, Volume 11, Issue 5, pp 701–715 | Cite as

Caspase redundancy and release of mitochondrial apoptotic factors characterize interdigital apoptosis

  • V. Zuzarte-Luis
  • M. T. Berciano
  • M. Lafarga
  • J. M. HurléEmail author


Here we show a detailed analysis of cellular and molecular events during in vivo apoptotic cell death in the INZs (interdigital necrotic zones) of the embryonic limb. As the apoptotic mechanisms proceed, the transcriptionally active chromatin and nuclear traffic of RNAs are disrupted, cytoskeletal components are disorganized and the adhesive properties of cells are compromised as Paxillin, a clue member of the focal adhesion complex, decreases in early apoptotic cells. Activation of effector caspases 3 and 7 follow nuclear degradation. In addition, active caspase2 is localized in the nuclei and cytoplasm of early apoptotic cells suggesting a major role in physiological conditions supported by its down-regulation in tissue survival experiments. However in caspase 2 siRNA assays we observed translocation of caspase 3 to the nuclei suggesting functional redundancy. We also observed release of cytochrome c and AIF from the mitochondria, and interestingly AIF becomes intranuclear in a caspase independent manner.


AIF caspase 2 cytochrome c limb development programmed cell death 


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  1. 1.
    Zuzarte-Luis V, Hurle JM. Programmed cell death in the developing limb. Int J Dev Biol 2002; 46: 871–876.Google Scholar
  2. 2.
    Fallon JF, Cameron J. Interdigital cell death during limb development of the turtle and lizard with an interpretation of evolutionary significance. J Embryol Exp Morphol 1977; 40: 285–289.PubMedGoogle Scholar
  3. 3.
    Milligan CE, Prevette D, Yaginuma H, et al. Peptide inhibitors of the ICE protease family arrest programmed cell death of motoneurons in vivo and in vitro. Neuron 1995; 15: 385–393.PubMedCrossRefGoogle Scholar
  4. 4.
    Jacobson MD, Weil M, Rafff MC. Role of Ced-3/ICE-family proteases in staurosporine-induced programmed cell death. J Cell Biol 1996; 133: 1041–1051.CrossRefGoogle Scholar
  5. 5.
    Umpierre CC, Little SA, Mirkes PE. Co-localization of active caspase-3 and DNA fragmentation (TUNEL) in normal and hyperthermia-induced abnormal mouse development. Teratology 2001; 63: 134–143.PubMedCrossRefGoogle Scholar
  6. 6.
    Huang C, Hales BF. Role of caspases in murine limb bud cell death induced by 4-hydroperoxycyclophosphamide, an activated analog of cyclophosphamide. Teratology 2002; 66: 288–299.PubMedCrossRefGoogle Scholar
  7. 7.
    Kuida K, Zheng TS, Na S, et al. Decreased apoptosis in the brain and premature lethality in CPP32-deficient mice. Nature 1996; 384: 368–372.PubMedCrossRefGoogle Scholar
  8. 8.
    Chautan M, Chazal G, Cecconi F, Gruss P, Golstein P. Interdigital cell death can occur through a necrotic and caspase-independent pathway. Curr Biol 1999; 9: 967–970.PubMedCrossRefGoogle Scholar
  9. 9.
    Wride MA, Lapchak PH, Sanders EJ. Distribution of TNF alpha-like proteins correlates with some regions of programmed cell death in the chick embryo. Int J Dev Biol 1994; 38: 673–682.PubMedGoogle Scholar
  10. 10.
    Wang J, Zheng L, Lobito A, et al. Inherited human Caspase 10 mutations underlie defective lymphocyte and dendritic cell apoptosis in autoimmune lymphoproliferative syndrome type II. Cell 1999; 98: 47–58.PubMedCrossRefGoogle Scholar
  11. 11.
    Chun HJ, Zheng L, Ahmad M, et al. Pleiotropic defects in lymphocyte activation caused by caspase-8 mutations lead to human immunodeficiency. Nature 2002; 419: 395–399.PubMedCrossRefGoogle Scholar
  12. 12.
    Dupe V, Ghyselinck NB, Thomazy V, et al. Essential roles of retinoic acid signalling in interdigital apoptosis and control of BMP-7 expression in mouse autopods. Dev Biol 1999; 208: 30–43.PubMedCrossRefGoogle Scholar
  13. 13.
    Salas-Vidal E, Lomeli H, Castro-Obregon S, Cuervo R, Escalante-Alcalde D, Covarrubias L. Reactive oxygen species participate in the control of mouse embryonic cell death. Exp Cell Res 1998; 238: 136–147.PubMedCrossRefGoogle Scholar
  14. 14.
    Lindsten T, Ross AJ, King A, et al. The combined functions of proapoptotic Bcl-2 family members bak and bax are essential for normal development of multiple tissues. Mol Cell 2000; 6: 1389–1399.PubMedCrossRefGoogle Scholar
  15. 15.
    Jurgensmeier JM, Xie Z, Deveraux Q, Ellerby L, Bredesen D, Reed JC. Bax directly induces release of cytochrome c from isolated mitochondria. Proc Natl Acad Sci USA 1998; 95: 4997–5002.PubMedCrossRefGoogle Scholar
  16. 16.
    Kuida K, Haydar TF, Kuan CY, et al. Reduced apoptosis and cytochrome c-mediated caspase activation in mice lacking caspase 9. Cell 1998; 94: 325–337.PubMedCrossRefGoogle Scholar
  17. 17.
    Drouin R, Lemieux N, Richer CL. Chromosome condensation from prophase to late metaphase: Relationship to chromosome bands and their replication time. Cytogenet Cell Genet 1991; 57: 91–99.PubMedCrossRefGoogle Scholar
  18. 18.
    Rubio E, Valenciano AI, Segundo C, Sanchez N, de Pablo F, de la Rosa EJ. Programmed cell death in the neurulating embryo is prevented by the chaperone heat shock cognate 70. Eur J Neurosci 2002; 15: 1646–1654.PubMedCrossRefGoogle Scholar
  19. 19.
    Jansen RP, Hurt EC, Kern H, Lehtonen H, Carmo-Fonseca M. Evolutionary conservation of the human nucleolar protein fibrillarin and its functional expression in yeast. J Cell Biol 1991; 113: 715–729.PubMedCrossRefGoogle Scholar
  20. 20.
    Pena E, Berciano MT, Fernandez R, Ojeda JL, Lafarga M. Neuronal body size correlates with the number of nucleoli and Cajal bodies, and with the organization of the splicing machinery in rat trigeminal ganglion neurons. J Comp Neurol 2001; 430: 250–263.PubMedCrossRefGoogle Scholar
  21. 21.
    Macias D, Ganan Y, Ros MA, Hurle JM. In vivo inhibition of programmed cell death by local administration of FGF-2 and FGF-4 in the interdigital areas of the embryonic chick leg bud. Anat Embryol (Berl) 1996; 193: 533–541.Google Scholar
  22. 22.
    Berciano MT, Villagra NT, Ojeda JL, et al. Oculopharyngeal muscular dystrophy-like nuclear inclusions are present in normal magnocellular neurosecretory neurons of the hypothalamus. Hum Mol Genet 2004; 13: 829–838.PubMedCrossRefGoogle Scholar
  23. 23.
    Mirkes PE, Little SA. cytochrome c release from the mitochondria of early postimplantation murine embryos exposed to 4-hydroperoxycyclophosphamide, heat shock and staurosporine. Tox App Pharma 2000; 162: 197–206.CrossRefGoogle Scholar
  24. 24.
    Kihlmark M, Imreh G, Hallberg E. Sequential degradation of proteins from the nuclear envelope during apoptosis. J Cell Sci 2001; 114: 3643–3653.PubMedGoogle Scholar
  25. 25.
    Gregory PD, Wagner K, Horz W. Histone acetylation and chromatin remodeling. Exp Cell Res 2001; 265: 195–202.PubMedCrossRefGoogle Scholar
  26. 26.
    Buendia B, Santa-Maria A, Courvalin JC. Caspase-dependent proteolysis of integral and peripheral proteins of nuclear membranes and nuclear pore complex proteins during apoptosis. J Cell Sci 1999; 112: 1743–1753.PubMedGoogle Scholar
  27. 27.
    Stott NS, Jiang TX, Chuong CM. Successive formative stages of precartilaginous mesenchymal condensations in vitro: Modulation of cell adhesion by Wnt-7A and BMP-2. J Cell Physiol 1999; 180: 314–324.PubMedCrossRefGoogle Scholar
  28. 28.
    Merino R, Macias D, Ganan Y, et al. Control of digit formation by activin signalling. Development 1999; 126: 2161–2170.PubMedGoogle Scholar
  29. 29.
    Pedersen MV, Kohler LB, Ditlevsen DK, Li S, Berezin V, Bock E. Neuritogenic and survival-promoting effects of the P2 peptide derived from a homophilic binding site in the neural cell adhesion molecule. J Neurosci Res 2004; 75: 55–65.PubMedCrossRefGoogle Scholar
  30. 30.
    Hanks SK, Ryzhova L, Shin NY, Brabek J. Focal adhesion kinase signaling activities and their implications in the control of cell survival and motility. Front Biosci 2003; 8: 982–996.Google Scholar
  31. 31.
    Chay KO, Park SS, Mushinski JF. Linkage of caspase-mediated degradation of paxillin to apoptosis in Ba/F3 murine pro-B lymphocytes. J Biol Chem 2002; 277: 14521–14529.PubMedCrossRefGoogle Scholar
  32. 32.
    Longuet M, Serduc R, Riva C. Implication of bax in apoptosis depends on microtubule network mobility. Int J Oncol 2004; 25: 309–317.PubMedGoogle Scholar
  33. 33.
    Byun Y, Chen F, Chang R, Trivedi M, Green KJ, Cryns VL. Caspase cleavage of vimentin disrupts intermediate filaments and promotes apoptosis. Cell Death Differ 2001; 8: 443–450.PubMedCrossRefGoogle Scholar
  34. 34.
    Martin DN, Baehrecke EH. Caspases function in autophagic programmed cell death in Drosophila. Development 2004; 131: 275–284.PubMedCrossRefGoogle Scholar
  35. 35.
    Guicciardi ME, Leist M, Gores GJ. Lysosomes in cell death. Oncogene 2004; 23: 2881–2890.PubMedCrossRefGoogle Scholar
  36. 36.
    Hurle J, Hinchcliffe JR. Cell death in the posterior necrotic zone (PNZ) of the chick wing-bud: A stereoscan and ultrastructural survey of autolysis and cell fragmentation. J Embryol Exp Morphol 1978; 43: 123–136.PubMedGoogle Scholar
  37. 37.
    Stewart S, Yi S, Kassabian G, Mayo M, Sank A, Shuler C. Changes in expression of the lysosomal membrane glycoprotein, LAMP-1 in interdigital regions during embryonic mouse limb development, in vivo and in vitro. Anat Embryol (Berl) 2000; 201: 483–490.CrossRefGoogle Scholar
  38. 38.
    Salvesen GS, Abrams JM. Caspase activation—Stepping on the gas or releasing the brakes? Lessons from humans and flies. Oncogene 2004; 23: 2774–2784.PubMedCrossRefGoogle Scholar
  39. 39.
    Zuzarte-Luis V, Hurle JM. Programmed cell death in the embryonic vertebrate limb. Sem Cell Dev Biol 2005; (in press).Google Scholar
  40. 40.
    Hong SJ, Dawson TM, Dawson VL. Nuclear and mitochondrial conversations in cell death: PARP-1 and AIF signaling. Trends Pharmacol Sci 2004; 25: 259–264.PubMedCrossRefGoogle Scholar
  41. 41.
    Grossmann J, Walther K, Artinger M, Kiessling S, Scholmerich J. Apoptotic signaling during initiation of detachment-induced apoptosis (“anoikis”) of primary human intestinal epithelial cells. Cell Growth Differ 2001; 12: 147–155.PubMedGoogle Scholar
  42. 42.
    De Arcangelis A, Mark M, Kreidberg J, Sorokin L, Georges-Labouesse E. Synergistic activities of alpha3 and alpha6 integrins are required during apical ectodermal ridge formation and organogenesis in the mouse. Development 1999; 126: 3957–3968.PubMedGoogle Scholar
  43. 43.
    Hurle JM, Corson G, Daniels K, Reiter RS, Sakai LY, Solursh M. Elastin exhibits a distinctive temporal and spatial pattern of distribution in the developing chick limb in association with the establishment of the cartilaginous skeleton. J Cell Sci 1994; 107: 2623–2634.PubMedGoogle Scholar
  44. 44.
    Miner JH, Cunningham J, Sanes JR. Roles for laminin in embryogenesis: Exencephaly, syndactyly, and placentopathy in mice lacking the laminin alpha5 chain. J Cell Biol 1998; 143: 1713–1723.PubMedCrossRefGoogle Scholar
  45. 45.
    Arteaga-Solis E, Gayraud B, Lee SY, Shum L, Sakai L, Ramirez F. Regulation of limb patterning by extracellular microfibrils. J Cell Biol 2001; 154: 275–281.PubMedCrossRefGoogle Scholar
  46. 46.
    Debeer P, Schoenmakers EF, Twal WO, et al. The fibulin-1 gene (FBLN1) is disrupted in a t(12; 22) associated with a complex type of synpolydactyly. J Med Genet 2002; 39: 98–104.PubMedCrossRefGoogle Scholar
  47. 47.
    Smyth I, Du X, Taylor MS, Justice MJ, Beutler B, Jackson IJ. The extracellular matrix gene Frem1 is essential for the normal adhesion of the embryonic epidermis. Proc Natl Acad Sci USA 2004; 101: 13560–13565.PubMedCrossRefGoogle Scholar
  48. 48.
    Takamiya K, Kostourou V, Adams S, et al. A direct functional link between the multi-PDZ domain protein GRIP1 and the Fraser syndrome protein Fras1. Nat Genet 2004; 36: 172–177.PubMedCrossRefGoogle Scholar
  49. 49.
    Levine B, Klionsky DJ. Development by self-digestion: Molecular mechanisms and biological functions of autophagy. Dev Cell 2004; 6: 463–477.PubMedCrossRefGoogle Scholar
  50. 50.
    Hurle JM, Colvee E, Fernandez-Teran MA. Vascular regression during the formation of the free digits in the avian limb bud: A comparative study in chick and duck embryos. J Embryol Exp Morphol 1985; 85: 239–250.PubMedGoogle Scholar
  51. 51.
    Kihlmark M, Rustum C, Eriksson C, Beckman M, Iverfeldt K, Hallberg E. Correlation between nucleocytoplasmic transport and caspase-3-dependent dismantling of nuclear pores during apoptosis. Exp Cell Res 2004; 293: 346–356.PubMedCrossRefGoogle Scholar
  52. 52.
    Ferrando-May E, Cordes V, Biller-Ckovric I, Mirkovic J, Gorlich D, Nicotera P. Caspases mediate nucleoporin cleavage, but not early redistribution of nuclear transport factors and modulation of nuclear permeability in apoptosis. Cell Death Differ 2001; 8: 495–505.PubMedCrossRefGoogle Scholar
  53. 53.
    Yasuhara N, Eguchi Y, Tachibana T, Imamoto N, Yoneda Y, Tsujimoto Y. Essential role of active nuclear transport in apoptosis. Genes Cells 1997; 2: 55–64.PubMedCrossRefGoogle Scholar
  54. 54.
    Zheng TS, Hunot S, Kuida K, Flavell RA. Caspase knockouts: Matters of life and death. Cell Death Differ 1999; 6: 1043–1053.PubMedCrossRefGoogle Scholar
  55. 55.
    Fischer U, Janicke RU, Schulze-Osthoff K. Many cuts to ruin: A comprehensive update of caspase substrates. Cell Death Differ 2003; 10: 76–100.PubMedCrossRefGoogle Scholar
  56. 56.
    Miyazaki K, Yoshida H, Sasaki M, et al. Caspase-independent cell death and mitochondrial disruptions observed in the Apaf1-deficient cells. J Biochem (Tokyo) 2001; 129: 963–969.Google Scholar
  57. 57.
    Joza N, Susin SA, Daugas E, et al. Essential role of the mitochondrial apoptosis-inducing factor in programmed cell death. Nature 2001; 410: 549–554.PubMedCrossRefGoogle Scholar
  58. 58.
    Sanders EJ, Parker E. Ablation of axial structures activates apoptotic pathways in somite cells of the chick embryo. Anat Embryol (Berl) 2001; 204: 389–398.CrossRefGoogle Scholar
  59. 59.
    Guo Y, Srinivasula SM, Druilhe A, Fernandes-Alnemri T, Alnemri ES. Caspase-2 induces apoptosis by releasing proapoptotic proteins from mitochondria. J Biol Chem 2002; 277: 13430–13437.PubMedCrossRefGoogle Scholar
  60. 60.
    Cregan SP, Fortin A, MacLaurin JG, et al. Apoptosis-inducing factor is involved in the regulation of caspase-independent neuronal cell death. J Cell Biol 2002; 158: 507–517.PubMedCrossRefGoogle Scholar
  61. 61.
    Broker LE, Kruyt FAE, Giaccone G. Cell Death Independent of Caspases: A review. Clin Cancer Res 2005; 11: 3155–3162.PubMedCrossRefGoogle Scholar
  62. 62.
    Nakanishi K, Maruyama M, Shibata T, Morishima N. Identification of a caspase-9 substrate and detection of its cleavage in programmed cell death during mouse development. J Biol Chem 2001; 276: 41237–41244.PubMedCrossRefGoogle Scholar
  63. 63.
    Bergeron L, Perez GI, Macdonald G, et al. Defects in regulation of apoptosis in caspase-2-deficient mice. Genes Dev 1998; 12: 1304–1314.PubMedGoogle Scholar
  64. 64.
    Troy CM, Shelanski ML. Caspase-2 redux. Cell Death Differ 2003; 10: 101–107.PubMedCrossRefGoogle Scholar
  65. 65.
    Wagner KW, Engels IH, Deveraux QL. Caspase-2 can function upstream of bid cleavage in the TRAIL apoptosis pathway. J Biol Chem 2004; 279: 35047–35052.PubMedCrossRefGoogle Scholar
  66. 66.
    Zhivotovsky B, Orrenius S. Caspase-2 function in response to DNA damage. Biochem Biophys Res Commun 2005; 331: 859–867.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science + Business Media, Inc. 2006

Authors and Affiliations

  • V. Zuzarte-Luis
    • 1
  • M. T. Berciano
    • 1
  • M. Lafarga
    • 1
  • J. M. Hurlé
    • 1
    • 2
    Email author
  1. 1.Departamento de Anatomía y Biología Celular, Facultad de MedicinaUniversidad de CantabriaSantanderSpain
  2. 2.Departamento de Anatomía y Biología Celular, Facultad de MedicinaC/Cardenal Herrera Oria s/nSantanderSpain

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