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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
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
Trowell OA (1959) The culture of mature organs in a synthetic medium. Exp Cell Res 16:118–147
Grobstein C (1953) Inductive epithelio-mesenchymal interaction in cultured organ rudiments of the mouse. Science 118:52–55
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
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
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
Nogawa H, Ito T (1995) Branching morphogenesis of embryonic mouse lung epithelium in mesenchyme-free culture. Development 121:1015–1022
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
Saxén L (1966) The effect of tetracycline on osteogenesis in vitro. J Exp Zool 162:269–294
Thesleff I, Lehtonen E, Wartiovaara J, Saxén L (1977) Interference of tooth differentiation with interposed filters. Dev Biol 58:197–203
Partanen AM, Ekblom P, Thesleff I (1985) Epidermal growth factor inhibits tooth morphogenesis and differentiation. Dev Biol 111:84–94
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
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
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
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
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
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
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
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
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
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
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
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
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
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
Tümmers M, Yamashiro T, Thesleff I (2007) Modulation of epithelial cell fate of the root in vitro. J Dent Res 86:1063–1067
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
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
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
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
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
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
Download citation
DOI: https://doi.org/10.1007/978-1-62703-125-7_24
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-124-0
Online ISBN: 978-1-62703-125-7
eBook Packages: Springer Protocols