Isolation and Differentiation of Xenopus Animal Cap Cells

Takashi Ariizumi1, Shuji Takahashi1, Te‐chuan Chan2, Yuzuru Ito3, Tatsuo Michiue1, Makoto Asashima2,3,1

1 University of Tokyo, Tokyo, Japan, 2 Japan Science and Technology Agency, Tokyo, Japan, 3 Organ Development Research Laboratory, National Institute of Advanced Industrial Science and Technology, Ibaraki, Japan
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1D.5
DOI:  10.1002/9780470151808.sc01d05s9
Online Posting Date:  April, 2009
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Abstract

Xenopus is used as a model animal for investigating the inductive events and organogenesis that occur during early vertebrate development. Given that they are easy to obtain in high numbers and are relatively large in size, Xenopus embryos are excellent specimens for performing manipulations such as microinjection and microsurgery. The animal cap, which is the area around the animal pole of the blastula, is destined to form the ectoderm during normal development. However, these cells retain pluripotentiality and upon exposure to specific inducers, the animal cap can differentiate into neural, mesodermal, and endodermal tissues. In this sense, the cells of the animal cap are equivalent to mammalian embryonic stem cells. In this unit, the isolation and differentiation of animal cap cells, the so-called animal cap assay, is described. Useful methods for analyzing the mechanism of animal cap differentiation at the molecular level are also described. Curr. Protoc. Stem Cell Biol. 9:1D.5.1-1D.5.31. © 2009 by John Wiley & Sons, Inc.

Keywords: animal cap; pluripotency; activin; retinoic acid; induction; organogenesis; Xenopus laevis

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Animal Cap Assay
  • Support Protocol 1: Obtaining Fertilized Eggs and Membrane Removal
  • Support Protocol 2: In Vitro Fertilization and Rapid Removal of the Jelly Coat
  • Support Protocol 3: Preparation of Micromanipulation Tools
  • Alternate Protocol 1: Multiple Treatments of Animal Caps for Kidney and Pancreas Induction
  • Alternate Protocol 2: Dissociation/Reaggregation of Animal Caps for Heart Induction
  • Basic Protocol 2: Microinjection of mRNA for Animal Cap Assay
  • Support Protocol 4: Histological Examination of Animal Cap Explants
  • Support Protocol 5: RT-PCR for Analyzing Gene Expression in Animal Cap Cells
  • Support Protocol 6: Immunohistochemistry of the Induced Animal Cap Cells
  • Support Protocol 7: Whole-Mount In Situ Hybridization
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Animal Cap Assay

 Materials
  • Blastula embryos at developmental stages 8 or 9 (Fig. 1D.5.2)
  • Steinberg's solution (SS; see recipe)
  • 0.1% (w/v) bovine serum albumin in SS (pH 7.4; 0.1% BSA-SS)
  • Test solutions (e.g., such as activin and fibroblast growth factor dissolved in 0.1% BSA-SS)
  • Operating dishes, transfer pipets, and tungsten needles (see Support Protocol 3)
  • Low-adhesion, 24-well tissue culture plate (Sumitomo Bakelite, cat. no. MS-80240)
  • 20° to 22°C incubator
     FigureFigure 1D.5.2 Temperature-dependent early development of Xenopus embryos. Within the normal tolerance range (18° to 24°C), it is possible to retard or accelerate the rate of embryonic development without altering the developmental processes.

Support Protocol 1: Obtaining Fertilized Eggs and Membrane Removal

 Materials
  • hCG dissolved in saline (0.9% NaCl) at a concentration of 2000 U/ml
  • Fully mature male and female frogs (Xenopus laevis or X. borealis)
  • Steinberg's solution (SS; see recipe)
  • Dejelling solution (CSS): 4.5% (w/v) cysteine-HCl in SS (pH 7.8), prepare fresh
  • Sterilized 1-ml syringe with 26-G needle
  • 10- to 15-liter container
  • Thin plastic card
  • Large-bore pipet (~5-mm diameter)
  • Sterilized beakers (100-ml)
  • Operating dishes, transfer pipets, and two pairs of watchmaker's forceps (see Support Protocol 3)

Support Protocol 2: In Vitro Fertilization and Rapid Removal of the Jelly Coat

 Materials
  • Fully mature male and hCG-primed female frogs (Xenopus laevis)
  • Anesthetic: 0.1% (w/v) ethyl 3-aminobenzoate methanesulfonate salt (Tricaine/MS222; Sigma) in tap water (not distilled water)
  • DeBoer's solution (DB; see recipe)
  • FBS
  • Dejelling solution: 1% (w/v) sodium thioglycollate in SS (pH 6.0)
  • 1 M NaOH
  • Steinberg's solution (SS; see recipe)
  • Surgical board
  • Forceps
  • Scissors
  • 60-mm dishes
  • 15-ml conical tubes
  • Pasteur pipets with tipfused by a flame

Alternate Protocol 1: Multiple Treatments of Animal Caps for Kidney and Pancreas Induction

 Materials
  • Late-blastula embryos at developmental stage 9 (Fig. 1D.5.2)
  • 0.1% (w/v) BSA in SS, pH 7.4 (0.1% BSA-SS; see recipe for SS)
  • Retinoic acid stock solution (10–2 M): 3 mg all-trans retinoic acid (Sigma, cat. no. R2625) dissolved in 1 ml DMSO or ethanol
  • Test solution 1: 10 µl retinoic acid stock solution plus 990 µl of 10 ng/ml activin in 0.1% BSA-SS
  • Test solution 2: 100 ng/ml activin in 0.1% BSA-SS
  • Test solution 3: 10 µl retinoic acid plus 990 µl of 0.1% BSA-SS
  • Operating dishes, transfer pipets, and two pairs of watchmaker's forceps (see Support Protocol 3)
  • Low-adhesion, 24-well tissue culture plate (Sumitomo Bakelite, cat. no. MS-80240)
  • 20°C incubator

Alternate Protocol 2: Dissociation/Reaggregation of Animal Caps for Heart Induction

 Materials
  • Mid-blastula embryos at developmental stage 8 (Fig. 1D.5.2)
  • Steinberg's solution (SS; see recipe)
  • 0.1% (w/v) bovine serum albumin in SS, pH 7.4 (0.1% BSA-SS)
  • 0.1% (w/v) BSA in Ca2+/Mg2+-free SS, pH 7.4 (0.1% BSA-CMFSS)
  • Activin solution: 100 ng/ml activin dissolved in 0.1% BSA-SS
  • Operating dishes, transfer pipets, and tungsten needles (see Support Protocol 3)
  • Low-adhesion, 96-well tissue culture plates with concave (U-shaped)-well bottoms (Sumitomo Bakelite, cat. no. MS-30960)

Basic Protocol 2: Microinjection of mRNA for Animal Cap Assay

 Materials (see Fig. 1D.5.7)
  • Synthetic RNA of interest
  • 5% (w/v) Ficoll in SS
  • In vitro fertilized eggs (see Support Protocol 2)
  • Steinberg's solution (SS; see recipe)
  • Glass needles
  • Microloader tip (Eppendorf, cat. no. 5242 956.003)
  • Microinjection capillary (e.g., Narishige G-1)
  • Micromanipulator (e.g., Marzhauser MM33) and support base (Drummond Scientific)
  • Microinjector (e.g., PLI-100/-90 Pico-Injector, Harvard/Medical Systems)
  • Microscope
  • Air compressor (e.g., oil-free BEBICON, Hitachi or N2 gas cylinder)
  • 60-mm glass dishes
  • Stainless-steel mesh
  • Pasteur pipets
  • Hair loop or polished forceps
  • 6-well plates, optional
     FigureFigure 1D.5.7 Equipment for microinjection and artificial insemination. (A) Equipment for microinjection: (1) microinjector, (2) binocular microscope and illuminator, (3) manipulator, (4) air compressor, (5) Ficoll solution and Steinberg's solution, (6) tissue culture plate. (B) A macrophotograph of the end of the glass needle. (C) The instruments needed for microinjection and in vitro fertilization are: (1); Pasteur pipet with flame-fused tip for spreading the fertilized eggs on the dish; (2) transfer pipet for handling embryos; (3) a stainless steel mesh for aligning the embryos; (4) scissors and watchmaker's forceps.

Support Protocol 4: Histological Examination of Animal Cap Explants

 Materials
  • Animal cap explants
  • Steinberg's solution (SS; see recipe)
  • Bouin's solution: 15 ml picric acid, 5 ml formalin, 1 ml acetic acid, prepare fresh
  • 70% ethanol
  • Xylene
  • Paraffin
  • Delafield's hematoxylin solution (Sigma, cat. no. 03971)
  • Eosin Y solution (Sigma, cat. no. HT 110216)
  • Canada balsam (Sigma, cat. no. 03984)
  • Special basket, consisting of a glass tube (1 cm × 1 cm) with the bottom covered with a nylon mesh (148-µm grids)
  • Paraffin molds
  • 56° to 58°C paraffin oven
  • Heated wide-bore pipet
  • Microtome
  • Glass microscope slides
  • 45°C oven
  • Coverslips

Support Protocol 5: RT-PCR for Analyzing Gene Expression in Animal Cap Cells

 Materials
  • Animal caps
  • ISOGEN RNA purification reagent (Nippon Gene)
  • Chloroform
  • 2-Propanol
  • 70% (v/v) ethanol, RNase-free
  • RNase-free water
  • Oligo(dT)15 (Roche cat. no. 814-270)
  • 0.1 M DTT
  • dNTP mixture (2.5 mM each)
  • Ribonuclease inhibitor (Takara)
  • Superscript II reverse transcriptase and buffer (Invitrogen cat. no. 18064-022)
  • ExTaq polymerase and 10× ExTaq buffer (Takara cat. no. RR001A)
  • Specific primer sets for detecting target genes (10 pmol/µl each)
  • 200-µl micropipettor
  • 1.5-ml tubes
  • Spectrophotometer
  • 1.5-ml microcentrifuge tubes
  • 42°, 60°, and 70°C heating blocks
  • 200-µl PCR tubes
  • Thermal cycler

Support Protocol 6: Immunohistochemistry of the Induced Animal Cap Cells

 Materials
  • Induced animal caps
  • Fixation solution: 4% (w/v) paraformaldehyde in PBS (see recipe)
  • 25%, 55%, 75%, and 100% methanol
  • Bleaching solution (see recipe)
  • PBT: 0.1% (v/v) Triton X-100 in PBS (see recipe for PBS)
  • Blocking solution (see recipe)
  • Primary antibody
  • Secondary antibody, alkaline phosphatase (AP)–conjugated
  • AP reaction buffer (see recipe)
  • Color solution: 4.5 µg/ml NBT, 3.5 µg/ml BCIP in AP reaction buffer
  • Screw-cap glass vial
  • Incline shaker
  • Dish
  • Aluminum foil
  • Fluorescent light source
  • Pasteur pipet

Support Protocol 7: Whole-Mount In Situ Hybridization

 Materials
  • Plasmid containing target clone
  • Appropriate restriction enzyme
  • Phenol/chloroform
  • 100% ethanol
  • RNase-free water
  • T3 RNA polymerase (Roche cat. no. 1031163), T7 RNA polymerase (Roche cat. no. 881767), or SP6 RNA polymerase (Roche cat. no. 810274) and 10× transcription buffer
  • Dig RNA labeling mix (Roche cat. no. 1277073)
  • RNase inhibitor (Takara cat. no. 2310A)
  • DNase I (Invitrogen cat. no. 18068-015)
  • Stop solution (see recipe)
  • Hydrolysis buffer (see recipe)
  • 3 M sodium acetate, pH 5.2
  • MEMFA (see recipe)
  • 50% and 75% ethanol in RNase-free water
  • 25% ethanol in PTw
  • PTw (see recipe)
  • 10 µg/ml Proteinase K (see recipe)
  • 0.1 M TEA (see recipe)
  • 4% PFA (see recipe)
  • Hybridization buffer (see recipe)
  • 0.2× and 2× SSC (see recipes)
  • RNase in 2× SSC (see recipe)
  • MAB (see recipe)
  • MAB+BR (see recipe)
  • MAB+BR+SS (see recipe)
  • Anti-digoxigenin-AP, Fab fragment (Roche cat. no. 1093274)
  • AP buffer (see recipe)
  • BM Purple (Roche cat. no. 1442074)
  • 70% and 100% methanol
  • Bleaching solution (see recipe)
  • Spectrophotometer
  • 37° and 60°C water bath
  • 5-ml screw-cap glass vial
  • Pipet
  • Mild shaker
  • Hybridization incubator
  • 24-well plate
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Figures

  •  FigureFigure 1D.5.1 Outline of the animal cap assay. An animal cap removed from a blastula is immersed in a saline solution that contains various concentrations of inducer. In the absence of inducer, the cap forms a cluster of epidermis, termed atypical epidermis. The differentiation of mesodermal tissues, such as the notochord and muscle, indicates the mesoderm-inducing activity of the inducer, whereas the differentiation of neural tissues, such as the brain and eyes, indicates the neural-inducing activity of the inducer.
    View Image
  •  FigureFigure 1D.5.2 Temperature-dependent early development of Xenopus embryos. Within the normal tolerance range (18° to 24°C), it is possible to retard or accelerate the rate of embryonic development without altering the developmental processes.
    View Image
  •  FigureFigure 1D.5.3 Obtaining eggs by hormone-stimulated natural mating. (A) Fertilized eggs are obtained by the injection of hCG into the dorsal lymph sacs of the male and female. (1) A 1-ml syringe filled with hCG; (2) “stitch” marks (indicated by a white dotted line); (3) the dorsal lymph sac; and (4) the cloaca. (B) The laying of fertilized eggs begins at the bottom of the container ~12 hr after hCG injection. (1) Male; (2) female; (3) a large-bore pipet; and (4) a thin plastic card for egg collection.
    View Image
  •  FigureFigure 1D.5.4 Obtaining fertilized eggs by in vitro fertilization. (A) Confirm that hCG-primed female is laying eggs from the cloaca. Hold the frog gently with both hands and push the region near the cloaca with the thumb and forefinger. Eggs are collected in a 60-mm dish. (B) After adding two or three drops of the sperm suspension and a few drops of DB to the collected eggs, mix and spread them into a single layer on the dish using a Pasteur pipet with flame-fused tip.
    View Image
  •  FigureFigure 1D.5.5 Instruments for removing and handling the animal caps. The instruments needed for the animal cap assay are: (1) clean bench; (2) binocular microscope; (3) fiber-optic light; (4) Steinberg's solution; (5) operating dish; (6) small dishes; (7) samples; (8) tissue culture plate; (9) watchmaker's forceps for removing the vitelline membrane; (10) tungsten needles for dissecting animal cap tissues; (11) transfer pipets for handling embryos and animal caps.
    View Image
  •  FigureFigure 1D.5.6 In vitro heart induction using the dissociation/reaggregation protocol. The cellular adhesion of the caps is loosened in CMFSS and the cells can be dispersed by gentle pipetting. The dissociated cells begin to form a reaggregate in SS that contains Ca2+ and 100 ng/ml activin.
    View Image
  •  FigureFigure 1D.5.7 Equipment for microinjection and artificial insemination. (A) Equipment for microinjection: (1) microinjector, (2) binocular microscope and illuminator, (3) manipulator, (4) air compressor, (5) Ficoll solution and Steinberg's solution, (6) tissue culture plate. (B) A macrophotograph of the end of the glass needle. (C) The instruments needed for microinjection and in vitro fertilization are: (1); Pasteur pipet with flame-fused tip for spreading the fertilized eggs on the dish; (2) transfer pipet for handling embryos; (3) a stainless steel mesh for aligning the embryos; (4) scissors and watchmaker's forceps.
    View Image
  •  FigureFigure 1D.5.8 Equipment for histological analyses of the differentiation of animal cap explants. The instruments needed for embedding the explants are: (1) special baskets that consist of a glass tube with a nylon mesh on the bottom; (2) watchmaker's forceps; (3) transfer pipet for handling explants; (4) paraffin molds for embedding the explants in paraffin.
    View Image
  •  FigureFigure 1D.5.9 Handling of animal cap explants on the whole-mount in situ hybridization. (A) The screw-cap glass vial is partially filled with solution (arrow). (B) Stratified acetic anhydrate diffuses gradually in 0.1 M TEA (see Day 1, Support Protocol 7, step 23).
    View Image

Literature Cited

Literature Cited
    Ariizumi, T. and Asashima, M. 1994. In vitro control of the embryonic form of Xenopus laevis by activin A: Time and dose-dependent inducing properties of activin-treated ectoderm. Develop. Growth Differ. 36:499-507.
    Ariizumi, T., Sawamura, K., Uchiyama, H., and Asashima, M. 1991a. Dose- and time-dependent mesoderm induction and outgrowth formation by activin A in Xenopus laevis. Int. J. Dev. Biol. 35:407-414.
    Ariizumi, T., Moriya, N., Uchiyama, H., and Asashima, M. 1991b. Concentration-dependent inducing activity of activin A. Roux's Arch. Dev. Biol. 200:230-233.
    Ariizumi, T., Kinoshita, M., Yokota, C., Takano, K., Fukuda, K., Moriyama, N., Malacinski, G.M., and Asashima, M. 2003. Amphibian in vitro heart induction: A simple and reliable model for the study of vertebrate cardiac development. Int. J. Dev. Biol. 47:405-410.
    Asashima, M., Michiue, T., and Kurisaki, A. 2008. Elucidation of the role of activin in organogenesis using a multiple organ induction system with amphibian and mouse undifferentiated cells in vitro. Develop. Growth Differ. 50:S35-S45.
    Green, J.B. and Smith, J.C. 1990. Graded changes in dose of a Xenopus activin A homologue elicit stepwise transitions in embryonic cell fate. Nature 347:337-338.
    Green, J.B., New, H.V., and Smith, J.C. 1992. Responses of embryonic Xenopus cells to activin and FGF are separated by multiple dose thresholds and correspond to distinct axes of the mesoderm. Cell 71:731-739.
    Harland, R.M. 1991. In situ hybridization: An improved whole-mount method for Xenopus embryos. Methods Cell Biol. 36:685-695.
    Kay, B.K. and Peng, H.B., eds. 1991. Methods in Cell Biology. Xenopus laevis: Practical Use in Cell and Molecular Biology. Academic Press, San Diego, California.
    Kobayashi, H., Michiue, T., Yukita, A., Danno, H., Sakurai, K., Fukui, A., Kikuchi, A., and Asashima, M. 2005. Novel Daple-like protein positively regulates both the Wnt/beta-catenin pathway and the Wnt/JNK pathway in Xenopus. Mech. Dev. 122:1138-1153.
    Moriya, H., Uchiyama, H., and Asashima, M. 1993. Induction of pronephric tubules by activin and retinoic acid in presumptive ectoderm of Xenopus laevis. Develop. Growth Differ. 35:123-128.
    Moriya, N., Komazaki, S., Takahashi, S., Yokota, C., and Asashima, M. 2000. In vitro pancreas formation from Xenopus ectoderm treated with activin and retinoic acid. Develop. Growth Differ. 42:593-602.
    Nieuwkoop, P.D. 1969. The formation of mesoderm in urodelan amphibians, Pt. 1: Induction by the endoderm. Roux' Arch. Entwicklungsmech. Org. 162:341-373.
    Nieuwkoop, P.D. and Faber, J. 1967. Normal Table of Xenopus laevis (Daudin). North-Holland Publishing, Amsterdam.
    Sambrook, J. and Russell, D.W. 2001. Molecular Cloning. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, New York.
    Sive, H.L., Grainger, R.M., and Harland, R.M. 2000. Early development of Xenopus laevis. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, New York.
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