Name: Tissue Determination Using the Animal Cap Transplant (ACT) Assay in Xenopus laevis (Video) | JoVE Journal
Uploaded: 2010-05-16T17:00:00.000+00:00
Duration: 11 min
Description: Learn tissue determination using the animal cap transplant assay in Xenopus laevis embryos. This method tests gene function in specifying primitive derm fate through RNA overexpression and transplantation techniques.

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5 minutes

This work was supported by grants from Research to Prevent Blindness (Career Development Awards to MEZ and ASV and an unrestricted grant to the Department of Ophthalmology), the E. Matilda Zeigler Foundation (MEZ and ASV) and the National Eye Institute/NIH (MEZ, grants R01EY015748 and R01EY017964).

Part I. Set-up for the ACT Assay
<ol>
	<li>Generate capped RNA for injection as suggested by the manufacturer, using their phenol:chloroform purification method (e.g. mMessage mMachine Kit; Applied Biosystems/Ambion, Austin, TX). RNA encoding a fluorescent protein is used as a tracer. We transcribe either yellow fluorescent protein (YFP) or mCherry 5 cDNA, which is in a pCS2+ expression vector.</li>
	<li>Pull borosilicate glass capillary tubes to form tapered glass tips using a micropipette puller. We use Sutter Micropipette Puller, P-97 (Sutter Instruments, Inc., Novato, CA), as previously described 6.</li>
	<li>Make sterile solutions in Table 1 from a 10 X Marc's Modified Ringer's (MMR) solution stock (10 mM MgCl2 ; 20 mM KCl; 20 mM CaCl2 ; 50 mM HEPES; 1 M NaCl; adjusted to pH 7.5, autoclaved and stored at RT).</li>
	<li>Isolate testes from a male frog and store the tissue at 4°C in a sterile solution of 1X MMR/gentamicin (50 μg/mL gentamicin sulfate; BioWhittaker, Cat # 17-518Z). This can be done up to a week before the start of the experiment but it is looses its efficacy over time.</li>
	<li>Egg laying solution (1X: 150 mM NaCl, 2.62 mM KCl, 0.8 mM Na2HPO4, 20 mM Trizma base, 2.62 mM NaHCO3 and 2.75 mM MgSO4; adjusted to pH 7.6) can be made as an 8X stock, which can be stored at 4°C for up to two weeks. Note: watch out for contamination in this solution, as this will affect the quality of your eggs.</li>
	<li>Generate injection plates. To do this, melt 1% agarose in 0.4X MMR in a microwave, add ~6 mL melted agarose solution into 60 mm plates, gently unroll an elastomer mold (Figure 1) on top of the solution to prevent bubbles and let cool for about 20 minutes at RT. To determine the number needed, consider the number of experimental conditions (e.g. 1, YFP; 2, YFP+Noggin) and the number of transplanted host embryos needed. For our experiments, we generate at least 20 transplanted host embryos for each experimental condition. At 18-19°C, embryos will be at stage 15 for about an hour. If it takes one hour to perform 20 transplants, then two fertilization time points are needed, assuming the host embryos accept all transplants. Therefore, four injection dishes will be needed (2 exp conditions x 2 fertilization time points = 4). Store the plates at 4°C for up to one week.</li>
	<li>Generate cap isolation plates using 1% agarose + 0.7X MMR with molds as for the injection plate. Make the same number of cap isolation as injection plates plus one extra per fertilization time point (4 + 2 fertilization time points = 6).</li>
	<li>Inject females with 500 units human chorionic gonadotropin late in the day on the day before microinjection. Store females at 16-18°C overnight.</li>
</ol>
Part II. RNA Microinjection of Xenopus laevis embryos
A. Preparation for microinjection
<ol>
	<li>Remove hormonally induced female and place them in individual tanks containing 1X egg laying solution (ELS). Note: we don't use eggs that have been in the ELS more than two hours as the quality of eggs is reduced. Therefore, about an hour before in vitro fertilization, we discard all eggs from the tank so that only freshly laid eggs are collected.</li>
	<li>Prepare the needle for microinjection by snipping off the tip to obtain a 25-35 μm diameter. Place the needle onto the picoinjector and calibrate it so that it dispenses 10 nL per injection.</li>
	<li>Set an incubator to 14°C.</li>
	<li>Once females have laid eggs for about an hour, remove the eggs from the tank using a transfer pipette and place them into 60 mm Petri dishes. Remove as much ELS from the eggs as possible and replace with 1X MMR. The eggs can be fertilized right away or stay in 1X MMR for up to an hour. For the transplant assay, it is best to fertilize two dishes, 80% full of a single layer of eggs in up to three separate batches at around 11 am, noon and 1 pm. After injection, they will be placed at 14°C so that they will grow to the desired stages for cap isolation (stage 8.5-9) and transplantation (stage 15; see Part I, #6 above).</li>
	<li>To check the quality of the testes, we perform an early morning fertilization (~8 am), which allows us to extract testes from another male if the sperm from the first is not viable. Note: we homogenize ~0.25 testes in 1 mL 1X MMR and add approximately 5 to 7 drops per plate of oocytes.</li>
	<li>In vitro fertilize the eggs with homogenized testes and, an hour later, remove the jelly coat on the eggs using de-jelly solution (164μl 1M DTT + 10mL 1M Tris, pH 8.8 up to 50 mL autoclaved nanopure water). Wait until they reach two-cell stage to begin sorting, which takes about 2 hours at 18°C or 1.5 hours at room temperature (22°C).</li>
	<li>Using a transfer pipette, sort uniformly pigmented and evenly dividing embryos, as shown in the left animal cap in Figure 3, into the well of an injection dish filled with 0.4X MMR + 6% Ficoll.</li>
</ol>
B. Microinjection
<ol>
	<li>Stretch Parafilm over the top of a 60 mm Petri dish lid. Pipette 2 μl of your first RNA sample onto the Parafilm and aspirate using the PLI-100 to fill your injection needle. Be sure to check that the needle is still dispensing 10 nL of sample before starting injection.</li>
	<li>Inject 20-30 two-cell stage embryos (both blastomeres) with 500 pg YFP RNA in one dish and your gene of interest (e.g. 20 pg Noggin RNA) plus YFP RNA in another dish 7.</li>
	<li>Place 100-200 non-injected embryos per time point in another injection dish with the Ficoll solution. Move them in and out of the incubator at the same time as the injected embryos. These are the hosts to be used in Part IV.</li>
	<li>After injection, incubate all embryos at 14°C overnight. Repeat for all time points.</li>
</ol>
Part III. Isolate animal caps from injected embryos
<ol>
	<li>Arrive early in the morning to stage the embryos. The earliest time point should be at stage 9. Remove those that did not survive. Proceed with the following steps working with each time point in batches.</li>
	<li>Pour off the Ficoll solution from each plate and add back 0.7X MMR/gentamicin solution. To a cap isolation dish, add a generous amount of 0.7X MMR/gentamicin solution. Dislodge embryos from their injection dish wells using a transfer pipette and place in the freshly prepared cap isolation dish.</li>
	<li>Isolate animal caps in 0.7X MMR + gentamicin solution, keeping 2 or 3 intact embryos for staging. Either the Gastromaster, fitted with a yellow tip bent to 400 μm, or a pair of sharp number 5 forceps can be used.</li>
	<li>Put caps back in 14°C incubator until the next day. The host embryos should be removed from the incubator at the same time as the caps to keep them at the same developmental stage as the donor tissue.</li>
</ol>
Part IV. Transplantation of caps onto flank
<ol>
	<li>Remove caps and embryos from the first time point from the 14°C incubator and stage the embryos. Sort for uniform yellow or red fluorescent-positive caps, depending on the tracer injected (Figure 2).</li>
	<li>Count the number of caps and remove double the number of stage 12-13 embryos to a freshly prepared injection dish with 0.7X MMR/gentamicin solution. With sharp #5 forceps, remove the vitelline membrane from each. Transfer them to the plate with the caps, but be careful. Without their vitelline membranes, embryos can quickly dissociate at the water's surface.</li>
	<li>Using the gastromaster (yellow tip, 200-250μm bent wire, highest setting) or #5 forceps, remove a 200 μm square on the side of the embryo at stage 14-15 (Figure 3). Then, cut a cap in half (with the gastromaster or the #5 forceps) and place the tissue in the hole made in the host embryo. Rotate the embryo against the well to allow healing. Do this for as many embryos as you can while they are at stage 15. Repeat for all time points.</li>
	<li>The embryo will usually heal within 30 minutes. Remove the dead cells (white) with a gentle brushing motion with a #5 forceps. Grow them at 18°C overnight.</li>
	<li>Under the dissecting fluorescence microscope, sort embryos with fluorescently-labeled, transplanted tissue. Put them back at 18°C to grow until stage 42-43.</li>
	<li>Anesthetize the animals in tricaine. At this point, you can take pictures under a fluorescence dissecting microscope. After you are done, fix, section and immunostain or perform in situ hybridization using markers for your tissue of interest.</li>
</ol>
<img src="/files/ftp_upload/1932/1932fig1.jpg" alt="Multiplex microarray diagram with 8x12 grid, used for high-throughput DNA or protein analysis." data-altupdatedat="1788235205000" data-alt="Figure 1"> Figure 1. Design of Plexiglass for casting the elastomer mold used for making embryo holding dishes. A 38 x 38 mm round-cornered square (~5 mm deep) has been routed out of a piece of Plexiglass that is 50 x 50 mm square and 25 mm thick. Circular, evenly spaced wells (1.5 mm diameter) have been drilled 1 mm deep into the plastic. To make the elastomer mold for the microinjection dishes, we poured Sylgard elastomer into the Plexiglass and allowed it to solidify, as per manufacturer instructions. This elastomer mold is used to make injection and cap isolation plates.
<img src="/files/ftp_upload/1932/1932fig2.jpg" alt="Brightfield, mCherry fluorescence, merge; microscopy image of cell observation process." data-altupdatedat="1788235205000" data-alt="Figure 2"> Figure 2. Animal cap sorting prior to transplantation is important, as expression can vary. Animal cap on the left expresses mCherry evenly throughout tissue while the explant on the right has spotty expression and will be discarded.
<img src="/files/ftp_upload/1932/1932fig3.jpg" alt="Fish lateral line system study; microscopy image; sensory organ detail; biological research." data-altupdatedat="1788235205000" data-alt="Figure 3"> Figure 3. A host tadpole has developed an eye-like structure on its flank from transplanted animal cap cells. A fluorescent image has been overlayed on the brightfield image.

<ol><li>Smith, W. C., Harland, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expression+cloning+of+noggin%2C+a+new+dorsalizing+factor+localized+to+the+Spemann+organizer+in+Xenopus+embryos%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Expression cloning of noggin, a new dorsalizing factor localized to the Spemann organizer in Xenopus embryos.</a> Cell. 70, 829-840 (1992).</li>
<li>Lamb, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neural+induction+by+the+secreted+polypeptide+noggin%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Neural induction by the secreted polypeptide noggin.</a> Science. 262, 713-718 (1993).</li>
<li>Green, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+Methods+in+Developmental+Biology%3A+Xenopus+%26+Zebrafish%5BTitle%5D)+AND+%22Methods+in+Developmental+Biology%22%5BJournal%5D)">Molecular Methods in Developmental Biology: Xenopus & Zebrafish.</a> Methods in Developmental Biology. , Humana Press. Towata, N.J. (1999).</li>
<li>Viczian, A. S., Solessio, E. C., Lyou, Y., Zuber, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+functional+eyes+from+pluripotent+cells%5BTitle%5D)+AND+%22PLoS+Biol%22%5BJournal%5D)">Generation of functional eyes from pluripotent cells.</a> PLoS Biol. 7, e1000174-e1000174 (2009).</li>
<li>Shaner, N. C., Steinbach, P. A., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+guide+to+choosing+fluorescent+proteins%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">A guide to choosing fluorescent proteins.</a> Nat Methods. 2, 905-909 (2005).</li>
<li>Brown, A. L., Johnson, B. E., Goodman, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Making+patch-pipettes+and+sharp+electrodes+with+a+programmable+puller%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Making patch-pipettes and sharp electrodes with a programmable puller.</a> J Vis Exp. , (2008).</li>
<li>Lan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Noggin+Elicits+Retinal+Fate+in+Xenopus+Animal+Cap+Embryonic+Stem+Cells%5BTitle%5D)+AND+%22Stem+Cells%22%5BJournal%5D)">Noggin Elicits Retinal Fate in Xenopus Animal Cap Embryonic Stem Cells.</a> Stem Cells. , (2009).</li>
</ol>

In this video, we have demonstrated our version of classic techniques used by Xenopus biologists, which we rearranged to create the animal cap transplant assay. At room temperature, Xenopus embryos develop very quickly through stages 9 and 15. By placing them at 14&deg;C, the development of the embryo is slowed down and many more transplants can be performed in one experiment. If it is necessary to grow the embryos to older stages (&gt;42/43), then we recommend using transgenic venus YFP animals, since ...

<table><tbody><tr><td>mMessage mMachine SP6 Kit</td><td>Applied Biosystems</td><td>AM1340</td><td></td></tr><tr><td>Borosilicate Capillary Tubes (1.0mm OD x 0.5mm ID)</td><td>FHC, Inc.</td><td>27-30-1</td><td></td></tr><tr><td>Sutter Micropipette Puller</td><td>Sutter Instrument Co.</td><td>P-97</td><td></td></tr><tr><td>Gentamicin sulfate [50 mg/ml]</td><td>Fisher Scientific</td><td>BW17-528Z</td><td></td></tr><tr><td>Sylgard/elastomer Kit 1.1lb</td><td>Fisher Scientific</td><td>NC9644388</td><td></td></tr><tr><td>60 x 15 mm polystyrene petri dish</td><td>USA Scientific, Inc.</td><td>8609-0160</td><td></td></tr><tr><td>human chorionic gonadotropin</td><td>Intervet Inc.</td><td>22219</td><td></td></tr><tr><td>Pico-Injector Microinjection Systems</td><td>Harvard Apparatus</td><td>650003</td><td></td></tr><tr><td>Torrey Pines Scientific ECHOtherm Incubator</td><td>Fisher Scientific</td><td>11-680-21</td><td></td></tr><tr><td>Transfer pipette</td><td>Krackeler Scientific, Inc.</td><td>6290-20635A</td><td></td></tr><tr><td>Dumostar #5 Forceps</td><td>Fisher Scientific</td><td>11295-10</td><td></td></tr></tbody></table>

tissue determination using the animal cap transplant (act) assay in xenopus laevis

Tissue Determination Using the Animal Cap Transplant (ACT) Assay in Xenopus laevis

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development of a larger region of the embryo. This makes defining its role in a particular tissue difficult to analyze. For example, noggin overexpression in Xenopus laevis embryos causes the expansion of the entire anterior region, including the eye1,2. From this result, it is not known if Noggin plays a direct role in eye determination or that by causing an expansion of neural tissue, Noggin indirectly affects eye formation. Having this complex phenotype makes studying its eye-specific role in cell fate determination difficult to analyze. We have developed an assay that overcomes this problem. Taking advantage of the pluripotent nature of the Xenopus laevis animal cap 3, we have developed an assay to test the ability of gene product(s), like noggin or the eye field transcription factors (EFTFs), to transform caps into particular tissue or cell types by transplanting this tissue onto the side of the embryo 4. While we have found either Noggin protein treatment or a collection of transcription factors can determine retinal cell fate in animal caps, this procedure could be used to identify gene product(s) involved in specifying other tissues as well.

Animal caps overexpressing gene product(s) are transplanted to the flank of developing Xenopus laevis embryos in order to establish whether tissue is determined.

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development ...

The overall goal of the A CT assay is to test whether a gene or genes of interest can specify primitive derm toward a particular cell fate. The procedure starts by first over expressing the RNAs in coding of fluorescent reporter gene and the gene or genes of interest in developing XUS lavis embryos. The next step is to dissect the animal cap or primitive odum expressing the overexpressed RNAs and grow them to stage 15.The third step of the procedure is to remove tissue on the flank of a stage 15 host embryo. The final step is to transplant half the donor animal cap tissue onto the flank of the host embryo and grow the host to stage 42 to 43. The results show ectopic tissue formation such as eye like structures through sectioning and immuno staining for tissue specific markers.Hi, I'm and sine in the Department of Ophthalmology at Upstate Medical University. Today I'll show you procedure that we call the animal cap transplant assay that I developed in collaboration with Mike Suber. We use this assay in both of our laboratories to study the genes that are sufficient for eye formation.So let's get started. To begin this procedure, prepare the cap RNAs for injection transcribe noggin or another gene of interest and YFP to be used as a tracer. Also prepare the MMR based solutions, a stock of eight x egg laying solution and the needles for injecting the embryos.A custom made elastomer mold is used for making embryo holding dishes. Generate one injection plate per experimental condition by melting 1%AROS in 0.4 XMMR in a microwave. Transfer about six milliliters of the solution into a 60 millimeter plate.Gently place an elastomer mold on top of the solution to prevent bubbles and let cool for about 20 minutes. At room temperature, generate one cap isolation plate per experimental condition, using 1%AGROS plus 0.7 XMMR with molds as was just shown for the injection plates. Prepare extra plates for each time point.Consult the accompanying written protocol for details of calculating the time points. Next isolate. Test these from a male frog and store the tissue at four degrees Celsius in a sterile solution of one XMMR supplemented with 50 micrograms per milliliter of Gentamycin.This can be done up to a week before the start of the experiment, but note that the sperm loses efficacy over time, late in the day on the day before microinjection. Inject females with 500 units of human chorionic gonadotropin. Place females at 16 to 18 degrees Celsius overnight.Now all is ready for microinjection the following day. Start by removing the hormonally induced females and place them in individual tanks containing one XELS. Prepare the needle for microinjection by snipping off the tip.To obtain a 25 to 35 micrometer diameter, place the needle onto the PLI 100 pico injector and calibrate it so that it dispenses 10 nanoliters per injection. Set an incubator to 14 degrees Celsius by placing the exit this lower temperature after injecting them with RNA, the development of the embryo is slowed down and many more transplants can be performed in one experiment about an hour before fertilization, remove the eggs from the tank. Once females have laid eggs for an additional hour, use a transfer pipette to remove the newly laid eggs from the tank.Note not to use eggs that have been in the ELS solution for more than two hours. As the quality of the eggs is reduced, place the eggs into 60 millimeter petro dishes. The dish is about 80%full of a single layer of eggs.Remove as much ELS from the eggs as possible and replace with one XMMR. The eggs can stay in one XMMR for up to an hour. Next in vitro fertilize the eggs with homogenized testes.It is best to fertilize the embryos in three separate batches at around 11:00 AM noon and 1:00 PM so that they can grow into the desired stage. 8.5 to nine for cap isolation and stage 15 for transplantation. An hour after fertilizing the eggs, remove the jelly coat on the eggs using D jelly Solution Wash with copious amounts of 0.1 XMMR and then wait until the fertilized eggs reach the two cell stage.To begin sorting. Prepare an injection dish filled with 0.4 XMMR plus 6%F ccal. Once the embryos reach the two cell stage, use a transfer pipette to sort uniformly pigmented and evenly dividing embryos into the well of the injection dish.Proceed to Microinjection first stretch perfil over the top of a 60 millimeter petro dish lid. Pipet two microliters of the RNA sample onto the biofilm and aspirate. Using the pico injector to fill the injection needle.Be sure to check that the needle is still dispensing 10 liters of sample before starting injection. The RNA is injected into both blasters over two cell stage embryo. Inject 20 to 30 embryos each with 500 picograms of Y-F-P-R-N-A and place in one dish.Inject another 20 to 30 embryos each with 20 picograms of Nain, RNA premixed with Y-F-P-R-N-A and place in dish. In addition, place 100 to 200 unin injected embryos per time point in another injection dish containing the FICO solution. Move these embryos in and outta the incubator at the same time as the injected embryos.These are the hosts to be used for transplanting the caps after injection. Incubate all embryos at 14 degrees Celsius overnight. Repeat for all time points planned.Following the overnight incubation, the animal cap can be isolated. Arrive early in the morning to stage the embryos. Remember to remove the unin injected host embryos from the incubator as well.The earliest time point should be at stage nine. First, remove the embryos that did not survive. Proceed to isolate the animal caps from the viable embryos, working with each time point in batches to a cap isolation dish.Add a generous amount of 0.7 XMMR plus gentamycin solution. Then pour off the fol solution from each plate containing the injected embryos and add back 0.7 XMMR plus Gentamycin solution. Dislodge the embryos from their injection dish wells using a transfer pipette and place in the freshly prepared cap isolation dish.Use either the gastro master fitted with a yellow tip bent to 400 micrometers or a pair of sharp number five forceps to isolate animal caps in 0.7 XMMR plus Gentamycin solution. Keep two or three intact embryos for staging. After isolating the animal caps, put them back in the 14 degrees Celsius incubator along with the host embryos until the next day when the caps are transplanted onto the flank of the host embryos.Remove caps and host embryos from the first time point from the 14 degrees Celsius incubator and stage the embryos sort for uniform fluorescent positive caps. Count the number of caps and remove. Double the number of host embryos to a freshly prepared injection dish with 0.7 XMMR plus Gentamycin solution with sharp number five forceps.Remove the Lene membrane from each host embryo. Transfer them to the plate with the caps, but be careful without their lene membranes. Embryos can quickly dissociate at the water surface using the gastro master fitted with a yellow tip with a 200 to 250 micrometer bent wire at the highest setting or using number five forceps.Remove a 200 micrometer square on the side of the embryo at stage 14 to 15. Then again using the gastro master or the number five forceps. Cut a cap in half and place the tissue in the hole made in the host embryo.Rotate the embryo against the well to allow healing. Do this for as many embryos as you can while they're at stage 15. Repeat for all time points.The embryo usually heals within 30 minutes. And with number five forceps, remove the dead cells which appear white with the gentle brushing motion. Grow the transplanted embryos at 18 degrees Celsius overnight.After the overnight incubation, work under the dissecting fluorescence microscope to sort embryos with fluorescently labeled transplanted tissue. Put the selected embryos back at 18 degrees Celsius to grow until stage 42 to 43. When the animals reach the desired stage, anesthetize them in trica and examine under a fluorescence dissecting microscope and take pictures.The transplanted embryos can then be fixed, sectioned and immuno stained, or one can perform in situ hybridization using markers for a tissue of interest. Here are representative results of a host tadpole with donor tissue expressing a fluorescent tracer and noggin one day after transplantation. Notice how the tissue balls up on the side of the tadpole.The host embryos receiving controlled donor tissue expressing only the fluorescent tracer already start incorporating the animal cap cells onto their flank. A few days later, the tadpoles receiving nain expressing donor cells form eye like structures on their side while the tadpoles receiving the control donor tissue have fluorescent cells on the surface of their skin. We've just shown you how to perform the animal cap transplant assay in Zaps Lavis.When doing this assay. It's important to remember to have everything ready prior to that day's procedure so the embryos don't develop faster than you can get to them. So that's it.Thanks for watching and good luck with your experiments.

tissue-determination-using-animal-cap-transplant-act-assay-xenopus

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Tissue Determination Using the Animal Cap Transplant (ACT) Assay in Xenopus laevis

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