Journal of Molecular Neuroscience

, Volume 54, Issue 3, pp 331–341 | Cite as

Structural and Morphometric Comparison of the Molar Teeth in Pre-eruptive Developmental Stage of PACAP-Deficient and Wild-Type Mice

  • B. Sandor
  • K. Fintor
  • Sz. Felszeghy
  • T. Juhasz
  • D. Reglodi
  • L. Mark
  • P. Kiss
  • A. Jungling
  • B. D. Fulop
  • A. D. Nagy
  • H. Hashimoto
  • R. Zakany
  • A. Nagy
  • A. TamasEmail author


Pituitary adenylate cyclase activating polypeptide (PACAP) is a pleiotropic neuropeptide with widespread distribution. It plays pivotal role in neuronal development. PACAP-immunoreactive fibers have been found in the tooth pulp, and recently, it has been shown that PACAP may also play a role in the regeneration of the periodontium after luxation injuries. However, there is no data about the effect of endogenous PACAP on tooth development. Ectodermal organogenesis including tooth development is regulated by different members of bone morphogenetic protein (BMP), fibroblast growth factor (FGF), hedgehog (HH), and Wnt families. There is also a growing evidence to support the hypothesis that PACAP interacts with sonic hedgehog (SHH) receptor (PTCH1) and its downstream target (Gli1) suggesting its role in tooth development. Therefore, our aim was to study molar tooth development in mice lacking endogenous PACAP. In this study morphometric, immunohistochemical and structural comparison of molar teeth in pre-eruptive developmental stage was performed on histological sections of 7-day-old wild-type and PACAP-deficient mice. Further structural analysis was carried out with Raman microscope. The morphometric comparison of the 7-day-old samples revealed that the dentin was significantly thinner in the molars of PACAP-deficient mice compared to wild-type animals. Raman spectra of the enamel in wild-type mice demonstrated higher diversity in secondary structure of enamel proteins. In the dentin of PACAP-deficient mice higher intracrystalline disordering in the hydroxyapatite molecular structure was found. We also obtained altered SHH, PTCH1 and Gli1 expression level in secretory ameloblasts of PACAP-deficient mice compared to wild-type littermates suggesting that PACAP might play an important role in molar tooth development and matrix mineralization involving influence on SHH signaling cascade.


PACAP Deficient Raman Tooth Development SHH PTCH1 Gli1 



We are grateful to Tamas Papp and Tunde Palne Terdik for technical assistance. This work was supported by PTE-MTA “Lendulet” Program, Arimura Foundation, OTKA K104984, PD109644, TAMOP 4.2.2.A-11/1/KONV-2012-0024, TAMOP 4.2.4.A/2-11-1-2012-0001 “National Excellence Program”, Bolyai Scholarship, Grants from University of Debrecen (RH/885/2013), Grants-in-Aid for Scientific Research, KAKENHI, Grant Numbers 26293020 and 26670122.


  1. Allais A, Burel D, Isaac ER et al (2007) Altered cerebellar development in mice lacking pituitary adenylate cyclase-activating polypeptide. Eur J Neurosci 25:2604–2618PubMedCrossRefGoogle Scholar
  2. Arimura A, Somogyvari-Vigh A, Weill C et al (1994) PACAP functions as a neurotrophic factor. Ann N Y Acad Sci 739:228–243PubMedCrossRefGoogle Scholar
  3. Bartlett JD, Ganss B, Goldberg M et al (2006) Protein–protein interactions of the developing enamel matrix. Curr Top Dev Biol 74:57–115PubMedCrossRefGoogle Scholar
  4. Bei M (2009) Molecular genetics of tooth development. Curr Opin Genet Dev 19:504–510PubMedCentralPubMedCrossRefGoogle Scholar
  5. Blechman J, Levkowitz G (2013) Alternative splicing of the pituitary adenylate cyclase-activating polypeptide receptor PAC1: mechanisms of fine tuning of brain activity. Front Endocrinol (Lausanne) 4:55Google Scholar
  6. Butler WT, Ritchie H (1995) The nature and functional significance of dentin extracellular matrix proteins. Int J Dev Biol 39:169–179PubMedGoogle Scholar
  7. Christopher KJ, Wang B, Kong Y, Weatherbee SD (2012) Forward genetics uncovers transmembrane protein 107 as a novel factor required for ciliogenesis and Sonic hedgehog signaling. Dev Biol 368:382–392PubMedCentralPubMedCrossRefGoogle Scholar
  8. D’Amico AG, Scuderi S, Saccone S, Castorina A, Drago F, D’Agata V (2013) Antiproliferative effects of PACAP and VIP in serum-starved glioma cells. J Mol Neurosci 51:503–513PubMedCrossRefGoogle Scholar
  9. Dassule HR, Lewis P, Bei M, Maas R, McMahon AP (2000) Sonic hedgehog regulates growth and morphogenesis of the tooth. Development 127:4775–4785PubMedGoogle Scholar
  10. de Mul FF, Hottenhuis MH, Bouter P, Greve J, Arends J, ten Bosch JJ (1986) Micro-Raman line broadening in synthetic carbonated hydroxyapatite. J Dent Res 65:437–440PubMedCrossRefGoogle Scholar
  11. Dollish FR, Fateley WG, Bentley FF (1974) Characteristic Raman frequencies of organic compounds. Wiley, New YorkGoogle Scholar
  12. Featherstone JD, Lussi A (2006) Understanding the chemistry of dental erosion. Monogr Oral Sci 20:66–76PubMedCrossRefGoogle Scholar
  13. Felszeghy S, Modis L, Nemeth P, Nagy G, Zelles T, Agre P, Laurikkala J, Fejerskov O, Thesleff I, Nielsen S (2004) Expression of aquaporin isoforms during human and mouse tooth development. Arch Oral Biol 49:247–257PubMedCrossRefGoogle Scholar
  14. Fincham AG, Moradian-Oldak J, Simmer JP (1999) The structural biology of the developing dental enamel matrix. J Struct Biol 126:270–299PubMedCrossRefGoogle Scholar
  15. Frechilla D, Garcia-Osta A, Palacios S, Cenarruzabeitia E, Del Rio J (2001) BDNF mediates the neuroprotective effect of PACAP-38 on rat cortical neurons. Neuroreport 12:919–923PubMedCrossRefGoogle Scholar
  16. Gritli-Linde A, Bei M, Maas R, Zhang XM, Linde A, McMahon AP (2002) Shh signaling within the dental epithelium is necessary for cell proliferation, growth and polarization. Development 129:5323–5337PubMedCrossRefGoogle Scholar
  17. Grumolato L, Louiset E, Alexandre D et al (2003) PACAP and NGF regulate common and distinct traits of the sympathoadrenal lineage: effects on electrical properties, gene markers and transcription factors in differentiating PC12 cells. Eur J Neurosci 17:71–82PubMedCrossRefGoogle Scholar
  18. Guirland C, Buck KB, Gibney JA, DiCicco-Bloom E, Zheng JQ (2003) Direct cAMP signaling through G-protein-coupled receptors mediates growth cone attraction induced by pituitary adenylate cyclase-activating polypeptide. J Neurosci 23:2274–2283PubMedGoogle Scholar
  19. Hansel DE, May V, Eipper BA, Ronnett GV (2001) Pituitary adenylyl cyclase-activating peptides and alpha-amidation in olfactory neurogenesis and neuronal survival in vitro. J Neurosci 21:4625–4636PubMedGoogle Scholar
  20. Hashimoto H, Shintani N, Tanaka K et al (2001) Altered psychomotor behaviors in mice lacking pituitary adenylate cyclase activating polypeptide (PACAP). Proc Natl Acad Sci U S A 98:13355–13360PubMedCentralPubMedCrossRefGoogle Scholar
  21. Hashimoto H, Hashimoto R, Shintani N et al (2009) Depression-like behavior in the forced swimming test in PACAP-deficient mice: amelioration by the atypical antipsychotic risperidone. J Neurochem 110:595–602PubMedCrossRefGoogle Scholar
  22. Hirose M, Niewiadomski P, Tse G et al (2011) Pituitary adenylyl cyclase-activating peptide counteracts hedgehog-dependent motor neuron production in mouse embryonic stem cell cultures. J Neurosci Res 89:1363–1374PubMedCentralPubMedCrossRefGoogle Scholar
  23. Ichikawa H, Sugimoto T (2003) Pituitary adenylate cyclase-activating polypeptide-immunoreactive nerve fibers in rat and human tooth pulps. Brain Res 980:288–292PubMedCrossRefGoogle Scholar
  24. Iwamoto T, Yamada A, Arakaki M et al (2011) Expressions and functions of neurotrophic factors in tooth development. J Oral Biosci 53:13–21CrossRefGoogle Scholar
  25. Jernvall J, Thesleff I (2000) Reiterative signaling and patterning during mammalian tooth morphogenesis. Mech Dev 92:19–29PubMedCrossRefGoogle Scholar
  26. Jernvall J, Thesleff I (2012) Tooth shape formation and tooth renewal: evolving with the same signals. Development 139:3487–3497PubMedCrossRefGoogle Scholar
  27. Khonsari RH, Seppala M, Pradel A et al (2013) The buccohypophyseal canal is an ancestral vertebrate trait maintained by modulation in sonic hedgehog signaling. BMC Biol 11:27PubMedCentralPubMedCrossRefGoogle Scholar
  28. Lakshminarayanan R, Fan D, Du C, Moradian-Oldak J (2007) The role of secondary structure in the entropically driven amelogenin self-assembly. Biophys J 93:3664–3674PubMedCentralPubMedCrossRefGoogle Scholar
  29. Lee FS, Rajagopal R, Kim AH, Chang PC, Chao MV (2002) Activation of Trk neurotrophin receptor signaling by pituitary adenylate cyclase activating polypeptides. J Biol Chem 277:9096–9102PubMedCrossRefGoogle Scholar
  30. Lelievre V, Ghiani CA, Seksenyan A, Gressens P, de Vellis J, Waschek JA (2006) Growth factor-dependent actions of PACAP on oligodendrocyte progenitor proliferation. Regul Pept 137:58–66PubMedCrossRefGoogle Scholar
  31. Liu M, Zhao S, Wang XP (2014) YAP overexpression affects tooth morphogenesis and enamel knot patterning. J Dent Res 93:469–474PubMedCrossRefGoogle Scholar
  32. Luukko K, Kettunen P (2014) Coordination of tooth morphogenesis and neuronal development through tissue interactions: lessons from mouse models. Exp Cell Res 325:72–77PubMedCrossRefGoogle Scholar
  33. Luukko K, Moshnyakov M, Sainio K, Saarma M, Sariola H, Thesleff I (1996) Expression of neurotrophin receptors during rat tooth development is developmentally regulated, independent of innervation, and suggests functions in the regulation of morphogenesis and innervation. Dev Dyn 206:87–99PubMedCrossRefGoogle Scholar
  34. Luukko K, Arumae U, Karavanov A et al (1997) Neurotrophin mRNA expression in the developing tooth suggests multiple roles in innervation and organogenesis. Dev Dyn 210:117–129PubMedCrossRefGoogle Scholar
  35. Maasz G, Pirger Z, Reglodi D et al (2014) Comparative protein composition of the brains of PACAP-deficient mice using mass spectrometry-based proteomic analysis. J Mol Neurosci PMID:24643519Google Scholar
  36. Margolis HC, Beniash E, Fowler CE (2006) Role of macromolecular assembly of enamel matrix proteins in enamel formation. J Dent Res 85:775–793PubMedCrossRefGoogle Scholar
  37. Matthaus C, Bird B, Miljkovic M, Chernenko T, Romeo M, Diem M (2008) Infrared and Raman microscopy in cell biology (Chapter 10). Methods Cell Biol 89:275–308PubMedCentralPubMedCrossRefGoogle Scholar
  38. Mitsiadis TA, Luukko K (1995) Neurotrophins in odontogenesis. Int J Dev Biol 39:195–202PubMedGoogle Scholar
  39. Moradian-Oldak J, Du C, Falini G (2006) On the formation of amelogenin microribbons. Eur J Oral Sci 114(Suppl 1):289–296, discussion 327–329, 382PubMedCrossRefGoogle Scholar
  40. Nagatomo KJ, Tompkins KA, Fong H et al (2008) Transgenic overexpression of gremlin results in developmental defects in enamel and dentin in mice. Connect Tissue Res 49:391–400PubMedCentralPubMedCrossRefGoogle Scholar
  41. Nanci A (2008a) Enamel: composition, formation, and structure in Ten Cate’s oral histology: development, structure, and function, 7th edn. Mosby, St. Louis, pp 268–289Google Scholar
  42. Nanci A (2008b) Dentin–pulp complex in Ten Cate’s oral histology: development, structure, and function, 7th edn. Mosby, St. Louis, pp 191–238Google Scholar
  43. Nicot A, Lelievre V, Tam J, Waschek JA, DiCicco-Bloom E (2002) Pituitary adenylate cyclase-activating polypeptide and sonic hedgehog interact to control cerebellar granule precursor cell proliferation. J Neurosci 22:9244–9254PubMedGoogle Scholar
  44. Niewiadomski P, Zhujiang A, Youssef M, Waschek JA (2013) Interaction of PACAP with Sonic hedgehog reveals complex regulation of the hedgehog pathway by PKA. Cell Signal 25:2222–2230PubMedCentralPubMedCrossRefGoogle Scholar
  45. Nonaka S, Kitaura H, Kimura K, Ishida M, Takano-Yamamoto T (2013) Expression of pituitary adenylate cyclase-activating peptide (PACAP) and PAC1 in the periodontal ligament after tooth luxation. Cell Mol Neurobiol 33:885–892PubMedCrossRefGoogle Scholar
  46. Nosrat CA, Fried K, Ebendal T, Olson L (1998) NGF, BDNF, NT3, NT4 and GDNF in tooth development. Eur J Oral Sci 106(Suppl 1):94–99PubMedGoogle Scholar
  47. Nosrat I, Seiger A, Olson L, Nosrat CA (2002) Expression patterns of neurotrophic factor mRNAs in developing human teeth. Cell Tissue Res 310:177–187PubMedCrossRefGoogle Scholar
  48. Otto C, Schutz G, Niehrs C, Glinka A (2000) Dissecting GHRH- and pituitary adenylate cyclase activating polypeptide-mediated signalling in Xenopus. Mech Dev 94:111–116PubMedCrossRefGoogle Scholar
  49. Penel G, Leroy G, Rey C, Sombret B, Huvenne JP, Bres E (1997) Infrared and Raman microspectrometry study of fluor–fluor-hydroxy and hydroxy-apatite powders. J Mater Sci Mater Med 8:271–276PubMedCrossRefGoogle Scholar
  50. Penel G, Leroy G, Rey C, Bres E (1998) MicroRaman spectral study of the PO4 and CO3 vibrational modes in synthetic and biological apatites. Calcif Tissue Int 63:475–481PubMedCrossRefGoogle Scholar
  51. Puceat E, Reynard B, Lecuyer C (2004) Can crystallinity be used to determine the degree of chemical alteration of biogenic apatites? Chem Geol 205:83–97CrossRefGoogle Scholar
  52. Reglodi D, Kiss P, Tamas A, Lengvari I (2003) The effects of PACAP and PACAP antagonist on the neurobehavioral development of newborn rats. Behav Brain Res 140:131–139PubMedCrossRefGoogle Scholar
  53. Reglodi D, Kiss P, Lubics A, Tamas A (2011) Review on the protective effects of PACAP in models of neurodegenerative diseases in vitro and in vivo. Curr Pharm Des 17:962–972PubMedCrossRefGoogle Scholar
  54. Reglodi D, Kiss P, Szabadfi K et al (2012) PACAP is an endogenous protective factor-insights from PACAP-deficient mice. J Mol Neurosci 48:482–492PubMedCrossRefGoogle Scholar
  55. Shen S, Gehlert DR, Collier DA (2013) PACAP and PAC1 receptor in brain development and behavior. Neuropeptides 47:421–430PubMedCrossRefGoogle Scholar
  56. Tajiri M, Hayata-Takano A, Seiriki K et al (2012) Serotonin 5-HT (7) receptor blockade reverses behavioral abnormalities in PACAP-deficient mice and receptor activation promotes neurite extension in primary embryonic hippocampal neurons: therapeutic implications for psychiatric disorders. J Mol Neurosci 48:473–481PubMedCrossRefGoogle Scholar
  57. Tamas A, Szabadfi K, Nemeth A et al (2012) Comparative examination of inner ear in wild type and pituitary adenylate cyclase activating polypeptide (PACAP)-deficient mice. Neurotox Res 21:435–444PubMedCrossRefGoogle Scholar
  58. Thesleff I (2006) The genetic basis of tooth development and dental defects. Am J Med Genet A 140:2530–2535PubMedCrossRefGoogle Scholar
  59. Thesleff I (2014) Current understanding of the process of tooth formation: transfer from the laboratory to the clinic. Aust Dent J 59(Suppl 1):48–54PubMedCrossRefGoogle Scholar
  60. Thomas DB, Fordyce RE, Frew RD, Gordon KC (2007) A rapid, non-destructive method of detecting diagenetic alteration in fossil bone using Raman spectroscopy. J Raman Spectrosc 38:1533–1537CrossRefGoogle Scholar
  61. Thomas DB, McGoverin CM, Fordyce RE, Frew RD, Gordon KC (2011) Raman spectroscopy of fossil bioapatite — a proxy for diagenetic alteration of the oxygen isotope composition. Palaeogeogr Palaeocl 310:62–70CrossRefGoogle Scholar
  62. Tuson M, He M, Anderson KV (2011) Protein kinase A acts at the basal body of the primary cilium to prevent Gli2 activation and ventralization of the mouse neural tube. Development 138:4921–4930Google Scholar
  63. Vaudry D, Gonzalez BJ, Basille M, Fournier A, Vaudry H (1999) Neurotrophic activity of pituitary adenylate cyclase-activating polypeptide on rat cerebellar cortex during development. Proc Natl Acad Sci U S A 96:9415–9420PubMedCentralPubMedCrossRefGoogle Scholar
  64. Vaudry D, Falluel-Morel A, Bourgault S et al (2009) Pituitary adenylate cyclase-activating polypeptide and its receptors: 20 years after the discovery. Pharmacol Rev 61:283–357PubMedCrossRefGoogle Scholar
  65. Vincze A, Reglodi D, Helyes Z, Hashimoto H, Shintani N, Abraham H (2011) Role of endogenous pituitary adenylate cyclase activating polypeptide (PACAP) in myelination of the rodent brain: lessons from PACAP-deficient mice. Int J Dev Neurosci 29:923–935PubMedCrossRefGoogle Scholar
  66. Walker MP, Fricke BA (2006) Dentin–enamel junction of human teeth. In: Akay M (ed) Wiley encyclopedia of biomedical engineering. Wiley, Hoboken, pp 1061–1064Google Scholar
  67. Wang Y, Spencer P, Walker MP (2007) Chemical profile of adhesive/caries-affected dentin interfaces using Raman microspectroscopy. J Biomed Mater Res A 81:279–286PubMedCentralPubMedCrossRefGoogle Scholar
  68. Waschek JA (2002) Multiple actions of pituitary adenylyl cyclase activating peptide in nervous system development and regeneration. Dev Neurosci 24:14–23PubMedCrossRefGoogle Scholar
  69. Watanabe J, Nakamachi T, Matsuno R et al (2007) Localization, characterization and function of pituitary adenylate cyclase-activating polypeptide during brain development. Peptides 28:1713–1719PubMedCrossRefGoogle Scholar
  70. Wojcieszak J, Zawilska JB (2014) PACAP38 and PACAP6-38 exert cytotoxic activity against human retinoblastoma Y79 cells. J Mol Neurosci PMID:24515671Google Scholar
  71. Wopenka B, Pasteris JD (2005) A mineralogical perspective on the apatite in bone. Mater Sci Eng 25:131–143CrossRefGoogle Scholar
  72. Xu C, Yao X, Walker MP, Wang Y (2009) Chemical/molecular structure of the dentin–enamel junction is dependent on the intratooth location. Calcif Tissue Int 84:221–228PubMedCentralPubMedCrossRefGoogle Scholar
  73. Yamada K, Matsuzaki S, Hattori T et al (2010) Increased stathmin1 expression in the dentate gyrus of mice causes abnormal axonal arborizations. PLoS One 5:e8596PubMedCentralPubMedCrossRefGoogle Scholar
  74. Yan Y, Zhou X, Pan Z, Ma J, Waschek JA, DiCicco-Bloom E (2013) Pro- and anti-mitogenic actions of pituitary adenylate cyclase-activating polypeptide in developing cerebral cortex: potential mediation by developmental switch of PAC1 receptor mRNA isoforms. J Neurosci 33:3865–3878PubMedCentralPubMedCrossRefGoogle Scholar
  75. Ye L, MacDougall M, Zhang S et al (2004) Deletion of dentin matrix protein-1 leads to a partial failure of maturation of predentin into dentin, hypomineralization, and expanded cavities of pulp and root canal during postnatal tooth development. J Biol Chem 279:19141–19148PubMedCrossRefGoogle Scholar
  76. Zhang L, Hua F, Yuan GH, Zhang YD, Chen Z (2008) Sonic hedgehog signaling is critical for cytodifferentiation and cusp formation in developing mouse molars. J Mol Histol 39:87–94PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • B. Sandor
    • 1
    • 2
  • K. Fintor
    • 3
  • Sz. Felszeghy
    • 4
    • 5
  • T. Juhasz
    • 4
  • D. Reglodi
    • 2
  • L. Mark
    • 6
  • P. Kiss
    • 2
  • A. Jungling
    • 2
  • B. D. Fulop
    • 2
  • A. D. Nagy
    • 2
  • H. Hashimoto
    • 7
  • R. Zakany
    • 4
  • A. Nagy
    • 1
  • A. Tamas
    • 2
    Email author
  1. 1.Department of Dentistry, Oral and Maxillofacial Surgery, Medical SchoolUniversity of PecsPecsHungary
  2. 2.Department of Anatomy, PTE-MTA “Lendulet” PACAP Research Team, Medical SchoolUniversity of PecsPecsHungary
  3. 3.Department of Mineralogy Geochemistry and Petrology, Faculty of Science and InformaticsUniversity of SzegedSzegedHungary
  4. 4.Department of Anatomy, Histology and Embryology, Faculty of MedicineUniversity of DebrecenDebrecenHungary
  5. 5.Faculty of DentistryUniversity of DebrecenDebrecenHungary
  6. 6.Department of Analytical Biochemistry, Institute of Biochemistry and Medical Chemistry, Imaging Center for Life and Material Sciences, Janos Szentagothai Research Center, Medical SchoolUniversity of PecsPecsHungary
  7. 7.Graduate School of Pharmaceutical SciencesOsaka UniversityOsakaJapan

Personalised recommendations