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

Summary

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

Abstract

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 eye^1,2. 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.

Protocol

Part I. Set-up for the ACT Assay

1. 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.
2. 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.
3. Make sterile solutions in T....

Discussion

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°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 (>42/43), then we recommend using transgenic venus YFP animals, since .......

Materials

List of materials used in this article

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