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).</li>
	<li>Dejelly embryos obtained with a 2% cysteine solution at room temperature (Table of Materials)16.
	<ol>
		<li>Collect 50&#8722;100 embryos in a large Petri dish. Remove the solution the embryos are in by decanting or using a plastic transfer pipette. Add 25 mL of 2% cysteine solution (0.5 g cysteine in 25 mL ddH2O, pH to 8.0) to the dish containing the embryos.</li>
		<li>Gently swirl the Petri dish containing the embryos in the cysteine solution until the jelly coats of the embryos fall off and the embryos collect in a clump in the center of the dish (5&#8722;10 min). At this point, slowly and gently pour off the cysteine solution into a waste beaker. Take care not to pour too many of the embryos in the waste beaker along with the cysteine solution.</li>
		<li>Rinse the embryos in the Petri dish 6x with 10% modified Mark&#8217;s Ringer solution (MMR) or other suitable solution (e.g., modified Barth&#8217;s Solution, MBS), swirling the dish each time the solution is replaced.</li>
	</ol>
	</li>
	<li>Culture the embryos in 10% MMR until they reach developmental stages 22&#8722;2417. Xenopus embryos can be incubated at temperatures between 15&#8722;25 &#176;C. The rate of development of the embryos depends on the temperature they are incubated at17. 
	NOTE: Xenopus embryos ordered from a catalogue usually arrive in the laboratory at developmental stages 20&#8722;24, so they can be dejellied and microinjected right away.</li>
</ol>
2. Preparing DNA Plasmids and Making a DNA/DOTAP Mixture
<ol>
	<li>Subclone DNA expression constructs into Xenopus expression vectors pCS2+ or pCS2+MT or derivatives thereof (originally constructed by D. Turner and R. Rupp)5,6,7. pCS2+ vectors contain a modified cytomegalovirus (CMV) promoter that facilitates gene expression in frogs.</li>
	<li>Amplify pCS2 plasmids containing GFP and/or genes of interest with miniprep kits (Table of Materials) following the standard procedure. In the final elution step of the miniprep protocol, perform a sequential elution of the DNA into ddH2O to yield a final concentration of &#62;1 &#181;g/&#181;L.</li>
	<li>Store all pCS2 plasmids at -80 &#176;C until ready to perform a microinjection/lipofection experiment, i.e., when embryos are at developmental stages 22&#8722;24.</li>
	<li>Thaw DNA plasmids to be lipofected at room temperature. Immediately prior to lipofection, briefly centrifuge DNA plasmids. This will prevent precipitate from forming in the DNA/DOTAP mixture that could clog the tip of the microcapillary pipette.</li>
	<li>Combine DNA plasmids with the DOTAP liposomal transfection reagent (Table of Materials) at a 1:3 (w/v) ratio9,10. For example, transfer 2 &#181;g of DNA to a 1.5 mL microcentrifuge tube and add 6 &#181;L of DOTAP, or transfer 3 &#181;g of DNA in a microcentrifuge tube and add 9 &#181;L of DOTAP.</li>
	<li>Once the DNA and DOTAP are combined, gently flick the microcentrifuge tube to mix the solution. The DNA/DOTAP solution should become slightly opaque after mixing.</li>
	<li>If two plasmids are to be lipofected together (e.g., pCS2-GFP with a second pCS2 plasmid containing a truncated or full-length version of a gene) in optic neurons, first combine the two plasmids (after briefly centrifuging both of them) at a 1:1 ratio, and then add DOTAP at a 1:3 (w/v) ratio. For example, combine 1 &#181;g of pCS2-GFP with 1 &#181;g of a second pCS2 plasmid and then add 6 &#181;L of DOTAP. 
	NOTE: Studies have shown that lipofection of two plasmids into eyebuds of Xenopus embryos at these developmental stages will result in their co-expression in individual optic neurons9,10.</li>
</ol>
3. Loading a Microinjection Needle with DNA/DOTAP
<ol>
	<li>Gently clip the tip of a pulled glass microcapillary pipette with fine forceps (Table of Materials).</li>
	<li>Backfill the glass microcapillary pipette with mineral oil using a microfil such that a tiny drop of mineral oil appears at the clipped tip of the micropipette. Fill the microcapillary pipette halfway with mineral oil.</li>
	<li>Load the pulled glass microcapillary pipette that is now filled with mineral oil into a suitable injection holder connected to an injector. If using an injector (Table of Materials), eject the plunger halfway before loading the microcapillary pipette onto it. Once the microcapillary pipette is securely attached to the injector, extend the plunger to the full extent to confirm that the microcapillary pipette is strongly attached to the injector and does not move with the extension of the plunger.</li>
	<li>Transfer a 3 &#956;L drop of the DNA/DOTAP mixture onto a cut square (1 inch square) sheet of paraffin paper.</li>
	<li>Under a stereo dissecting microscope, move the tip of the glass microcapillary pipette into the DNA/DOTAP drop.</li>
	<li>Slowly suck the DNA/DOTAP drop into the glass microcapillary pipette using the fill option on the injection apparatus. As the liquid is loaded into the microcapillary pipette, the drop will get smaller. Due to the slight opacity of the DNA/DOTAP solution, the boundary between the mineral oil and the DNA/DOTAP solution should be visible in the glass microcapillary pipette (Figure 1). If needed, stop filling the microcapillary pipette periodically to allow the pressure in the glass microcapillary pipette to recalibrate.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60123/60123fig01v2.jpg" /> 
Figure 1: Images of microcapillary pipette.&#160;Images show a microcapillary pipette on the injection apparatus, before (A), and after (B) filling with DNA/DOTAP. Open arrows, tip of plunger in the microcapillary pipette (A,B). Closed arrow, line between mineral oil and DNA/DOTAP in the filled microcapillary pipette (B). Scale bar = 1 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60123/60123fig01v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Microinjecting DNA/DOTAP into Eyebuds of 1 Day Old Xenopus Embryos
<ol>
	<li>Manually devitellinize ten stage 20&#8722;24 Xenopus embryos with fine forceps in a 10 mm Petri dish filled with 0.1x MMR. Grasp the vitelline envelope at the waist to avoid injuring the embryos. With forceps in both the experimenter&#8217;s left and right hands, pop the bubble of the vitelline envelope and release the embryo from the vitelline envelope. Take care not to injure the embryos when removing the vitelline envelope. 
	NOTE: Beginning at stage 20, Xenopus embryos develop an indentation or &#8216;waist&#8217; between the anterior and posterior halves of the embryo. This waist allows a gap to form between the vitelline envelope and the embryo at this position.</li>
	<li>Use a plastic transfer pipette with a cut tip to transfer 5&#8722;10 devitellinized stage 22&#8722;24 embryos to a 10 mm Petri dish filled with 1x MMR. 
	NOTE: The higher salt solution in 1x MMR facilitates healing of puncture wounds that will result from the microinjection.</li>
	<li>Under the stereomicroscope, grasp one of the devitellinized embryos in the Petri dish with forceps in each hand, and arrange the embryo so that its anterior pole is pointed up in the field of view. Orient the embryo so that it is lying laterally and one of its eyebuds (left or right) is facing upwards.</li>
	<li>Under the stereomicroscope, hold the embryo with the forceps in the experimenter&#8217;s non-dominant hand, and with the experimenter&#8217;s dominant hand introduce the tip of the glass micropipette into the eyebud (from the ventral or the dorsal side, depending on the half of the embryo that is currently being injected), just beneath the epidermis (Figure 2). Inject between 70&#8722;210 nL of the DNA/DOTAP solution. This can be done in several pulses, depending on the size of the pulse the injector is set to (usually 70 nL). 
	NOTE: The deepness of injection is very important for lipofection into optic neurons. If the position of the tip of the microcapillary is correctly inserted very superficially into the eyebud, then following microinjection, the grey epidermis overlying the eyebud will swell. If the position is too deep, the grey eyebud will not show any changes, and the frequency of expression of DNA in optic neurons will be lower.</li>
	<li>Turn the embryo around and perform the same microinjection into the eyebud on the contralateral side of the embryo.</li>
	<li>Inject both eyebuds of 6&#8722;10 (or more) embryos in each experiment.</li>
	<li>After microinjection, store embryos in a Petri dish with 1x MMR for approximately 30 min to facilitate wound healing.</li>
	<li>After 30 min, transfer injected embryos into a 0.1x MMR solution with 0.001% bleaching agent (phenylthiocarbamide) to reduce pigmentation. Culture the embryos covered for approximately five days, until the embryos have developed into tadpoles at stages 46&#8722;4716.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60123/60123fig02v2.jpg" /> 
Figure 2: Demarcation of eyebud region for microinjection.&#160;Schematic (A) and photomicrograph (B) of X. laevis embryo at developmental stages 22/23 show eyebud region that should be targeted for microinjection (red highlights). Scale bar = 1 mm. Panel A has been modified from Zahn et al.18. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60123/60123fig02v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
5. Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Tadpoles
NOTE: When tadpoles that were lipofected with DNA reach developmental stages 46&#8722;47, they are ready for imaging.
<ol>
	<li>Prior to imaging, tadpoles must be anesthetized. To anesthetize tadpoles, transfer the lipofected tadpoles into a 0.02% tricaine solution in ddH2O in a 10 mm Petri dish. Wait 5&#8722;10 min until tadpoles become immobile. Verify that tadpoles are still alive by observing their beating hearts under a stereo dissecting microscope.</li>
	<li>Place one anesthetized tadpole into a custom-made silicone chamber on a glass slide and seal with a coverslip. The tadpole should tilt slightly so that one side (left or right) of its head is angled upwards and just barely touches the cover slip.</li>
	<li>Screen the half of the dorsal tectal midbrain of the tadpole that is tilted upwards at low magnification for GFP expressing optic axonal arbors. 
	NOTE: A widefield upright microscope equipped with an epilfuorescence illumination and an apochromatic objective lens (Table of Materials) can be used to screen for fluorescent arbors.</li>
	<li>If the tectal hemisphere contains between one to three GFP expressing optic axonal arbors, capture a z-series of images of these arbors using a high contrast 40x air long working distance objective (Table of Materials). For each axonal arbor, capture 10&#8722;20 z-series slices at 1.5 &#181;m intervals.</li>
	<li>In order to view axon arbors on the other side of the tectal midbrain, reload the tadpole in the silicon chamber so that it tilts to the other side and seal with a cover slip. Then repeat steps 5.3 and 5.4.</li>
</ol>
6. Reconstruction and Quantification of Optic Axonal Arbor Morphology
<ol>
	<li>Select an image stack that contains between one to three GFP expressing optic axonal arbors.</li>
	<li>Use the freehand drawing tool in graphic editing software (Table of Materials) to trace the portion of each optic axonal arbor visible in each z-slice. Tracing through the pieces of each arbor evident in each z-slice will create an accurate 2D projection of the arbor. Different colors can be used to trace distinct GFP expressing optic axonal arbors.</li>
	<li>Make all morphometric measurements on 2D reconstructions of the axonal arbors, with reference to the original z-series of images when needed7. Using Image J software (Table of Materials), measure morphological parameters such as number of branches (i.e., number of branch tips or branch points), total arbor branch length, length per branch, length and width of arbor, overall shape of arbor (L/W ratio, circularity), and angle of branches7.</li>
</ol>

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The authors have nothing to disclose.

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 mixture into eyebud primordia of one day old Xenopus embryos, using a technique first developed in the laboratory of Christine Holt to study optic axon pathfinding in young tadpoles9,10,19,20,21. We and others have also applied this DNA microinjection/lipofection technique to study molecular mechanisms that regulate optic axon arborization in older intact, living X. laevis tadpoles5,6,7,8. This inexpensive, simple procedure for transient, cell specific transgenesis allows determination of cell-autonomous gene function in developing optic axonal arbors in a living vertebrate model system.
There are several important factors to consider for best practices when microinjecting DNA/DOTAP into eyebuds of Xenopus embryos. First, as noted in other reports, the DNA concentration should be greater than 1 &#181;g/&#181;L9,10. DNA concentrations between 1&#8722;3 &#181;g/&#181;L are best, but DNA concentrations as low as 0.7 &#181;g/&#181;L can also be used. Second, microinjecting DNA into stage 22&#8722;24 Xenopus embryos is optimal for experiments in which the goal is to examine mechanisms regulating optic axonal arborization. In embryos at these developmental stages, eyebuds are morphologically differentiated and can be more easily targeted for injection14. Most optic neurons in the eyebud primordia of stage 22&#8722;24 embryos are also post-mitotic, which results in a smaller number of optic neurons expressing GFP, which in turn allows for better resolution when imaging individual GFP optic axonal arbors in tadpoles. Finally, previous studies have shown that exogenous genes are expressed ~8 h after lipofection9. Therefore, expressing gene constructs in eyebuds of stage 22&#8722;24 embryos means that the genes will perturb neither cell fate selection of optic neurons nor the initial outgrowth of their axons. A third factor that will ensure success when microinjecting DNA into Xenopus embryo eyebuds is that the DNA should be injected into a relatively superficial region of the eyebud9,10. Injecting into more deep tissues in or around the eyebud will result in a lower percentage of embryos that contain optic neurons expressing the exogenous DNA10.
There are also several issues to be aware of when imaging and reconstructing GFP expressing optic axonal arbors in intact, living Xenopus tadpoles. First, researchers should only capture images of tectal hemispheres that contain between one to three GFP expressing optic axonal arbors. If a single image contains more than three GFP expressing optic axonal arbors, the arbors are likely to have overlapping branches, which will make it difficult to define the individual arbors during the reconstruction process. Another issue to be aware of is that the reconstruction and quantification of optic axonal arbor morphology are the most time-consuming steps in this protocol. We estimate that reconstructing and quantifying optic axonal arbor morphology require approximately 80&#8722;90% of the total time of the experiment. Although techniques have been developed to automate the reconstruction of tectal neuron dendritic arbors, these computational methods have not yet been applied to optic axonal arbors as well22. Although laborious, the process of reconstructing optic arbor morphology dramatically increases researchers&#8217; comprehension of the details of optic axonal arbor morphology. This added detail, in turn, significantly improves the quality of images of GFP expressing arbors these researchers are able to capture in future experiments.

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&quot; Micropipettes</td><td>Drummond Scientific</td><td>3-000-203 - G/X</td><td></td></tr><tr><td>&amp;mu;-manager software (Version 1.4)</td><td>Vale Lab</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>ImageJ (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><td></td></tr><tr><td></td><td></td><td>3-000-024-A</td><td></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>&lt;em&gt;Xenopus laevis&lt;/em&gt; embryos</td><td>Nasco</td><td>LM00490</td><td></td></tr></tbody></table>

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&#946;-cat) into pCS2 plasmids, and co-injected these plasmids together with a pCS2-GFP labelling plasmid into eyebuds of one day old Xenopus embryos. Figure 4 shows results of several quantitative measurements we made on reconstructions of the control and APC mutant axonal arbors, including number of branches, total arbor branch length, and mean length of branches.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60123/60123fig03v2.jpg" /> 
Figure 3: Representative images of GFP expressing control and mutant optic axonal arbors.&#160;(A) Schematic of mutants of APC N-terminal and central domain mutants that were cloned into pCS2 plasmids. (B) Representative confocal images of single GFP and GFP-APC mutant optic axonal arbors in tecta of intact, living Xenopus tadpoles. (C) Reconstructions of z-series images of GFP control and APC mutant optic axonal arbors. Scale bars = 30 &#181;m (B), 40 &#181;m (C). This figure has been modified from Jin et al.7. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60123/60123fig03v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60123/60123fig04.jpg" /> 
Figure 4: Quantification of morphologies of reconstructions of control and mutant axonal arbors.&#160;Plots of number of branches (A), total arbor branch length (B), and mean branch length (C) confirm observed differences between control and APC mutant expressing axonal arbors. Data in panels A&#8722;C is shown as percent of control mean with SEM. *above data bar or line indicates p &#60; 0.05. Additional scatter plots of number of branches versus mean branch length with regression lines show inverse correlation between these parameters in optic axonal arbors expressing APC domains (D, E). Sample numbers: (A) GFP-12, APCNTERM&#8211;18 APC&#946;-cat-25; (B) GFP-12, APCNTERM-16, APC&#946;-cat-25; (C) GFP-11, APCNTERM-16, APC&#946;-cat-25. This figure has been modified from Jin et al.7. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60123/60123fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

microinjection-dna-into-eyebuds-xenopus-laevis-embryos-imaging-gfp

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

6.2K Views.  Touro University California. In deze video laten we zien hoe exogene DNA-constructies kunnen worden uit te drukken in optische neuronen en hoe individuele GFP-uitdrukkende axonale arbors in intacte, levende Xenopus laevis kikkervisjes kunnen worden afgebeeld. Dit is een goedkope, eenvoudige procedure voor voorbijgaande celspecifieke transgenese die het mogelijk maakt celautonoome genfunctie in individuele optische axonale prieel arbors, ontwikkelen in een levend gewerveld modelsysteem. Het demonstreren van de procedure z...

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

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the large size of their cells in early embryos, make Xenopus laevis a useful model for visualising GFP-tagged ligands. Synthetic mRNAs are efficiently translated after injection into early stage Xenopus embryos, and injections can be targeted to a single cell. When combined with a lineage tracer such as membrane tethered RFP, the injected cell (and its descendants) that are producing the overexpressed protein can easily be followed. This protocol describes a method for the production of fluorescently tagged Wnt and Shh ligands from injected mRNA. The methods involve the micro dissection of ectodermal explants (animal caps) and the analysis of ligand diffusion in multiple samples. By using confocal imaging, information about ligand secretion and diffusion over a field of cells can be obtained. Statistical analyses of confocal images provide quantitative data on the shape of ligand gradients. These methods may be useful to researchers who want to test the effects of factors that may regulate the shape of morphogen gradients.

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating morphogen gradients.

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the ...

Single Cell Electroporation in vivo within the Intact Developing Brain

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. The distinct advantage of this technique is that experimental manipulations may be performed on individual cells while leaving the surrounding tissue unaltered, thereby distinguishing cell-autonomous effects from those resulting from global treatments. When combined with advanced in vivo imaging techniques, SCE of fluorescent markers permits direct visualization of cellular morphology, cell growth, and intracellular events over timescales ranging from seconds to days. While this technique is used in a variety of in vivo and ex vivo preparations, we have optimized this technique for use in Xenopus laevis tadpoles. In this video article, we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the albino Xenopus tadpole. We also discuss methods to optimize yield, and show examples of live two-photon fluorescence imaging of neurons fluorescently labeled by SCE.

Single-cell electroporation (SCE) is a specialized technique allowing delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. Here we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the Xenopus laevis tadpole.

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact ...

Biology

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The efficient in vivo visualization of blood vessels and the reliable quantification of their complexity are key to understanding the biology and disease of the vascular network. Here, we provide a detailed method to visualize blood vessels with a commercially available fluorescent dye, human plasma acetylated low density lipoprotein DiI complex (DiI-AcLDL), and to quantify their complexity in Xenopus tropicalis. Blood vessels can be labeled by a simple injection of DiI-AcLDL into the beating heart of an embryo, and blood vessels in the entire embryo can be imaged in live or fixed embryos. Combined with gene perturbation by the targeted microinjection of nucleic acids and/or the bath application of pharmacological reagents, the roles of a gene or of a signaling pathway on vascular development can be investigated within one week without resorting to sophisticated genetically engineered animals. Because of the well-defined venous system of Xenopus and its stereotypic angiogenesis, the sprouting of pre-existing vessels, vessel complexity can be quantified efficiently after perturbation experiments. This relatively simple protocol should serve as an easily accessible tool in diverse fields of cardiovascular research.

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

This protocol demonstrates a fluorescence-based method to visualize the vasculature and to quantify its complexity in Xenopus tropicalis. Blood vessels can be imaged minutes after the injection of a fluorescent dye into the beating heart of an embryo after genetic and/or pharmacological manipulations to study cardiovascular development in vivo.

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The ...

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Pant&eacute;, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus. 
Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.

Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

Neural Explant Cultures from Xenopus laevis

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During axon outgrowth, the growth cone must integrate multiple sources of guidance cue information to modulate its cytoskeleton in order to propel the growth cone forward and accurately navigate to find its specific targets1. How this integration occurs at the cytoskeletal level is still emerging, and examination of cytoskeletal protein and effector dynamics within the growth cone can allow the elucidation of these mechanisms. Xenopus laevis growth cones are large enough (10-30 microns in diameter) to perform high-resolution live imaging of cytoskeletal dynamics (e.g.2-4 ) and are easy to isolate and manipulate in a lab setting compared to other vertebrates. The frog is a classic model system for developmental neurobiology studies, and important early insights into growth cone microtubule dynamics were initially found using this system5-7 . In this method8, eggs are collected and fertilized in vitro, injected with RNA encoding fluorescently tagged cytoskeletal fusion proteins or other constructs to manipulate gene expression, and then allowed to develop to the neural tube stage. Neural tubes are isolated by dissection and then are cultured, and growth cones on outgrowing neurites are imaged. In this article, we describe how to perform this method, the goal of which is to culture Xenopus laevis growth cones for subsequent high-resolution image analysis. While we provide the example of +TIP fusion protein EB1-GFP, this method can be applied to any number of proteins to elucidate their behaviors within the growth cone.

Neural Explant Cultures from Xenopus laevis

Culturing neural explants from dissected Xenopus laevis embryos that express fluorescent fusion proteins allows for imaging of growth cone cytoskeletal dynamics.

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During ...

Neuroscience

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 ...

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking axonal projection of spinal interneurons in vertebrates, germ line-targeted reporter genes yield bilaterally symmetric labeling. Therefore, it is hard to distinguish between the ipsi- and contra-laterally projecting axons. Unilateral electroporation into the chick neural tube provides a useful means to restrict expression of a reporter gene to one side of the central nervous system, and to follow axonal projection on both sides 1 ,2-5. This video demonstrates first how to handle the eggs prior to injection. At HH stage 18-20, DNA is injected into the sacral level of the neural tube, then tungsten electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator. The egg is sealed with tape and placed back into an incubator for further development. Three days later (E6) the spinal cord is removed as an open book preparation from embryo, fixed, and processed for whole mount antibody staining. The stained spinal cord is mounted on slide and visualized using confocal microscopy.

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

This video demonstrates how to visualize axonal pathways of genetically defined groups of neurons in the embryonic chick spinal cord utilizing in ovo electroporation of reporter genes under the control of specific enhancer elements.

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking ...

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During this process, a portion of neural stem cells undergo differentiation and give rise to the first populations of differentiated primary neurons within the nascent central nervous system. Several vertebrate model organisms have been used to explore the mechanisms of neural cell fate specification, patterning, and differentiation. Among these is the African clawed frog, Xenopus, which provides a powerful system for investigating the molecular and cellular mechanisms responsible for primary neurogenesis due to its rapid and accessible development and ease of embryological and molecular manipulations. Here, we present a convenient and rapid method to observe the different populations of neuronal cells within Xenopus central nervous system. Using antibody staining and immunofluorescence on sections of Xenopus embryos, we are able to observe the locations of neural stem cells and differentiated primary neurons during primary neurogenesis.

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

This article presents a convenient and rapid method for visualizing different neuronal cell populations in the central nervous system of Xenopus embryos using immunofluorescent staining on sections.

Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During ...

Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling

Cell polarity is a fundamental property of eukaryotic cells that is dynamically regulated by both intrinsic and extrinsic factors during embryonic development 1, 2. One of the signaling pathways involved in this regulation is the Wnt pathway, which is used many times during embryogenesis and critical for human disease3, 4, 5. Multiple molecular components of this pathway coordinately regulate signaling in a spatially-restricted manner, but the underlying mechanisms are not fully understood. Xenopus embryonic epithelial cells is an excellent system to study subcellular localization of various signaling proteins. Fluorescent fusion proteins are expressed in Xenopus embryos by RNA microinjection, ectodermal explants are prepared and protein localization is evaluated by epifluorescence. In this experimental protocol we describe how subcellular localization of Diversin, a cytoplasmic protein that has been implicated in signaling and cell polarity determination6, 7 is visualized in Xenopus ectodermal cells to study Wnt signal transduction8. Coexpression of a Wnt ligand or a Frizzled receptor alters the distribution of Diversin fused with red fluorescent protein, RFP, and recruits it to the cell membrane in a polarized fashion 8, 9. This ex vivo protocol should be a useful addition to in vitro studies of cultured mammalian cells, in which spatial control of signaling differs from that of the intact tissue and is much more difficult to analyze.

Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling

Xenopus embryonic ectoderm has become an attractive model for studies of cell polarity. An assay is described, in which subcellular distribution of fluorescent proteins is assessed in ectoderm cells. This protocol will help address questions related to spatial control of signaling.

Cell polarity is a fundamental property of eukaryotic cells that is dynamically regulated by both intrinsic and extrinsic factors during embryonic ...

Facial Transplants in Xenopus laevis Embryos

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. To identify where signaling pathways and tissues act during patterning of the developing face, a 'face transplant' technique has been developed in embryos of the frog Xenopus laevis. A region of presumptive facial tissue (the "Extreme Anterior Domain" (EAD)) is removed from a donor embryo at tailbud stage, and transplanted to a host embryo of the same stage, from which the equivalent region has been removed. This can be used to generate a chimeric face where the host or donor tissue has a loss or gain of function in a gene, and/or includes a lineage label. After healing, the outcome of development is monitored, and indicates roles of the signaling pathway within the donor or surrounding host tissues. Xenopus is a valuable model for face development, as the facial region is large and readily accessible for micromanipulation. Many embryos can be assayed, over a short time period since development occurs rapidly. Findings in the frog are relevant to human development, since craniofacial processes appear conserved between Xenopus and mammals.

Facial Transplants in Xenopus laevis Embryos

A technique for transplanting "Extreme Anterior Domain" facial tissue between Xenopus laevis embryos has been developed. Tissue can be moved from one gene expression background into another, allowing the study of local requirements for craniofacial development and for signaling interactions between facial regions.

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. ...

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

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