Name: microinjection of dna into eyebuds in xenopus laevis embryos and imaging of gfp expressing optic axonal arbors in intact, living xenopus tadpoles
Uploaded: 2019-09-04T15:00:00
Description: Watch this Scientific Journal Video about Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles at JoVE.com

We thank Touro University California College of Osteopathic Medicine for supporting our research. We acknowledge previous students in the laboratory (Esther Wu, Gregory Peng, Taegun Jin, John Lim) who helped implement this microinjection technique in our laboratory. We are grateful to Dr. Christine Holt, in whose laboratory this DNA microinjection/lipofection technique in Xenopus embryos was first developed.

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Touro University California (Protocol # TUCA003TE01X).
1. Obtaining X. laevis Embryos
<ol>
	<li>Obtain X. laevis embryos by natural mating of pairs of male and female adult frogs primed with human chorionic gonadotropin (HCG), by in vitro fertilization of eggs shed from female adult frogs primed with HCG, or by ordering directly (Table of Materials).<...

<ol>
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E., Bonhoeffer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+axons+with+and+without+their+somata,+growing+to+and+arborizing+in+the+tectum+of+Xenopus+embryos:+a+time-lapse+video+study+of+single+fibres+in+vivo.">Retinal axons with and without their somata, growing to and arborizing in the tectum of Xenopus embryos: a time-lapse video study of single fibres in vivo.</a> Development. 101 (1), 123-133 (1987).</li><li>Sakaguchi, D. S., Murphey, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Map+formation+in+the+developing+Xenopus+retinotectal+system:+an+examination+of+ganglion+cell+terminal+arborizations.">Map formation in the developing Xenopus retinotectal system: an examination of ganglion cell terminal arborizations.</a> Journal of Neuroscience. 5 (12), 3228-3245 (1985).</li><li>Elul, T. 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M., Dirks, R., Martens, G. J., Cohen-Cory, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-autonomous+TrkB+signaling+in+presynaptic+retinal+ganglion+cells+mediates+axon+arbor+growth+and+synapse+maturation+during+the+establishment+of+retinotectal+synaptic+connectivity.">Cell-autonomous TrkB signaling in presynaptic retinal ganglion cells mediates axon arbor growth and synapse maturation during the establishment of retinotectal synaptic connectivity.</a> Journal of Neuroscience. 27 (10), 2444-2456 (2007).</li><li>Holt, C. E., Garlick, N., Cornel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipofection+of+cDNAs+in+the+Embryonic+Vertebrate+Central+Nervous+System.">Lipofection of cDNAs in the Embryonic Vertebrate Central Nervous System.</a> Neuron. 4 (2), 203-214 (1990).</li><li>Ohnuma, S. I., Mann, F., Boy, S., Perron, M., Harris, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipofection+strategy+for+the+study+of+Xenopus+retinal+development.">Lipofection strategy for the study of Xenopus retinal development.</a> Methods. 28 (4), 411-419 (2002).</li><li>Joesch, M., Meister, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+neuronal+circuit+for+colour+vision+based+on+rod-cone+opponency.">A neuronal circuit for colour vision based on rod-cone opponency.</a> Nature. 532 (7598), 236-239 (2016).</li><li>Meyer, M. P., Smith, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+from+in+vivo+imaging+that+synaptogenesis+guides+the+growth+and+branching+of+axonal+arbors+by+two+distinct+mechanisms.">Evidence from in vivo imaging that synaptogenesis guides the growth and branching of axonal arbors by two distinct mechanisms.</a> Journal of Neuroscience. 26 (13), 3604-3614 (2006).</li><li>Li, X., Monckton, E. A., Godbout, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ectopic+expression+of+transcription+factor+AP-2&#948;+in+developing+retina:+effect+on+PSA-NCAM+and+axon+routing.">Ectopic expression of transcription factor AP-2&#948; in developing retina: effect on PSA-NCAM and axon routing.</a> Journal of Neurochemistry. 129 (1), 72-84 (2014).</li><li>Haas, K., Jensen, K., Sin, W. C., Foa, L., Cline, H. 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In this article, we demonstrate how to express exogenous DNA constructs in single or small numbers of optic neurons and how to image individual GFP expressing optic axonal arbors with normal and altered molecular signaling in intact, living tadpoles of the frog X. laevis. We also explain how to reconstruct and quantify the morphology of GFP expressing optic axonal arbors from images captured in vivo. To express exogenous DNA plasmids in small number of optic neurons, we microinject a DNA/lipofection reagent mixt...

During development of the nervous system, axons of presynaptic neurons navigate through diverse regions of the brain to reach their target areas. When axons invade their target tissues, they establish synaptic connections with postsynaptic target neurons. In many types of neurons, axons increase the number and spatial extent of synaptic connections they can make by elaborating networks of terminal branches or arbors1. The retino-tectal projection of tadpoles of the aquatic frog Xenopus laevis is a powerful vertebrate model for examining mechanisms underlying terminal axon arborization and synaptic connectivity2,3,4. Individual GFP expressing optic axonal arbors with normal and altered molecular signaling can be observed directly in intact, living Xenopus tadpoles5,6,7,8. To express GFP alone or together with full-length or truncated versions of genes in small number of optic neurons, we use a technique involving microinjection/lipofection of DNA into eyebuds of one day old Xenopus embryos9,10. This technique was originally developed to study mechanisms of optic axon pathfinding in young Xenopus tadpoles, and has since been applied by us and others to determine cell-autonomous molecular mechanisms underlying optic axon arborization in Xenopus tadpoles5,6,7,8,9,10.
Alternate techniques to express exogenous genes in a small number of optic neurons have been developed in other model species, as well as in X. laevis. However, each of these approaches presents challenges and limitations when compared to microinjection of DNA/lipofection reagent in eyebuds of Xenopus embryos. In mice, transgenesis can be used to express genes in a small number of optic neurons, but the generation of transgenic mice is costly and time consuming and transgenic mice often present with undesirable side effects11. Transgenic zebrafish that express exogenous genes in optic neurons can also be created by injecting plasmids into early cleavage stage embryos12. However, this process requires cloning of a specific promoter to express genes in a mosaic pattern in optic neurons in zebrafish larvae12. The frequency of expression of exogenous DNA in optic neurons in transgenic zebrafish is also somewhat lower (&#60;30%) compared to Xenopus tadpoles that were microinjected with DNA/liposomal reagent (30&#8722;60%)12. In ovo electroporation has also been used to express genes in small numbers of optic neurons in chicks13. However, this procedure has failed to fully characterize mechanisms that establish optic projections because optic axon arborization cannot be imaged in intact, living chick embryos. Finally, several laboratories have used electroporation to transfect genes into small number of optic neurons in Xenopus tadpoles14,15. Yet, electroporation requires optimization of equipment and protocols (stimulator, electrodes, spatial and temporal patterns of wave pulses) beyond that used for microinjection of DNA/lipofection reagent into eyebuds of Xenopus embryos.
We and others previously used the technique of microinjection/lipofection of DNA into eyebuds of Xenopus embryos to determine cell autonomous signaling mechanisms that establish optic axon arborization5,6,7,8. We initially used this approach to dissect the functions of the Cadherin and Wnt adaptor protein &#946;-catenin in optic axonal arborization in Xenopus tadpoles5,6. In one study, we showed that &#946;-catenin binding to &#945;-catenin and to PDZ is required, respectively, for initiating and shaping optic axonal arbors in vivo5. In a second report, we demonstrated that the &#946;-catenin binding domains for &#945;-catenin and GSK-3&#946; oppositely modulate projection patterns of ventral optic axonal arbors6. More recently, we identified roles for the Wnt factor, adenomatous poliposis coli (APC), in regulating morphological features of optic axonal arbors in Xenopus tadpoles7. By co-expressing the N-terminal and central domains of APC that modulate &#946;-catenin stability and microtubule organization together with GFP in individual optic neurons, we determined shared and distinct roles for these APC interaction domains on branch number, length, and angle in optic axonal arbors in vivo7. Another laboratory used the microinjection/lipofection technique to determine cell autonomous roles for signaling by the BDNF receptor, TrkB, in optic axonal arbors in Xenopus tadpoles8. This group showed that expression of a dominant-negative TrkB perturbed branching and synaptic maturation in individual optic axon arbors in vivo8. Overall, the lipofection technique in Xenopus has already illuminated the specific roles of different genes in optic axon branching in the native environment.

<table>
	<tbody>
		<tr>
			<td>3.5&#34; Micropipettes</td>
			<td>Drummond Scientific</td>
			<td>3-000-203 - G/X</td>
			<td></td>
		</tr>
		<tr>
			<td>&#956;-manager software (Version )</td>
			<td></td>
			<td></td>
			<td>www.micro-manager.org</td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td>Scion Corporation</td>
			<td>CFW-1312 M</td>
			<td></td>
		</tr>
		<tr>
			<td>Chorulon (Human Chorionic Gonadotropin)</td>
			<td>AtoZ Vet Supply</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Cysteine</td>
			<td>Sigma-Aldrich</td>
			<td>168149-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>DOTAP</td>
			<td>Sigma-Aldrich</td>
			<td>11202375001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont Forceps #5</td>
			<td>Fine Science Tools</td>
			<td>11250-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Eclipse E800 epifluoresence microscope</td>
			<td>Nikon</td>
			<td></td>
			<td>Objectives: Nikon Plan Apo 20X/0.75, Nikon Plan Fluor 40/0.75</td>
		</tr>
		<tr>
			<td>GNU Image Manipulation Program (Version 2.10.10)</td>
			<td>GIMP</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Illustrator (2017 Creative Cloud)</td>
			<td>Adobe</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Image J (Version 1.46r)</td>
			<td>NIH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microfil</td>
			<td>World Precision Instruments</td>
			<td>MF 34G-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator with universal adaptor and support base</td>
			<td>Drummond Scientific</td>
			<td>3-000-024-R</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>3-000-025-SB</td>
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		<tr>
			<td></td>
			<td></td>
			<td>3-000-024-A</td>
		</tr>
		<tr>
			<td>Micropipette Puller</td>
			<td>Sutter Instrument</td>
			<td>P-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Miniprep Kit</td>
			<td>Qiagen</td>
			<td>27104</td>
			<td></td>
		</tr>
		<tr>
			<td>Motorized z-stage</td>
			<td>Applied Scientific Instrumentation</td>
			<td>MFC-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanoject II injector</td>
			<td>Drummond Scientific</td>
			<td>3-000-204</td>
			<td></td>
		</tr>
		<tr>
			<td>Powerpoint (Version 15.31)</td>
			<td>Microsoft</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xenopus laevis embryos</td>
			<td>Nasco</td>
			<td>LM00490</td>
			<td></td>
		</tr>
	</tbody>
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microinjection of dna into eyebuds in xenopus laevis embryos and imaging of gfp expressing optic axonal arbors in intact, living xenopus tadpoles

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the development of neuronal connectivity. During establishment of the retino-tectal projection, optic axons extend from the eye and navigate through distinct regions of the brain to reach their target tissue, the optic tectum. Once optic axons enter the tectum, they elaborate terminal arbors that function to increase the number of synaptic connections they can make with target interneurons in the tectum. Here, we describe a method to express DNA encoding green fluorescent protein (GFP), and gain- and loss-of-function gene constructs, in optic neurons (retinal ganglion cells) in Xenopus embryos. We explain how to microinject a combined DNA/lipofection reagent into eyebuds of one day old embryos such that exogenous genes are expressed in single or small numbers of optic neurons. By tagging genes with GFP or co-injecting with a GFP plasmid, terminal axonal arbors of individual optic neurons with altered molecular signaling can be imaged directly in brains of intact, living Xenopus tadpoles several days later, and their morphology can be quantified. This protocol allows for determination of cell-autonomous molecular mechanisms that underlie the development of optic axon arborization in vivo.

The protocol described in this article yields a success rate of 30&#8722;60% of injected Xenopus embryos expressing GFP (alone or together with an additional DNA constructs) in one to ten optic axonal arbors. In Figure 3, we show representative confocal images of GFP expressing control and mutant optic axonal arbors in intact Xenopus tadpoles from our recently published study7. For this study, we cloned two domain mutants of APC (APCNTERM and APC&#94...

Watch this Scientific Journal Video about Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles at JoVE.com

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

This protocol aims to demonstrate how to microinject a DNA/DOTAP mixture into eyebuds of one day old Xenopus laevis embryos, and how to image and reconstruct individual green fluorescent protein (GFP) expressing optic axonal arbors in tectal midbrains of intact, living Xenopus tadpoles.

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the ...

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