was replaced with Dextran dye in the mix (see discussion for further details), the mRNA seems to have been trapped by the Dextran dye and injection of this mix never led to fluorescent signal in the embryo.
Using this microinjection technique and our constructs (Tables 2 and 3, Supplementary File 1) (see discussion), we can thus reproducibly produce homogenous fluorescent labeling throughout the embryo with mCherry or eGFP in the nucleus and with eGFP at the membrane (Figure 4). The imaging protocol used to generate Figure 4 will be described elsewhere (Faure et al., currently in preparation). Depending on the amount of mRNA injected, fluorescent protein expression typically becomes detectable between 16-cell (Figure 4A) and 64-cell stages and stays detectable at least up to the late neurula stage (data not shown). Later stages have not been tested. Nuclear signal typically appears earlier than membrane signal, potentially because of the intrinsically diffuse nature of the membrane compared to the compact nature of the nucleus (data not shown). Although it has not been monitored systematically, embryos injected with both a nuclear and a membrane label appear to be less healthy than embryos injected exclusively with a nuclear eGFP label. Intriguingly, this effect seems to be independent of the total amount of mRNA injected as the total amount of injected mRNA is the same, whether one or two mRNA species are included to the mix (data not shown).
<img alt="Figure 1" src="/files/ftp_upload/52042/52042fig1highres.jpg" /> 
Figure 1: Construct details. Sketches of (A) the membrane-targeted eGFP (eGFP:CAAX box), (B) the nuclear-targeted eGFP (H2B:eGFP) and (C) the nuclear-targeted mCherry (H2B:mCherry) constructs are shown.
<img alt="Figure 2" src="/files/ftp_upload/52042/52042fig2highres.jpg" /> 
Figure 2: Shape of a representative injection needle. (A) The taper is about 2 cm in length and the needle curves at the tip. Scale bar = 2 mm. (B) Magnification of the boxed area in A. The position for clipping the needle (arrow) is in the curve of needle. The tip of the needle is indicated by the arrowhead. Scale bar = 500 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52042/52042fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52042/52042fig3highres.jpg" /> 
Figure 3: Interactions of mRNA and selected fluorescent dyes. (A) Result of the migration of a mix containing mRNA and Phenol Red in lane 1 and of mRNA and Texas Red dextran (at a final concentration of 3.3 mg/ml) in lane 2. (B) Result of the migration of a mix containing mRNA and Oregon Green dextran (at a final concentration of 3.3 mg/ml) in lane 3, of mRNA and FITC dextran (at a final concentration of 3.3 mg/ml) in lane 4 and of mRNA and Rhodamine dextran (at a final concentration of 3.3 mg/ml) in lane 5. Gel migration time in A and B was different. For the sake of clarity, the black rectangle in A masks a light reflection and the dashed lines delineate the migration lanes. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52042/52042fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52042/52042fig4highres.jpg" /> 
Figure 4: 3D rendering (Amira software) of amphioxus embryos injected with mRNAs coding for fluorescent proteins. Images are extracted from 3D time-lapses acquired on an optimized Leica SP5 2-photon laser-scanning microscope. (A) 16-cell stage embryo expressing nuclear H2B:eGFP. (B) Gastrula stage embryo expressing nuclear H2B:mCherry and the membrane-targeted eGFP:CAAX box. (C) Gastrula stage embryo expressing nuclear H2B:mCherry and the membrane-targeted eGFP:CAAX box. The optical section shows internal cells with nuclear mCherry and membrane-associated eGFP signals. Scale bar = 50 &#181;m.
<table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>Experiment number</td>
			<td>Injected construct mRNA</td>
			<td>Number of injected embryos</td>
			<td>Number of labeled embryos</td>
		</tr>
		<tr>
			<td>1</td>
			<td>H2B:eGFP</td>
			<td>53</td>
			<td>23</td>
		</tr>
		<tr>
			<td>2</td>
			<td>H2B:eGFP</td>
			<td>50</td>
			<td>31</td>
		</tr>
		<tr>
			<td>3</td>
			<td>H2B:eGFP</td>
			<td>48</td>
			<td>20</td>
		</tr>
		<tr>
			<td>4</td>
			<td>H2B:eGFP</td>
			<td>54</td>
			<td>24</td>
		</tr>
		<tr>
			<td>5</td>
			<td>H2B:eGFP</td>
			<td>114</td>
			<td>47</td>
		</tr>
		<tr>
			<td>6</td>
			<td>H2B:eGFP</td>
			<td>80</td>
			<td>46</td>
		</tr>
		<tr>
			<td rowspan="2">7</td>
			<td>H2B:eGFP or</td>
			<td rowspan="2">62</td>
			<td rowspan="2">24</td>
		</tr>
		<tr>
			<td>H2B:mCherry + eGFP:CAAX box</td>
		</tr>
		<tr>
			<td>8</td>
			<td>H2B:eGFP</td>
			<td>87</td>
			<td>60</td>
		</tr>
		<tr>
			<td>9</td>
			<td>H2B:eGFP</td>
			<td>77</td>
			<td>45</td>
		</tr>
		<tr>
			<td rowspan="2">10</td>
			<td>eGFP:CAAX box or</td>
			<td rowspan="2">110</td>
			<td rowspan="2">51</td>
		</tr>
		<tr>
			<td>H2B:mCherry + eGFP:CAAX box</td>
		</tr>
		<tr>
			<td>11</td>
			<td>H2B:mCherry + eGFP:CAAX box</td>
			<td>45</td>
			<td>26</td>
		</tr>
		<tr>
			<td>12</td>
			<td>H2B:mCherry + eGFP:CAAX box</td>
			<td>52</td>
			<td>16</td>
		</tr>
		<tr>
			<td rowspan="2">13</td>
			<td>eGFP:CAAX box or</td>
			<td rowspan="2">74</td>
			<td rowspan="2">35</td>
		</tr>
		<tr>
			<td>H2B:mCherry + eGFP:CAAX box</td>
		</tr>
		<tr>
			<td></td>
			<td>Total</td>
			<td>906</td>
			<td>448</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>Success rate</td>
			<td>49%</td>
		</tr>
	</tbody>
</table>
Table 1: Injection success rates. The injected constructs, the number of injected embryos that survived the injection and the number of labeled (or successfully injected) embryos are indicated, as is the overall percentage of successful injections.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Construct</td>
			<td>Position in the pCS2+ vector</td>
			<td>Original sequence</td>
			<td>Mutated sequence</td>
		</tr>
		<tr>
			<td>pCS2+ eGFP:CAAX box</td>
			<td>79..87</td>
			<td>GGA TCC ACC</td>
			<td>ACC GTC AAC</td>
		</tr>
		<tr>
			<td>pCS2+ H2B:eGFP</td>
			<td>82..90</td>
			<td>TCC GAC ACG</td>
			<td>ACC GTC AAC</td>
		</tr>
		<tr>
			<td>pCS2+ H2B:mCherry</td>
			<td>82..90</td>
			<td>TCC GAC ACG</td>
			<td>ACC GTC AAC</td>
		</tr>
	</tbody>
</table>
Table 2: Tentative optimization of Kozak sequences. Original and mutated Kozak sequences are indicated for each construct. The introduced mutations recover the Kozak sequence of the B. lanceolatum actin gene, which very closely resembles that of the inferred theoretical preferred Kozak sequence of amphioxus (A/CGA/C G/TTC A/GAC atg TG/CT).
<table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">pCS2+ eGFP:CAAX box</td>
		</tr>
		<tr>
			<td>Original codon</td>
			<td>Mutated codon</td>
			<td colspan="2" rowspan="2">Position in the pCS2+ vector</td>
		</tr>
		<tr>
			<td>(usage level)</td>
			<td>(usage level)</td>
		</tr>
		<tr>
			<td>GTA</td>
			<td>GTG</td>
			<td rowspan="2">154..156</td>
			<td rowspan="2">eGFP sequence</td>
		</tr>
		<tr>
			<td>(0.08)</td>
			<td>(0.43)</td>
		</tr>
		<tr>
			<td>AGA</td>
			<td>AGG</td>
			<td rowspan="2">814..816</td>
			<td rowspan="2">Linker</td>
		</tr>
		<tr>
			<td>(0.09)</td>
			<td>(0.27)</td>
		</tr>
		<tr>
			<td colspan="4">pCS2+ H2B:eGFP</td>
		</tr>
		<tr>
			<td>Original codon</td>
			<td>Mutated codon</td>
			<td colspan="2" rowspan="2">Position in the pCS2+ vector</td>
		</tr>
		<tr>
			<td>(usage level)</td>
			<td>(usage level)</td>
		</tr>
		<tr>
			<td>GTA</td>
			<td>GTG</td>
			<td rowspan="2">223..225</td>
			<td rowspan="2">H2B sequence</td>
		</tr>
		<tr>
			<td>(0.08)</td>
			<td>(0.43)</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>289..291</td>
			<td>H2B sequence</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>469..471</td>
			<td>Linker</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>568..570</td>
			<td>eGFP sequence</td>
		</tr>
		<tr>
			<td>CTA</td>
			<td>CTG</td>
			<td rowspan="2">226..228</td>
			<td rowspan="2">H2B sequence</td>
		</tr>
		<tr>
			<td>(0.06)</td>
			<td>(0.51)</td>
		</tr>
		<tr>
			<td colspan="4">pCS2+ H2B:mCherry</td>
		</tr>
		<tr>
			<td>Original codon</td>
			<td>Mutated codon</td>
			<td colspan="2" rowspan="2">Position in the pCS2+ vector</td>
		</tr>
		<tr>
			<td>(usage level)</td>
			<td>(usage level)</td>
		</tr>
		<tr>
			<td>GTA</td>
			<td>GTG</td>
			<td rowspan="2">223..225</td>
			<td rowspan="2">H2B sequence</td>
		</tr>
		<tr>
			<td>(0.08)</td>
			<td>(0.43)</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>289..291</td>
			<td>H2B sequence</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>469..471</td>
			<td>Linker</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>913..915</td>
			<td>mCherry sequence</td>
		</tr>
		<tr>
			<td>CTA</td>
			<td>CTG</td>
			<td rowspan="2">226..228</td>
			<td rowspan="2">H2B sequence</td>
		</tr>
		<tr>
			<td>(0.06)</td>
			<td>(0.51)</td>
		</tr>
	</tbody>
</table>
Table 3: Tentative optimization of codon usage. Original and mutated codons are indicated for each construct. While this has not been experimentally tested, the introduced mutations are meant to optimize mRNA translation in amphioxus.
Supplementary File 1: Construct sequences. The nucleotide sequences of (A) the membrane-targeted eGFP (eGFP:CAAX box), (B) the nuclear-targeted eGFP (H2B:eGFP) and (C) the nuclear-targeted mCherry (H2B:mCherry) constructs are given in the context of the pCS2+ vector backbone. The main domains encoded by each of the constructs are indicated in color: eGFP (green), mCherry (red), membrane localization signal from human HRAS (CAAX box) (cyan blue), zebrafish histone 2B exon (H2B) (yellow).

Watch this Scientific Journal Video about Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes at JoVE.com

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

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.

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

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the ...

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Nanyang Technological University

Research

JoVE Journal

Developmental Biology

11.0K Views.  Institut Pasteur.  Wij rapporteren hier de robuuste en efficiënte expressie van fluorescerende eiwitten na mRNA injectie in onbevruchte eicellen van Branchiostoma lanceolatum. De ontwikkeling van de micro-injectie techniek in deze basale chordate zal de weg voor vergaande technische innovaties in deze opkomende model systeem, met inbegrip van in vivo beeldvorming en gen-specifieke manipulaties effenen. 

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

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion (FDAP)

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological systems. In FDAP experiments, the clearance of proteins within living organisms can be measured. A protein of interest is tagged with a photoconvertible fluorescent protein, expressed in vivo and photoconverted, and the decrease in the photoconverted signal over time is monitored. The data is then fitted with an appropriate clearance model to determine the protein half-life. Importantly, the clearance kinetics of protein populations in different compartments of the organism can be examined separately by applying compartmental masks. This approach has been used to determine the intra- and extracellular half-lives of secreted signaling proteins during zebrafish development. Here, we describe a protocol for FDAP experiments in zebrafish embryos. It should be possible to use FDAP to determine the clearance kinetics of any taggable protein in any optically accessible organism.

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion FDAP

Protein levels in cells and tissues are often tightly regulated by the balance of protein production and clearance. Using Fluorescence Decay After Photoconversion (FDAP), the clearance kinetics of proteins can be experimentally measured in vivo.

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological ...

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into the developing oocyte. Because these events rely on maternal products which typically act very soon after fertilization-that preexist inside the egg, standard approaches for expression and functional reduction involving the injection of reagents into the fertilized egg are typically ineffective. Instead, such manipulations must be performed during oogenesis, prior to or during the accumulation of maternal products. This article describes in detail a protocol for the in vitro maturation of immature zebrafish oocytes and their subsequent in vitro fertilization, yielding viable embryos that survive to adulthood. This method allows the functional manipulation of maternal products during oogenesis, such as the expression of products for phenotypic rescue and tagged construct visualization, as well as the reduction of gene function through reverse-genetics agents.

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

An optimized protocol for the in vitro maturation of zebrafish oocytes used for the manipulation of maternal gene products is presented here.

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into ...

Use of Single Molecule Fluorescent In Situ Hybridization (SM-FISH) to Quantify and Localize mRNAs in Murine Oocytes

Current methods routinely used to quantify mRNA in oocytes and embryos include digital reverse-transcription polymerase chain reaction (dPCR), quantitative, real-time RT-PCR (RT-qPCR) and RNA sequencing. When these techniques are performed using a single oocyte or embryo, low-copy mRNAs are not reliably detected. To overcome this problem, oocytes or embryos can be pooled together for analysis; however, this often leads to high variability amongst samples. In this protocol, we describe the use of fluorescence in situ hybridization (FISH) using branched DNA chemistry. This technique identifies the spatial pattern of mRNAs in individual cells. When the technique is coupled with Spot Finding and Tracking&#160;computer software, the abundance of mRNAs in the cell can also be quantified. Using this technique, there is reduced variability within an experimental group and fewer oocytes and embryos are required to detect significant differences between experimental groups. Commercially available branched-DNA SM-FISH kits have been optimized to detect mRNAs in sectioned tissues or adherent cells on slides. However, oocytes do not effectively adhere to slides and some reagents in the kit were too harsh resulting in oocyte lysis. To prevent this lysis, several modifications were made to the FISH kit. Specifically, oocyte permeabilization and wash&#160;buffers designed for the immunofluorescence of oocytes and embryos replaced the proprietary buffers. The permeabilization, washes, and incubations with probes and amplifier were performed in 6-well plates and oocytes were placed on slides at the end of the protocol using mounting media. These modifications were able to overcome the limitations of the commercially available kit, in particular, the oocyte lysis. To accurately and reproducibly count the number of mRNAs in individual oocytes, computer software was used. Together, this protocol represents an alternative to PCR and sequencing to compare the expression of specific transcripts in single cells.

Use of Single Molecule Fluorescent In Situ Hybridization SM-FISH to Quantify and Localize mRNAs in Murine Oocytes

To reproducibly count the numbers of mRNAs in individual oocytes, single molecule RNA fluorescence in situ hybridization (RNA-FISH) was optimized for non-adherent cells. Oocytes were collected, hybridized with the transcript specific probes, and quantified using an image quantification software.

Current methods routinely used to quantify mRNA in oocytes and embryos include digital reverse-transcription polymerase chain reaction (dPCR), ...

Genetics

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. The observation of Tribolium embryos with light sheet-based fluorescence microscopy has multiple advantages over conventional widefield and confocal fluorescence microscopy. Due to the unique properties of a light sheet-based microscope, three dimensional images of living specimens can be recorded with high signal-to-noise ratios and significantly reduced photo-bleaching as well as photo-toxicity along multiple directions over periods that last several days. With more than four years of methodological development and a continuous increase of data, the time seems appropriate to establish standard operating procedures for the usage of light sheet technology in the Tribolium community as well as in the insect community at large. This protocol describes three mounting techniques suitable for different purposes, presents two novel custom-made transgenic Tribolium lines appropriate for long-term live imaging, suggests five fluorescent dyes to label intracellular structures of fixed embryos and provides information on data post-processing for the timely evaluation of the recorded data. Representative results concentrate on long-term live imaging, optical sectioning and the observation of the same embryo along multiple directions. The respective datasets are provided as a downloadable resource. Finally, the protocol discusses quality controls for live imaging assays, current limitations and the applicability of the outlined procedures to other insect species.
This protocol is primarily intended for developmental biologists who seek imaging solutions that outperform standard laboratory equipment. It promotes the continuous attempt to close the gap between the technically orientated laboratories/communities, which develop and refine microscopy methodologically, and the life science laboratories/communities, which require 'plug-and-play' solutions to technical challenges. Furthermore, it supports an axiomatic approach that moves the biological questions into the center of attention.

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

Imaging the morphogenesis of insect embryos with light sheet-based fluorescence microscopy has become state of the art. This protocol outlines and compares three mounting techniques appropriate for Tribolium castaneum embryos, introduces two novel custom-made transgenic lines well suited for live imaging, discusses essential quality controls and indicates current experimental limitations.

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. ...

Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression studies. In zebrafish (Danio rerio), microinjection of RNA, DNA, proteins, antisense oligonucleotides and other small molecules into the developing embryo provides researchers a quick and robust assay for exploring gene function in vivo. In this video-article, we will demonstrate how to prepare and microinject in vitro synthesized EGFP mRNA and a translational-blocking morpholino oligo against pkd2, a gene associated with autosomal dominant polycystic kidney disease (ADPKD), into 1-cell stage zebrafish embryos. We will then analyze the success of the mRNA and morpholino microinjections by verifying GFP expression and phenotype analysis. Broad applications of this technique include generating transgenic animals and germ-line chimeras, cell-fate mapping and gene screening. Herein we describe a protocol for overexpression of EGFP and knockdown of pkd2 by mRNA and morpholino oligonucleotide injection.

Microinjection is a well-established and effective method for introducing foreign substances into fertilized zebrafish embryos. Here, we demonstrate a robust microinjection technique for performing mRNA overexpression, and morpholino oligonucleotide gene knockdown studies in zebrafish.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression ...

Biology

Fluorescent Protein Tagging in Zebrafish Larvae Using CRISPR/Cas9 Knock-In

Fluorescence Labeling to Visualize Low-Expressed Proteins in Zebrafish

CRISPR/Cas9-mediated knock-in (KI) technology allows for easier fluorescent-protein tagging in zebrafish (Danio rerio), a preferred model organism for in vivo imaging due to its transparency during the early developmental stage. Here, we provide a detailed protocol for performing high-efficiency fluorescence gene KI, rapid screening for KI founders, and low-abundance protein tracing in zebrafish larvae, which will lay a critical foundation for subsequent physio-pathological studies in zebrafish. The current protocol includes complete steps for the sgRNA design for the gene of interest, sgRNA in vitro transcription, Cas9 mRNA in vitro transcription, in vivo sgRNA screen for the one with the highest efficiency, donor plasmid design and construction, microinjection in zebrafish larvae, KI founder screen and zebrafish live imaging. Critical steps, troubleshooting tips, quality control methods, and advantages and applications of this protocol are included and discussed. This protocol assures quick and accurate results at a low cost and has been validated by multiple trials.

Here, we present a protocol to label proteins with a fluorescence protein tag in zebrafish larvae, a newly developed and modified in vivo system especially useful for visualizing low-abundance proteins in zebrafish.

CRISPR/Cas9-mediated knock-in (KI) technology allows for easier fluorescent-protein tagging in zebrafish (Danio rerio), a preferred model organism for in ...

Fast and Efficient Expression of Multiple Proteins in Avian Embryos Using mRNA Electroporation

We report that mRNA electroporation permits fluorescent proteins to label cells in living quail embryos more quickly and broadly than DNA electroporation. The high transfection efficiency permits at least 4 distinct mRNAs to be co-transfected with ~87% efficiency. Most of the electroporated mRNAs are degraded during the first 2 h post-electroporation, permitting time-sensitive experiments to be carried out in the developing embryo. Finally, we describe how to dynamically image live embryos electroporated with mRNAs that encode various subcellular targeted fluorescent proteins.

We report messenger RNA (mRNA) electroporation as a method that permits fast and efficient expression of multiple proteins in the quail embryo model system. This method can be used to fluorescently label cells and record their in vivo movements by time-lapse microscopy shortly after electroporation.

We report that mRNA electroporation permits fluorescent proteins to label cells in living quail embryos more quickly and broadly than DNA electroporation. ...

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.
Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.
Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.
Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

Live Imaging of Cell Extrusion from the Epidermis of Developing Zebrafish

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function. Our studies have found that dying cells send signals to their live neighbors to form and contract a ring of actin and myosin that ejects it out from the epithelial sheet while closing any gaps that might have resulted from its exit, a process termed cell extrusion1. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe a method to induce and image extrusion in the larval zebrafish epidermis. To visualize extrusion, we inject a red fluorescent protein labeled probe for F-actin into one-cell stage transgenic zebrafish embryos expressing green fluorescent protein in the epidermis and induce apoptosis by addition of G418 to larvae. We then use time-lapse imaging on a spinning disc confocal microscope to observe actin dynamics and epithelial cell behaviors during the process of apoptotic cell extrusion. This approach allows us to investigate the extrusion process in live epithelia and will provide an avenue to study disease states caused by the failure to eliminate apoptotic cells.

Dying cells are extruded from epithelial tissues by concerted contraction of neighboring cells without disrupting barrier function. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe methods to induce and image extrusion in the larval zebrafish epidermis at cellular resolution.

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function.  Our studies have found ...

High Throughput Microinjections of Sea Urchin Zygotes

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of treatment solution is loaded into a microinjection needle that is used to physically inject individual immobilized cells or embryos. Despite the need for initial training to perform this procedure for high-throughput delivery, microinjection offers maximum efficiency and reproducible delivery of a wide variety of treatment solutions (including complex mixtures of samples) into cells, eggs or embryos. Applications to microinjections include delivery of DNA constructs, mRNAs, recombinant proteins, gain of function, and loss of function reagents. Fluorescent or colorimetric dye is added to the injected solution to enable instant visualization of efficient delivery as well as a tool for reliable normalization of the amount of the delivered solution. The described method enables microinjection of 100-400 sea urchin zygotes within 10-15 min.

Microinjection is a common technique used to deliver DNA constructs, mRNAs, morpholino antisense oligonucleotides or other treatments into eggs, embryos, and cells of various species.

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of ...

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

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