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...

<ol>
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Biotechnol. 19, 506-510 (2008).</li><li>Stepicheva, N. A., Song, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+throughput+microinjections+of+sea+urchin+zygotes.">High throughput microinjections of sea urchin zygotes.</a> J. Vis. Exp. , e50841 (2014).</li><li>Schubert, M., Escriva, H., Xavier-Neto, J., Laudet, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphioxus+and+tunicates+as+evolutionary+model+systems.">Amphioxus and tunicates as evolutionary model systems.</a> Trends Ecol. Evol. 21, 269-277 (2006).</li><li>Bertrand, S., Escriva, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+crossroads+in+developmental+biology:+amphioxus.">Evolutionary crossroads in developmental biology: amphioxus.</a> Development. 138, 4819-4830 (2011).</li><li>Holland, L. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+new+characters+after+whole+genome+duplications:+insights+from+amphioxus.">Evolution of new characters after whole genome duplications: insights from amphioxus.</a> Semin. Cell Dev. Biol. 24, 101-109 (2013).</li><li>Lemaire, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+crossroads+in+developmental+biology:+the+tunicates.">Evolutionary crossroads in developmental biology: the tunicates.</a> Development. 138, 2143-2152 (2011).</li><li>Yu, J. K., Holland, L. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cephalochordate+(amphioxus)+embryos:+procurement,+culture,+and+basic+methods.">Cephalochordate (amphioxus) embryos: procurement, culture, and basic methods.</a> Methods Cell Biol. 74, 195-215 (2004).</li><li>Holland, L. Z., Onai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyses+of+gene+function+in+amphioxus+embryos+by+microinjection+of+mRNAs+and+morpholino+oligonucleotides.">Analyses of gene function in amphioxus embryos by microinjection of mRNAs and morpholino oligonucleotides.</a> Methods Mol. Biol. 770, 423-438 (2011).</li><li>Liu, X., Li, G., Feng, J., Yang, X., Wang, Y. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+microinjection+method+for+unfertilized+eggs+of+Asian+amphioxus+Branchiostoma+belcheri.">An efficient microinjection method for unfertilized eggs of Asian amphioxus Branchiostoma belcheri.</a> Dev. Genes Evol. 223, 269-278 (2013).</li><li>Harvey, K. J., Lukovic, D., Ucker, D. 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L., Weintraub, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+achaete-scute+homolog+3+in+Xenopus+embryos+converts+ectodermal+cells+to+a+neural+fate.">Expression of achaete-scute homolog 3 in Xenopus embryos converts ectodermal cells to a neural fate.</a> Genes Dev. 8, 1434-1447 (1994).</li><li>Nakagawa, S., Niimura, Y., Gojobori, T., Tanaka, H., Miura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+of+preferred+nucleotide+sequences+around+the+translation+initiation+codon+in+eukaryote+genomes.">Diversity of preferred nucleotide sequences around the translation initiation codon in eukaryote genomes.</a> Nucleic Acids Res. 36, 861-871 (2008).</li><li>Nakamura, Y., Gojobori, T., Ikemura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Codon+usage+tabulated+from+international+DNA+sequence+databases:+status+for+the+year+2000.">Codon usage tabulated from international DNA sequence databases: status for the year 2000.</a> Nucleic Acids Res. 28, 292 (2000).</li><li>Deheyn, D. D., 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=Endogenous+green+fluorescent+protein+(GFP)+in+amphioxus.">Endogenous green fluorescent protein (GFP) in amphioxus.</a> Biol. Bull. 213, 95-100 (2007).</li><li>Schubert, M., 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=Retinoic+acid+signaling+acts+via+Hox1+to+establish+the+posterior+limit+of+the+pharynx+in+the+chordate+amphioxus.">Retinoic acid signaling acts via Hox1 to establish the posterior limit of the pharynx in the chordate amphioxus.</a> Development. 132, 61-73 (2005).</li><li>Schubert, M., Holland, N. D., Laudet, V., Holland, L. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+retinoic+acid-Hox+hierarchy+controls+both+anterior/posterior+patterning+and+neuronal+specification+in+the+developing+central+nervous+system+of+the+cephalochordate+amphioxus.">A retinoic acid-Hox hierarchy controls both anterior/posterior patterning and neuronal specification in the developing central nervous system of the cephalochordate amphioxus.</a> Dev. Biol. 296, 190-202 (2006).</li><li>Yu, J. K., Holland, N. D., Holland, L. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-specific+expression+of+FoxD+reporter+constructs+in+amphioxus+embryos.">Tissue-specific expression of FoxD reporter constructs in amphioxus embryos.</a> Dev. Biol. 274, 452-461 (2004).</li><li>Beaster-Jones, L., Schubert, M., Holland, L. 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Z., 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=The+amphioxus+genome+illuminates+vertebrate+origins+and+cephalochordate+biology.">The amphioxus genome illuminates vertebrate origins and cephalochordate biology.</a> Genome Res. 18, 1100-1111 (2008).</li><li>Urasaki, A., Mito, T., Noji, S., Ueda, R., Kawakami, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transposition+of+the+vertebrate+Tol2+transposable+element+in+Drosophila+melanogaster.">Transposition of the vertebrate Tol2 transposable element in Drosophila melanogaster.</a> Gene. 425, 64-68 (2008).</li><li>Li, G., 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=Mutagenesis+at+specific+genomic+loci+of+amphioxus+Branchiostoma+belcheri+using+TALEN+method.">Mutagenesis at specific genomic loci of amphioxus Branchiostoma belcheri using TALEN method.</a> J. 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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 (...

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 border="0" cellpadding="0" cellspacing="0" width="1009">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Name of Material/ Equipment</td>
			<td style="width:156px;">Company</td>
			<td style="width:143px;">Catalog Number</td>
			<td style="width:413px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Consumables</td>
			<td style="width:156px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">35 mm Petri dishes</td>
			<td>Falcon</td>
			<td>353001</td>
			<td>culture-treated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Filtration unit (Stericup 1L)</td>
			<td style="width:156px;">Fisher</td>
			<td style="width:143px;">W21719</td>
			<td>0.22 micron filtration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Spin-X tubes</td>
			<td style="width:156px;">Costar</td>
			<td style="width:143px;">8160</td>
			<td>0.22 micron filtration tube</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:297px;">Needle storage jar for 1.2 mm diameter capillaries</td>
			<td style="width:156px;">WPI</td>
			<td style="width:143px;">E212</td>
			<td style="width:413px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Pasteur Pipettes</td>
			<td style="width:156px;"></td>
			<td style="width:143px;"></td>
			<td>230 mm long</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Aspiration tube</td>
			<td style="width:156px;">Dutscher</td>
			<td style="width:143px;">75056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Capillaries for injection needles</td>
			<td style="width:156px;">Sutter</td>
			<td style="width:143px;">BF 120-94-10</td>
			<td>Borosilicate glass with filament, OD 1.20 mm, ID 0.94 mm, length 10 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;"></td>
			<td style="width:156px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Reagents</td>
			<td style="width:156px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Low-melting agarose</td>
			<td style="width:156px;">SIGMA</td>
			<td style="width:143px;">A9414</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Phenol Red</td>
			<td style="width:156px;">SIGMA</td>
			<td style="width:143px;">114537</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Glycerol</td>
			<td style="width:156px;">SIGMA</td>
			<td style="width:143px;">G2025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Poly-L-Lysine hydrobromide</td>
			<td style="width:156px;">SIGMA</td>
			<td style="width:143px;">P9155</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">H2O Dnase, Rnase-free</td>
			<td style="width:156px;">Gibco</td>
			<td style="width:143px;">10977-035</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">mMessage mMachine SP6 Transcription kit</td>
			<td style="width:156px;">Ambion</td>
			<td style="width:143px;">AM1340</td>
			<td>mRNA synthesis kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Phenol pH8</td>
			<td style="width:156px;">SIGMA</td>
			<td style="width:143px;">P4557</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">24:1 chloroform:isoamylic alcohol</td>
			<td style="width:156px;">SIGMA</td>
			<td style="width:143px;">C0549</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">5:1 phenol pH4.7:chloroform</td>
			<td style="width:156px;">SIGMA</td>
			<td style="width:143px;">P1944</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Reef Crystal salts (200 kg)</td>
			<td style="width:156px;">Europrix&#160;</td>
			<td style="width:143px;"></td>
			<td>Commercial salts</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;"></td>
			<td style="width:156px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Equipment</td>
			<td style="width:156px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:297px;">Fluorescent dissecting scope with 200x magnification&#160;</td>
			<td style="width:156px;">Leica</td>
			<td style="width:143px;">MZ16F</td>
			<td>25x oculars, DSR and GFP2 filters</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Micromanipulator</td>
			<td style="width:156px;">Marzhauzer</td>
			<td style="width:143px;">M-33</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Injector</td>
			<td style="width:156px;">Picospritzer</td>
			<td style="width:143px;">model II or III</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Needle puller</td>
			<td style="width:156px;">Sutter</td>
			<td style="width:143px;">P97</td>
			<td>heating-filament needle puller</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:297px;">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 batc...

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 ...

expression-fluorescent-proteins-branchiostoma-lanceolatum-mrna

Research

JoVE Journal

Developmental Biology

10.9K Görüntüleme.  Institut Pasteur.  Biz Branchiostoma lanceolatum ve döllenmemiş oosit içine burada mRNA enjeksiyonundan sonra floresan proteinleri sağlam ve verimli bir ifade rapor. Bu bazal kordalısı içinde mikroenjeksiyon tekniği gelişimi, gelişmekte olan bu model sistemde teknik yenilikleri geniş kapsamlı vivo görüntüleme ve gen-spesifik manipülasyonlar dahil önünü açacak. 