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Analysis of Tissue Interactions in Ectodermal Organ Culture

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Epithelial Cell Culture Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 945))

Abstract

The morphogenesis of ectodermal organs is regulated by epithelial mesenchymal interactions mediated by conserved signaling molecules. Analyzing the roles of these molecules will increase our understanding of mechanisms regulating organogenesis, and organ culture methods provide powerful tools in this context. Here we present two organ culture methods for skin and tooth development: the hanging drop method for the short-term culture of small explants and the Trowell-type method for the long-term cultures of variable size explants. The latter allows manipulations such as combining separated epithelial and mesenchymal tissues and the use of signal-releasing beads. The effects of signaling molecules on morphogenesis can be observed during culture by using tissues from GFP-reporter mice. After culture, the effects of signals on gene expression can be analyzed by in situ hybridization or quantitative RT-PCR.

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References

  1. Tummers M, Thesleff I (2009) The importance of signal pathway modulation in all aspects of tooth development. J Exp Zool (Mol Dev Evol) 312B(4):309–319

    Article  Google Scholar 

  2. Trowell OA (1959) The culture of mature organs in a synthetic medium. Exp Cell Res 16:118–147

    Article  PubMed  CAS  Google Scholar 

  3. Grobstein C (1953) Inductive epithelio-mesenchymal interaction in cultured organ rudiments of the mouse. Science 118:52–55

    Article  PubMed  CAS  Google Scholar 

  4. Saxén I (1973) Effects of hydrocortisone on the development in vitro of the secondary palate in two inbred strains of mice. Arch Oral Biol 18:1469–1479

    Article  PubMed  Google Scholar 

  5. Saxén L, Lehtonen E, Karkinen-Jääskeläinen M, Nordling S, Wartiovaara J (1976) Morphogenetic tissue interactions: mediation by transmissible signal substances or through cell contacts? Nature 259:662–663

    Article  PubMed  Google Scholar 

  6. Nogawa H, Takahashi Y (1991) Substitution for mesenchyme by basement-membrane-like substratum and epidermal growth factor in inducing branching morphogenesis of mouse salivary epithelium. Development 112:855–861

    PubMed  CAS  Google Scholar 

  7. Nogawa H, Ito T (1995) Branching morphogenesis of embryonic mouse lung epithelium in mesenchyme-free culture. Development 121:1015–1022

    PubMed  CAS  Google Scholar 

  8. Kim H-J, Rice DPC, Kettunen PJ, Thesleff I (1998) FGF-, BMP- and Shh-mediated signaling pathways in the regulation of cranial suture morphogenesis and calvarial bone development. Development 125:1241–1251

    PubMed  CAS  Google Scholar 

  9. Saxén L (1966) The effect of tetracycline on osteogenesis in vitro. J Exp Zool 162:269–294

    Article  Google Scholar 

  10. Thesleff I, Lehtonen E, Wartiovaara J, Saxén L (1977) Interference of tooth differentiation with interposed filters. Dev Biol 58:197–203

    Article  PubMed  CAS  Google Scholar 

  11. Partanen AM, Ekblom P, Thesleff I (1985) Epidermal growth factor inhibits tooth morphogenesis and differentiation. Dev Biol 111:84–94

    Article  PubMed  CAS  Google Scholar 

  12. Vainio S, Karavanova I, Jowett A, Thesleff I (1993) Identification of BMP-4 as a signal mediating secondary induction between epithelial and mesenchymal tissues during early tooth development. Cell 75:45–58

    PubMed  CAS  Google Scholar 

  13. Jernvall J, Aberg T, Kettunen P, Keranen S, Thesleff I (1998) The life history of an embryonic signaling center: BMP-4 induces p21 and is associated with apoptosis in the mouse tooth enamel knot. Development 125:161–169

    PubMed  CAS  Google Scholar 

  14. Mitsiadis T, Muramatsu T, Muramatsu H, Thesleff I (1995) Midkine (MK), a heparing-binding growth/differentiation factor, is regulated by retinoic acid and epithelial-mesenchymal interactions in the developing mouse tooth, and affects cell proliferation and morphogenesis. J Cell Biol 129:267–281

    Article  PubMed  CAS  Google Scholar 

  15. Laurikkala J, Mikkola M, Mustonen T, Åberg T, Koppinen P, Pispa J, Nieminen P, Galceran J, Grosshedl R, Thesleff I (2001) TNF signaling via the ligand-receptor pair ectodysplasin and edar controls the function of epithelial signaling centers and is regulated by Wnt and activin during tooth organogenesis. Dev Biol 229:443–455

    Article  PubMed  CAS  Google Scholar 

  16. Wang X-P, Suomalainen M, Felszeghy S, Zelarayan LC, Alonso MT, Plikus MV, Maas RL, Chuong CM, Schimmang T, Thesleff I (2007) An integrated gene regulatory network controls stem cell proliferation in teeth. PLoS Biol 5:1324–1333

    CAS  Google Scholar 

  17. Järvinen E, Salaar-Ciudad I, Birchmeier W, Taketo MT, Jernvall J, Thesleff I (2006) Continuous tooth regeneration in mouse in induced by activated epithelial Wnt/β-catenin signaling. Proc Natl Acad Sci USA 103:18627–18632

    Article  PubMed  Google Scholar 

  18. Rice D, Åberg T, Chan Y-S, Kettunen P, Pakarinen L, Maxson RE, Thesleff I (2000) Integration of FGF and TWIST in calvarial bone and suture development. Development 127:1845–1855

    PubMed  CAS  Google Scholar 

  19. Michon F, Charveron M, Dhouailly D (2007) Dermal condensation formation in the chick embryo: requirement for integrin engagement and subsequent stabilization by a possible notch/integrin interaction. Dev Dyn 236:755–768

    Article  PubMed  CAS  Google Scholar 

  20. Fliniaux I, Mikkola ML, Lefebvre S, Thesleff I (2008) Identification of dkk4 as a target of Eda-A1/Edar pathway reveals an unexpected role of ectodysplasin as inhibitor of Wnt signalling in ectodermal placodes. Dev Biol 320:60–71

    Article  PubMed  CAS  Google Scholar 

  21. Wilkinson DG, Green J (1990) In situ hybridization and the three-dimensional reconstruction of serial sections. In: Copp AJ, Cockroft DL (eds) Postimplantation mammalian embryos: a practical approach. IRL, Oxford, pp 155–171

    Google Scholar 

  22. Vaahtokari A, Vainio S, Thesleff I (1991) Associations between transforming growth factor β1 RNA expression and epithelial-mesenchymal interactions during tooth morphogenesis. Development 113:985–994

    PubMed  CAS  Google Scholar 

  23. Kettunen P, Thesleff I (1998) Expression and function of FGFs-4, -8, and -9 suggest functional redundancy and repetitive use as epithelial signals during tooth morphogenesis. Dev Dyn 211:256–268

    Article  PubMed  CAS  Google Scholar 

  24. Mustonen T, Tümmers M, Mikami T, Itoh N, Zhang N, Gridley T, Thesleff I (2002) Lunatic fringe, FGF, and BMP regulate the Notch pathway during epithelial morphogenesis of teeth. Dev Biol 248:281–293

    Article  PubMed  CAS  Google Scholar 

  25. Laurikkala J, Kassai Y, Pakkasjärvi L, Thesleff I, Itoh N (2003) Identification of a secreted BMP antagonist, ectodin, interacting BMP, FGF, and SHH signals from the tooth enamel knot. Dev Biol 264:91–105

    Article  PubMed  CAS  Google Scholar 

  26. Tümmers M, Yamashiro T, Thesleff I (2007) Modulation of epithelial cell fate of the root in vitro. J Dent Res 86:1063–1067

    Article  PubMed  Google Scholar 

  27. Munne P, Tummers M, Järvinen E, Thesleff I, Jernvall J (2009) Tinkering with the inductive mesenchyme: Sostdc1 uncovers the role for dental mesenchyme in limiting tooth induction. Development 136:393–402

    Article  PubMed  CAS  Google Scholar 

  28. Chen Y, Gelfond JA, McManus LM, Shireman PK (2009) Reproducibility of quantitative RT-PCR array in miRNA expression profiling and comparison with microarray analysis. BMC Genomics 10:407

    Article  PubMed  Google Scholar 

  29. Närhi K, Järvinen E, Birchmeier W, Taketo MM, Mikkola ML, Thesleff I (2008) Sustained epithelial beta-catenin activity induces precocious hair development but disrupts hair follicle down-growth and hair shaft formation. Development 135:1019–1028

    Article  PubMed  Google Scholar 

  30. Rice R, Thesleff I, Rice DP (2005) Regulation of Twist, Snail, and Id1 is conserved between the developing murine palate and tooth. Dev Dyn 234:28–35

    Article  PubMed  CAS  Google Scholar 

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Correspondence to Frederic Michon .

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Munne, P.M., Närhi, K., Michon, F. (2012). Analysis of Tissue Interactions in Ectodermal Organ Culture. In: Randell, S., Fulcher, M. (eds) Epithelial Cell Culture Protocols. Methods in Molecular Biology, vol 945. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-125-7_24

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  • DOI: https://doi.org/10.1007/978-1-62703-125-7_24

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-124-0

  • Online ISBN: 978-1-62703-125-7

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