Sema3A chemorepellant regulates the timing and patterning of dental nerves during development of incisor tooth germ

Abstract

Semaphorin 3A (Sema3A) axon repellant serves multiple developmental functions. Sema3A mRNAs are expressed in epithelial and mesenchymal components of the developing incisor in a dynamic manner. Here, we investigate the functions of Sema3A during development of incisors using Sema3A-deficient mice. We analyze histomorphogenesis and innervation of mandibular incisors using immunohistochemistry as well as computed tomography and thick tissue confocal imaging. Whereas no apparent disturbances in histomorphogenesis or hard tissue formation of Sema3A −/− incisors were observed, nerve fibers were prematurely seen in the presumptive dental mesenchyme of the bud stage Sema3A −/− tooth germ. Later, nerves were ectopically present in the Sema3A −/− dental papilla mesenchyme during the cap and bell stages, whereas in the Sema3A +/+ mice the first nerve fibers were seen in the pulp after the onset of dental hard tissue formation. However, no apparent topographic differences in innervation pattern or nerve fasciculation were seen inside the pulp between postnatal and adult Sema3A +/+ or Sema3A −/− incisors. In contrast, an abnormally large number of nerves and arborizations were observed in the Sema3A −/− developing dental follicle target field and periodontium and, unlike in the wild-type mice, nerve fibers were abundant in the labial periodontium. Of note, the observed defects appeared to be mostly corrected in the adult incisors. The expressions of Ngf and Gdnf neurotrophins and their receptors were not altered in the Sema3A −/− postnatal incisor or trigeminal ganglion, respectively. Thus, Sema3A is an essential, locally produced chemorepellant, which by creating mesenchymal exclusion areas, regulates the timing and patterning of the dental nerves during the development of incisor tooth germ.

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References

  1. Airaksinen MS, Saarma M (2002) The GDNF family: signalling, biological functions and therapeutic value. Nat Rev Neurosci 3:383–394

    CAS  PubMed  Article  Google Scholar 

  2. Amar S, Karcherdjuricic V, Meyer JM, Ruch JV (1986) The lingual (root analog) and the labial (crown analog) mouse incisor dentin promotes ameloblast differentiation. Arch Anat Microsc Morphol 75:229–239

    Google Scholar 

  3. Becker PM, Tran TS, Delannoy MJ, He C, Shannon JM, McGrath-Morrow S (2011) Semaphorin 3A contributes to distal pulmonary epithelial cell differentiation and lung morphogenesis. PLoS ONE 6:11

    Google Scholar 

  4. Behar O, Golden JA, Mashimo H, Schoen FJ, Fishman MC (1996) Semaphorin III is needed for normal patterning and growth of nerves, bones and heart. Nature 383:525–528

    CAS  PubMed  Article  Google Scholar 

  5. Ben-Zvi A, Sweetat S, Behar O (2013) Elimination of aberrant DRG circuitries in Sema3A mutant mice leads to extensive neuronal deficits. PLoS ONE 8:e70085

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  6. Charoy C, Nawabi H, Reynaud F, Derrington E, Bozon M, Wright K, Falk J, Helmbacher F, Kindbeiter K, Castellani V (2012) gdnf activates midline repulsion by Semaphorin3B via NCAM during commissural axon guidance. Neuron 75:1051–1066

    CAS  PubMed  Article  Google Scholar 

  7. Charron F, Tessier-Lavigne M (2007) The Hedgehog, TGF-beta/BMP and Wnt families of morphogens in axon guidance. Adv Exp Med Biol 621:116–133

    PubMed  Article  Google Scholar 

  8. Cobourne MT, Sharpe PT (2003) Tooth and jaw: molecular mechanisms of patterning in the first branchial arch. Arch Oral Biol 48:1–14

    CAS  PubMed  Article  Google Scholar 

  9. Emmenlauer M, Ronneberger O, Ponti A, Schwarb P, Griffa A, Filippi A, Nitschke R, Driever W, Burkhardt H (2009) XuvTools: free, fast and reliable stitching of large 3D datasets. J Microsc (Oxford) 233:42–60

    CAS  Article  Google Scholar 

  10. Fornaro M, Lee JM, Raimondo S, Nicolino S, Geuna S, Giacobini-Robecchi M (2008) Neuronal intermediate filament expression in rat dorsal root ganglia sensory neurons: an in vivo and in vitro study. Neuroscience 153:1153–1163

    CAS  PubMed  Article  Google Scholar 

  11. Fried K, Lillesaar C, Sime W, Kaukua N, Patarroyo M (2007) Target finding of pain nerve fibers: neural growth mechanisms in the tooth pulp. Physiol Behav 92:40–45

    CAS  PubMed  Article  Google Scholar 

  12. Fujisawa H (2003) Discovery of semaphorin receptors, neuropilin and plexin, and their functions in neural development. J Neurobiol 59:24–33

    Article  Google Scholar 

  13. Fukuda T, Takeda S, Xu R, Ochi H, Sunamura S, Sato T, Shibata S, Yoshida Y, Gu ZR, Kimura A, Ma CS, Xu C, Bando W, Fujita K, Shinomiya K, Hirai T, Asou Y, Enomoto M, Okano H, Okawa A, Itoh H (2013) Sema3A regulates bone-mass accrual through sensory innervations. Nature 497:490–493

    CAS  PubMed  Article  Google Scholar 

  14. Ginty DD, Wickramasinghe SR, Alvania RS, Ramanan N, Wood JN, Mandai K (2008) Serum response factor mediates NGF-dependent target innervation by embryonic DRG sensory neurons. Neuron 58:532–545

    PubMed Central  PubMed  Article  Google Scholar 

  15. Goodman CS, Kolodkin AL, Luo Y, Puschel AW, Raper JA, Comm SN (1999) Unified nomenclature for the semaphorins collapsins. Cell 97:551–552

    Article  Google Scholar 

  16. Harada H, Kettunen P, Jung HS, Mustonen T, Wang YA, Thesleff I (1999) Localization of putative stem cells in dental epithelium and their association with Notch and FGF signaling. J Cell Biol 147:105–120

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  17. Haupt C, Kloos K, Faus-Kessler T, Huber AB (2010) Semaphorin 3A-Neuropilin-1 signaling regulates peripheral axon fasciculation and pathfinding but not developmental cell death patterns. Eur J Neurosci 31:1164–1172

    PubMed  Article  Google Scholar 

  18. Hayashi M, Nakashima T, Taniguchi M, Kodama T, Kumanogoh A, Takayanagi H (2012) Osteoprotection by semaphorin 3A. Nature 485:69–74

    CAS  PubMed  Article  Google Scholar 

  19. Hildebrand C, Fried K, Tuisku F, Johansson CS (1995) Teeth and tooth nerves. Prog Neurobiol 45:165–222

    CAS  PubMed  Article  Google Scholar 

  20. Juuri E, Saito K, Ahtiainen L, Seidel K, Tummers M, Hochedlinger K, Klein OD, Thesleff I, Michon F (2012) Sox2+ stem cells contribute to all epithelial lineages of the tooth via Sfrp5+ Progenitors. Dev Cell 23:317–328

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  21. 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

    CAS  PubMed  Article  Google Scholar 

  22. Kettunen P, Loes S, Furmanek T, Fjeld K, Kvinnsland IH, Behar O, Yagi T, Fujisawa H, Vainio S, Taniguchi M, Luukko K (2005) Coordination of trigeminal axon navigation and patterning with tooth organ formation: epithelial–mesenchymal interactions, and epithelial Wnt4 and Tgf{beta}1 regulate semaphorin 3a expression in the dental mesenchyme. Development 132:323–334

    CAS  PubMed  Article  Google Scholar 

  23. Kettunen P, Spencer-Dene B, Furmanek T, Kvinnsland IH, Dickson C, Thesleff I, Luukko K (2007) Fgfr2b mediated epithelial–mesenchymal interactions coordinate tooth morphogenesis and dental trigeminal axon patterning. Mech Dev 124:868–883

    CAS  PubMed  Article  Google Scholar 

  24. Kitsukawa T, Shimizu M, Sanbo M, Hirata T, Taniguchi M, Bekku Y, Yagi T, Fujisawa H (1997) Neuropilin-semaphorin III/D-mediated chemorepulsive signals play a crucial role in peripheral nerve projection in mice. Neuron 19:995–1005

    CAS  PubMed  Article  Google Scholar 

  25. Klein OD, Lyons DB, Balooch G, Marshall GW, Basson MA, Peterka M, Boran T, Peterkova R, Martin GR (2008) An FGF signaling loop sustains the generation of differentiated progeny from stem cells in mouse incisors. Development 135:377–385

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  26. Kvinnsland IH, Luukko K, Fristad I, Kettunen P, Jackson DL, Fjeld K, von Bartheld CS, Byers MR (2004) Glial cell line-derived neurotrophic factor (GDNF) from adult rat tooth serves a distinct population of large-sized trigeminal neurons. Eur J Neurosci 19:2089–2098

    PubMed  Article  Google Scholar 

  27. Lesot H, Brook AH (2009) Epithelial histogenesis during tooth development. Arch Oral Biol 54:S25–S33

    CAS  PubMed  Article  Google Scholar 

  28. Lillesaar C, Eriksson C, Johansson CS, Fried K, Hildebrand C (1999). Tooth pulp tissue promotes neurite outgrowth from rat trigeminal ganglia in vitro. J Neurocytol 28(8):663–670

    Google Scholar 

  29. Lillesaar C, Fried K (2004) Neurites from trigeminal ganglion explants grown in vitro are repelled or attracted by tooth-related tissues depending on developmental stage. Neuroscience 125:149–161

    CAS  PubMed  Article  Google Scholar 

  30. Loes S, Kettunen P, Kvinnsland IH, Taniguchi M, Fujisawa H, Luukko K (2001) Expression of class 3 semaphorins and neuropilin receptors in the developing mouse tooth. Mech Dev 101:191–194

    CAS  PubMed  Article  Google Scholar 

  31. Loes S, Kettunen P, Kvinnsland H, Luukko K (2002) Mouse rudimentary diastema tooth primordia are devoid of peripheral nerve fibers. Anat Embryol (Berl) 205:187–191

    Article  Google Scholar 

  32. Luukko K (1997) Immunohistochemical localization of nerve fibres during development of embryonic rat molar using peripherin and protein gene product 9.5 antibodies. Arch Oral Biol 42:189–195

    CAS  PubMed  Article  Google 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 206:87–99

    CAS  PubMed  Article  Google Scholar 

  34. Luukko K, Arumae U, Karavanov A, Moshnyakov M, Sainio K, Sariola H, Saarma M, Thesleff I (1997a) Neurotrophin mRNA expression in the developing tooth suggests multiple roles in innervation and organogenesis. Dev Dyn 210:117–129

    CAS  PubMed  Article  Google Scholar 

  35. Luukko K, Suvanto P, Saarma M, Thesleff I (1997b) Expression of GDNF and its receptors in developing tooth is developmentally regulated and suggests multiple roles in innervation and organogenesis. Dev Dyn 210:463–471

    CAS  PubMed  Article  Google Scholar 

  36. Luukko K, Kvinnsland IH, Kettunen P (2005) Tissue interactions in the regulation of axon pathfinding during tooth morphogenesis. Dev Dyn 234:482–488

    CAS  PubMed  Article  Google Scholar 

  37. Luukko K, Moe K, Sijaona A, Furmanek T, Hals Kvinnsland I, Midtbo M, Kettunen P (2008) Secondary induction and the development of tooth nerve supply. Ann Anat 190:178–187

    PubMed  Article  Google Scholar 

  38. Madduri S, Papaloizos M, Gander B (2009) Synergistic effect of GDNF and NGF on axonal branching and elongation in vitro. Neurosci Res 65:88–97

    CAS  PubMed  Article  Google Scholar 

  39. Maeda T, Ochi K, Nakakura-Ohshima K, Youn SH, Wakisaka S (1999) The Ruffini ending as the primary mechanoreceptor in the periodontal ligament: its morphology, cytochemical features, regeneration, and development. Crit Rev Oral Biol Med 10:307–327

    CAS  PubMed  Article  Google Scholar 

  40. Matsuo S, Ichikawa H, Henderson TA, Silos-Santiago I, Barbacid M, Arends JJ, Jacquin MF (2001) trkA modulation of developing somatosensory neurons in oro-facial tissues: tooth pulp fibers are absent in trkA knockout mice. Neuroscience 105:747–760

    CAS  PubMed  Article  Google Scholar 

  41. Mitsiadis TA, Dicou E, Joffre A, Magloire H (1992) Immunohistochemical localization of nerve growth factor (NGF) and NGF receptor (NGF-R) in the developing first molar tooth of the rat. Differentiation 49:47–61

    CAS  PubMed  Article  Google Scholar 

  42. Moe K, Kettunen P, Kvinnsland IH, Luukko K (2008) Development of the pioneer sympathetic innervation into the dental pulp of the mouse mandibular first molar. Arch Oral Biol 53:865–873

    CAS  PubMed  Article  Google Scholar 

  43. Moe K, Shrestha A, Kvinnsland IH, Luukko K, Kettunen P (2011) Developmentally regulated expression of Sema3A chemorepellant in the developing mouse incisor. Acta Odontol Scand 70:184–189

    PubMed  Article  Google Scholar 

  44. Moe K, Sijaona A, Shrestha A, Kettunen P, Taniguchi M, Luukko K (2012) Semaphorin 3A controls timing and patterning of the dental pulp innervation. Differentiation 84:371–379

    CAS  PubMed  Article  Google Scholar 

  45. Mohamed SS, Atkinson ME (1983) A histological study of the innervation of developing mouse teeth. J Anat 136(Pt 4):735–749

    CAS  PubMed Central  PubMed  Google Scholar 

  46. Naftel JP, Qian XB, Bernanke JM (1994) Effects of postnatal anti-nerve growth factor serum exposure on development of apical nerves of the rat molar. Brain Res Dev Brain Res 80:54–62

    CAS  PubMed  Article  Google Scholar 

  47. Naftel JP, Richards LP, Pan M, Bernanke JM (1999) Course and composition of the nerves that supply the mandibular teeth of the rat. Anat Rec 256:433–447

    CAS  PubMed  Article  Google Scholar 

  48. Nakakura-Ohshima K, Maeda T, Sato O, Takano Y (1993) Postnatal development of periodontal innervation in rat incisors: an immunohistochemical study using protein gene product 9.5 antibody. Arch Histol Cytol 56:385–398

    CAS  PubMed  Article  Google Scholar 

  49. Nosrat CA, Fried K, Lindskog S, Olson L (1997) Cellular expression of neurotrophin mRNAs during tooth development. Cell Tissue Res 290:569–580

    CAS  PubMed  Article  Google Scholar 

  50. Obara N, Takeda M (1997) Distribution of the neural cell adhesion molecule (NCAM) during pre- and postnatal development of mouse incisors. Anat Embryol (Berl) 195:193–202

    CAS  Article  Google Scholar 

  51. Padilla F, Couble ML, Coste B, Maingret F, Clerc N, Crest M, Ritter AM, Magloire H, Delmas P (2007) Expression and localization of the Nav1.9 sodium channel in enteric neurons and in trigeminal sensory endings: implication for intestinal reflex function and orofacial pain. Mol Cell Neurosci 35:138–152

    CAS  PubMed  Article  Google Scholar 

  52. Reichardt LF (2006) Neuratrophin-regulated signalling pathways. Philos Trans R Soc Lond B 361:1545–1564

    CAS  Article  Google Scholar 

  53. Sato O, Maeda T, Kobayashi S, Iwanaga T, Fujita T, Takahashi Y (1988) Innervation of periodontal-ligament and dental-pulp in the rat incisor—an immunohistochemical investigation of neurofilament protein and glia-specific S-100 protein. Cell Tissue Res 251:13–21

    CAS  PubMed  Article  Google Scholar 

  54. Serini G, Valdembri D, Zanivan S, Morterra G, Burkhardt C, Caccavari F, Zammataro L, Primo L, Tamagnone L, Logan M, Tesssier-Lavigne M, Taniguchi M, Puschel AW, Bussolino F (2003) Class 3 semaphorins control vascular morphogenesis by inhibiting integrin function. Nature 424:391–397

    CAS  PubMed  Article  Google Scholar 

  55. Sijaona A, Luukko K, Kvinnsland IH, Kettunen P (2011) Expression patterns of Sema3F, PlexinA4, -A3, neuropilin1 and -2 in the postnatal mouse molar suggest roles in tooth innervation and organogenesis. Acta Odontol Scand 70:140–148

    PubMed  Article  Google Scholar 

  56. Thesleff I, Tummers M (2009) Tooth organogenesis and regeneration. Stembook. Harvard Stem Cell Institute, Cambridge

    Google Scholar 

  57. Tran TS, Kolodkin AL, Bharadwaj R (2007) Semaphorin regulation of cellular morphology. Annu Rev Cell Dev Biol 23:263–292

    CAS  PubMed  Article  Google Scholar 

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

    Article  Google Scholar 

  59. Wang XP, Suomalainen M, Jorgez CJ, Matzuk MM, Werner S, Thesleff I (2004) Follistatin regulates enamel patterning in mouse incisors by asymmetrically inhibiting BMP signaling and ameloblast differentiation. Dev Cell 7:719–730

    CAS  PubMed  Article  Google Scholar 

  60. White FA, Behar O (2000) The development and subsequent elimination of aberrant peripheral axon projections in Semaphorin3A null mutant mice. Dev Biol 225:79–86

    CAS  PubMed  Article  Google Scholar 

  61. Yazdani U, Terman JR (2006) The semaphorins. Genome Biol 7:211

    PubMed Central  PubMed  Article  Google Scholar 

  62. Zhang JQ, Nagata K, Iijima T (1998) Scanning electron microscopy and immunohistochemical observations of the vascular nerve plexuses in the dental pulp of rat incisor. Anat Rec 251:214–220

    CAS  PubMed  Article  Google Scholar 

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Acknowledgments

Kjellfrid Haukanes is acknowledged for skilful technical assistance. We thank the staff of the animal facility for careful mouse husbandry. Confocal imaging and computed tomography were performed at the Molecular Imaging Centre (Fuge, Norwegian Research Council), University of Bergen. We thank Cecilie Brekke Rygh for help with the computed tomography. We also thank Stein Atle Lie (Department of Clinical Dentistry, University of Bergen) for help in statistical analysis. This study has been supported by grants from the Norwegian Cancer Society, L. Meltzer’s foundation and Norsk Dental Depot.

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The authors declare no conflict of interest.

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Correspondence to Paivi Kettunen.

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Anjana Shrestha and Kyaw Moe contributed equally to the article.

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Immunohistochemical localization of nerve fibers using peripherin antibody in 100-μm-thick tissue sections of PN7 Sema3A +/+ and Sema3A −/− mandibular incisors. Images shown in Fig 6a, b are shown as a movie (MOV 2938 kb)

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Shrestha, A., Moe, K., Luukko, K. et al. Sema3A chemorepellant regulates the timing and patterning of dental nerves during development of incisor tooth germ. Cell Tissue Res 357, 15–29 (2014). https://doi.org/10.1007/s00441-014-1839-3

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Keywords

  • Axon guidance
  • Incisor
  • Odontogenesis
  • Semaphorin 3A
  • Tooth innervation