Skip to main content
SpringerLink
Log in

Strategies to detect interdigital cell death in the frog, Xenopus laevis: T3 accerelation, BMP application, and mesenchymal cell cultivation

  • Published:
In Vitro Cellular & Developmental Biology - Animal Aims and scope Submit manuscript

Abstract

Fates of digits in amniotes, i.e., free or webbed digits, are determined by the size of programmed interdigital cell death (ICD) area. However, no (or very few) cell death has thus far been observed in developing limb buds of non-amniotic terrestrial vertebrates including other anuran or urodela amphibians. We speculate that the undetectable situation of amphibian ICD is the result of their less frequency due to slow developmental speed characteristic to most amphibian species. Here, we present three strategies for detecting difficult-to-find ICD in the frog, Xenopus laevis. (1) Addition of triiodo-L-thyronine (T3) accelerated two to three times the limb development and increased two to four times the appearance frequency of vital dye-stainable cells in limb buds of the accelerated tadpoles (stage 54 to 55). (2) Application of human bone morphogenetic protein-4 to the autopods of tadpoles at stage 53 to 54 enhanced digital cartilage formation and induced vital dye-stainable cells around the enhanced digital cartilages within 2 d. (3) In cell culture, T3 increased the chondrogenic and cell death activities of limb mesenchymal cells. The augmentation of both activities by T3 was stronger in the forelimb cells than in the hindlimb cells. This situation is well coincided with the limb fates of non-webbed forelimbs and webbed hindlimbs in X. laevis adulthood. Collectively, all three approaches showed that it become possible to detect X. laevis ICD with appropriate strategies.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Figure 2.
Figure 3.
Figure 4.
Fig. 5

Similar content being viewed by others

References

  • Ahrens P. B.; Solursh M.; Reiter R. S. Stage-related capacity for limb chondrogenesis in cell culture. Dev Biol 60: 69–82; 1977.

    Article  PubMed  CAS  Google Scholar 

  • Allen B. M. The effects of extirpation of the thyroid and pituitary glands upon the limb development of anuran. J Exp Zool 42: 13–30; 1925.

    Article  Google Scholar 

  • Baba K.; Okada Y. Frog development (Bufo vulgaris). (“Kaeru hassei” in Japanese). Iwanami Publishing Co, Tokyo; 1932.

    Google Scholar 

  • Bardot B.; Lecoin L.; Fliniaux I.; Huillard E.; Marx M.; Viallet J. P. Drm/Gremlin, a BMP antagonist, defines the interbud region during feather development. Int J Dev Biol 48: 149–156; 2004. doi:10.1387/ijdb.041804bb.

    Article  PubMed  CAS  Google Scholar 

  • Beck C. W.; Christen B.; Barker D.; Slack J. M. Temporal requirement for bone morphogenetic proteins in regeneration of the tail and limb of Xenopus tadpoles. Mech Dev 123: 674–688; 2006. doi:10.1016/j.mod.2006.07.001.

    Article  PubMed  CAS  Google Scholar 

  • Brincks E. L.; Kucaba T. A.; Legge K. L.; Griffith T. S. Influenza-induced expression of functional tumor necrosis factor-related apoptosis-inducing ligand on human peripheral blood mononuclear cells. Hum Immunol 69: 634–646; 2008. doi:10.1016/j.humimm.2008.07.012.

    Article  PubMed  CAS  Google Scholar 

  • Brown D. D.; Cai L.; Das B.; Marsh-Armstrong N.; Schreiber A. M.; Juste R. Thyroid hormone controls multiple independent programs required for limb development in Xenopus laevis metamorphosis. Proc Natl Acad Sci U S A 102: 12455–12458; 2005. doi:10.1073/pnas.0505989102.

    Article  PubMed  CAS  Google Scholar 

  • Cameron J. A.; Fallon J. F. The absence of cell death during development of free digits in amphibians. Dev Biol 55: 331–338; 1977.

    Article  PubMed  CAS  Google Scholar 

  • Daniels K.; Reiter R.; Solursh M. Micromass cultures of limb and other mesenchyme. Methods Cell Biol 51: 237–247; 1996.

    Article  PubMed  CAS  Google Scholar 

  • Das B.; Cai L.; Carter M. G.; Piao Y. L.; Sharov A. A.; Ko M. S.; Brown D. D. Gene expression changes at metamorphosis induced by thyroid hormone in Xenopus laevis tadpoles. Dev Biol 291: 342–355; 2006. doi:10.1016/j.ydbio.2005.12.032.

    Article  PubMed  CAS  Google Scholar 

  • Denker A. E.; Nicoll S. B.; Tuan R. S. Formation of cartilage-like spheroids by micromass cultures of murine C3H10T1/2 cells upon treatment with transforming growth factor-beta 1. Differentiation 59: 25–34; 1995. doi:10.1046/j.1432-0436.1995.5910025.x.

    Article  PubMed  CAS  Google Scholar 

  • Fallon J. F.; Cameron J. Interdigital cell death during limb development of the turtle and lizard with an interpretation of evolutionary significance. J Embryol Exp Morphol 40: 285–289; 1977.

    PubMed  CAS  Google Scholar 

  • Ganãn Y.; Macias D.; Duterque-Coquillaud M.; Ros M. A.; Hurle J. M. Role of TGF beta s and BMPs as signals controlling the position of the digits and the areas of interdigital cell death in the developing chick limb autopod. Development 122: 2349–2357; 1996.

    PubMed  Google Scholar 

  • Gavrieli Y.; Sherman Y.; Ben-Sasson S. A. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 119: 493–501; 1992.

    Article  PubMed  CAS  Google Scholar 

  • Guha U.; Gomes W. A.; Kobayashi T.; Pestell R. G.; Kessler J. A. In vivo evidence that BMP signaling is necessary for apoptosis in the mouse limb. Dev Biol 249: 108–120; 2002.

    Article  PubMed  CAS  Google Scholar 

  • Hamburger V.; Hamilton H. A series of normal stages in the development of the chick embryo. J Morphol 88: 49–92; 1951.

    Article  Google Scholar 

  • Hernandez-Martinez R.; Covarrubias L. Interdigital cell death function and regulation: new insights on an old programmed cell death model. Dev Growth Differ 53: 245–258; 2011. doi:10.1111/j.1440-169X.2010.01246.x;10.1111/j.1440-169X.2010.01246.x.

    Article  PubMed  Google Scholar 

  • Hinchliffe J. R.; Thorogood P. V. Genetic inhibition of mesenchymal cell death and the development of form and skeletal pattern in the limbs of talpid3 (ta3) mutant chick embryos. J Embryol Exp Morphol 31: 747–760; 1974.

    PubMed  CAS  Google Scholar 

  • Iwasawa H.; Kera Y. Normal stages of development of the Japanese lungless salamander, Onychodactylus japonicus (houttuyn). (in Japanese: Abstract in English). Jpn J Herpetol 8: 73–89; 1980.

    Google Scholar 

  • Kaltenback J. C. Local action of thyoxin on amphibian metamorphosis. I. Local metamorphosis in Rana pipiens larvae effected by thyroxin-cholesterol implants. J Exp Zool 122: 21–23; 1953.

    Article  Google Scholar 

  • Kerr J. F.; Harmon B.; Searle J. An electron-microscope study of cell deletion in the anuran tadpole tail during spontaneous metamorphosis with special reference to apoptosis of striated muscle fibers. J Cell Sci 14: 571–585; 1974.

    PubMed  CAS  Google Scholar 

  • Kim M.; Kim S. E.; Kang S. S.; Kim Y. H.; Tae G. The use of de-differentiated chondrocytes delivered by a heparin-based hydrogel to regenerate cartilage in partial-thickness defects. Biomaterials 32: 7883–7896; 2011. doi:10.1016/j.biomaterials.2011.07.015;2011.

    Article  PubMed  CAS  Google Scholar 

  • Lippens S.; Hoste E.; Vandenabeele P.; Agostinis P.; Declercq W. Cell death in the skin. Apoptosis 14: 549–569; 2009. doi:10.1007/s10495-009-0324-z.

    Article  PubMed  Google Scholar 

  • Macias D.; Ganan Y.; Sampath T. K.; Piedra M. E.; Ros M. A.; Hurle J. M. Role of BMP-2 and OP-1 (BMP-7) in programmed cell death and skeletogenesis during chick limb development. Development 124: 1109–1117; 1997.

    PubMed  CAS  Google Scholar 

  • Montero J. A.; Hurle J. M. Sculpturing digit shape by cell death. Apoptosis 15: 365–375; 2010. doi:10.1007/s10495-009-0444-5.

    Article  PubMed  Google Scholar 

  • Nieuwkoop P. D.; Faber J. Normal Table of Xenopus laevis (Daudin). North-Holland, Amsterdam; 1967.

    Google Scholar 

  • Nishikawa A.; Kaiho M.; Yoshizato K. Cell death in the anuran tadpole tail: thyroid hormone induces keratinization and tail-specific growth inhibition of epidermal cells. Dev Biol 131: 337–344; 1989.

    Article  PubMed  CAS  Google Scholar 

  • Nishikawa A.; Yoshizato K. Hormonal regulation of growth and life span of bullfrog tadpole tail epidermal cells cultured in vitro. J Exp Zool 237: 221–230; 1986. doi:10.1002/jez.1402370208.

    Article  PubMed  CAS  Google Scholar 

  • Nye H. L.; Cameron J. A. Strategies to reduce variation in Xenopus regeneration studies. Dev Dyn 234: 151–158; 2005. doi:10.1002/dvdy.20508.

    Article  PubMed  CAS  Google Scholar 

  • Omi M.; Sato-Maeda M.; Ide H. Role of chondrogenic tissue in programmed cell death and BMP expression in chick limb buds. Int J Dev Biol 44: 381–388; 2000.

    PubMed  CAS  Google Scholar 

  • Roberts L. M.; Hirokawa Y.; Nachtigal M. W.; Ingraham H. A. Paracrine-mediated apoptosis in reproductive tract development. Dev Biol 208: 110–122; 1999. doi:10.1006/dbio.1998.9190.

    Article  PubMed  CAS  Google Scholar 

  • Roth K. A.; D'Sa C. Apoptosis and brain development. Ment Retard Dev Disabil Res Rev 7: 261–266; 2001. doi:10.1002/mrdd.1036.

    Article  PubMed  CAS  Google Scholar 

  • Salas-Vidal E.; Valencia C.; Covarrubias L. Differential tissue growth and patterns of cell death in mouse limb autopod morphogenesis. Dev Dyn 220: 295–306; 2001. doi:10.1002/dvdy.1108.

    Article  PubMed  CAS  Google Scholar 

  • Samuels H. H.; Stanley F.; Casanova J. Depletion of L-3,5,3'-triiodothyronine and L-thyroxine in euthyroid calf serum for use in cell culture studies of the action of thyroid hormone. Endocrinology 105: 80–85; 1979.

    Article  PubMed  CAS  Google Scholar 

  • Satoh A.; Suzuki M.; Amano T.; Tamura K.; Ide H. Joint development in Xenopus laevis and induction of segmentations in regenerating froglet limb (spike). Dev Dyn 233: 1444–1453; 2005. doi:10.1002/dvdy.20484.

    Article  PubMed  CAS  Google Scholar 

  • Saunders Jr. J. W.; Fallon J. F. Cell death in morphogenesis. In: Locke M. (ed) Major problems in developmental biology. Academic Press, New York, pp 289–314; 1966.

    Google Scholar 

  • Saunders Jr. J. W.; Gasseling M. T. Cellular death in morphogenesis of the avian wing. Dev Biol 5: 147–178; 1962.

    Article  PubMed  Google Scholar 

  • Shibota Y.; Kaneko Y.; Kuroda M.; Nishikawa A. Larval-to-adult conversion of a myogenic system in the frog, Xenopus laevis, by larval-type myoblast-specific control of cell division, cell differentiation, and programmed cell death by triiodo-L-thyronine. Differentiation 66: 227–238; 2000. doi:10.1046/j.1432-0436.2000.660409.x.

    PubMed  CAS  Google Scholar 

  • Sohn S. J.; Thompson J.; Winoto A. Apoptosis during negative selection of autoreactive thymocytes. Curr Opin Immunol 19: 510–515; 2007. doi:10.1016/j.coi.2007.06.001.

    Article  PubMed  CAS  Google Scholar 

  • Tallheden T.; Karlsson C.; Brunner A.; Van Der Lee J.; Hagg R.; Tommasini R.; Lindahl A. Gene expression during redifferentiation of human articular chondrocytes. Osteoarthr Cartil 12: 525–535; 2004. doi:10.1016/j.joca.2004.03.004.

    Article  PubMed  Google Scholar 

  • Tang M. K.; Leung A. K.; Kwong W. H.; Chow P. H.; Chan J. Y.; Ngo-Muller V.; Li M.; Lee K. K. Bmp-4 requires the presence of the digits to initiate programmed cell death in limb interdigital tissues. Dev Biol 218: 89–98; 2000. doi:10.1006/dbio.1999.9578.

    Article  PubMed  CAS  Google Scholar 

  • Tesniere A.; Panaretakis T.; Kepp O.; Apetoh L.; Ghiringhelli F.; Zitvogel L.; Kroemer G. Molecular characteristics of immunogenic cancer cell death. Cell Death Differ 15: 3–12; 2008. doi:10.1038/sj.cdd.4402269.

    Article  PubMed  CAS  Google Scholar 

  • Theiler K. The house mouse: atlas of embryonic development. Springer-Verlag, New York; 1989.

    Google Scholar 

  • Togo S.; Sato T.; Sugiura H.; Wang X.; Basma H.; Nelson A.; Liu X.; Bargar T. W.; Sharp J. G.; Rennard S. I. Differentiation of embryonic stem cells into fibroblast-like cells in three-dimensional type I collagen gel cultures. In Vitro Cell Dev Biol Anim 47: 114–124; 2011. doi:10.1007/s11626-010-9367-2.

    Article  PubMed  CAS  Google Scholar 

  • Tone S.; Tanaka S.; Kato Y. The inhibitory effect of 5-bromodeoxyuridine on the programmed cell death in the chick limb. Dev Growth Differ 25: 381–391; 1983.

    Article  CAS  Google Scholar 

  • Yokouchi Y.; Sakiyama J.; Kameda T.; Iba H.; Suzuki A.; Ueno N.; Kuroiwa A. BMP-2/-4 mediate programmed cell death in chicken limb buds. Development 122: 3725–3734; 1996.

    PubMed  CAS  Google Scholar 

  • Yokoyama H.; Endo T.; Tamura K.; Yajima H.; Ide H. Multiple digit formation in Xenopus limb bud recombinants. Dev Biol 196: 1–10; 1998. doi:10.1006/dbio.1998.8856.

    Article  PubMed  CAS  Google Scholar 

  • Zou H.; Niswander L. Requirement for BMP signaling in interdigital apoptosis and scale formation. Science 272: 738–741; 1996.

    Article  PubMed  CAS  Google Scholar 

  • Zuzarte-Luis V.; Hurle J. M. Programmed cell death in the embryonic vertebrate limb. Semin Cell Dev Biol 16: 261–269; 2005. doi:10.1016/j.semcdb.2004.12.004.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. The authors express their thanks to Dr. Naoyuki Wada (Kawasaki Medical School) for his helpful advice on BMP application techniques.

Author information

Authors and Affiliations

  1. Department of Biological Science, Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue, Shimane, 690-8504, Japan

    Keiko Shimizu-Nishikawa & Akio Nishikawa

  2. Department of Molecular Biology, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama, 701-0192, Japan

    Shin-ichiro Nishimatsu

Authors
  1. Keiko Shimizu-Nishikawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Shin-ichiro Nishimatsu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Akio Nishikawa
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Akio Nishikawa.

Additional information

Editor: T. Okamoto

Rights and permissions

Reprints and permissions

About this article

Cite this article

Shimizu-Nishikawa, K., Nishimatsu, Si. & Nishikawa, A. Strategies to detect interdigital cell death in the frog, Xenopus laevis: T3 accerelation, BMP application, and mesenchymal cell cultivation. In Vitro Cell.Dev.Biol.-Animal 48, 313–325 (2012). https://doi.org/10.1007/s11626-012-9508-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11626-012-9508-x

Keywords

Search

Navigation