epithelial mesenchymal interaction in odontogenesis

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1. Epithelial mesenchymal interactionS in odontogenesis

2. What are epithelial mesenchymal interactions ? Epithelial mesenchymal interactions are a series of programmed, sequential and reciprocal (complex and multiphasic) communications between the epithelium and mesenchyme (two heterotypic cell populations ) resulting in the differentiation of one or both the cell populations involved. E-M interactions are the hallmark of odontogenesis .

3. Development of teeth and many other organs such as lungs, hair, kidneys, salivary glands, mammary glands, oral mucosa are characterized by such interactions. Development of tooth in mammals has served as a useful model for the study of Epithelial mesenchymal interactions. B Hemamalini , Issue 4 volume 1, January-February 2014, Epithelial –Mesenchymal Interactions in Tooth Development.

4. The system could be any one or the combination of all of the following :- 1 . Direct cell to cell communication involving the cytoplasmic processes and gap junctions. 2. Matrix vesicles between two cell populations. 3. Ions like K and Ca  

5. 4 . Extracellular matrix (ECM) components like collagen IV, 1 and III, fibronectin, tenascin, E- cadherin and laminin. 5. Molecular diffusion and transfer of information by substances like hormones and growth factors such as bone morphogenetic protein (BMP-2, 4, 6, 7), FGF, EGF and TGF.

6. 6. Autocrine and paracrine regulators 7. Messenger RNA B Arvind, T Jon, the epithelial mesenchymal interactions: insights into physiological and pathological aspects of oral tissues, Oncology reviews 2014, volume 8, pg no 239

7. The relative importance of epithelium and mesenchyme in E-M interactions are noted by many experimental recombinations of other non dental tissues in mice. E-M interactions have been studied using various methods in the recent past which include animal experiments, tissue culturing, immunohistochemistry, in situ hybridization, immunofluorescence, polymerization chain reaction and others.

8. HISTORY Manjunatha BS, Kumar BS, epithelial mesenchymal interactions in odontogenesis part-1, Oral and maxillofacial pathology, vol 9, issue 2, july-dec 2005.

9. 1887(Von Brunn ) Suggested that odontoblasts differentiate only in the presence of inner enamel epithelial cells and thus discovering the interesting phenomenon of epithelial mesenchymal interactions. 1934 (Huggins et al ) Recognized the fundamental role of E-M interactions. 1950(Billingham and Medawar) & 1957 ( Kadoya Y et al ) Evidenced similar interactions in skin and submandibular gland in their studies.

10. Del Moral (2010) Maintained the roles of the epithelium and the mesenchyme in signaling process. Lesot (2012) EMIs are mediated by the extracellular matrix components of the basement membrane, which contains the signaling molecules and enables a strict time and space regulation of cell kinetics, epithelial histogenesis and morphogenesis Angelova Volponi (2013) Epithelial cells derived from adult gingival tissue are capable of responding to tooth-inducing signals from the embryonic tooth mesenchyme and contribute to the development of the tooth.

11. Two hypothetical models have been proposed to explain how these different shapes are determined:- The first is the field model which proposes that the factors responsible for tooth shape reside within the ectomesenchyme in distinct graded and overlapping fields for each tooth family .

12. The clone model proposes that each tooth class is derived from a clone of ectomesenchymal cells programmed by epithelium to produce teeth of a given pattern . In support of this contention, isolated presumptive first molar tissues have been shown to continue development to form three molar teeth in their normal positional sequence.

13. Interactions in odontogenesis occur in two stages 1. The first specifies the dental nature of the mesenchyme (migration of neural crest cells). 2. The second specifies the tooth type and nature of products to be formed by the epithelium and mesenchyme.

14. Animal experimental studies Usually any stimulus (reciprocation and interaction) results in a cytoplasmic response in one group of cell and the same is carried over to the nucleus. Results in mitosis, histodifferentiation & functional changes in other group of cells .

15. Recombinations have been performed in pre migratory neural crest and early oral epithelium along with second arch epithelium and mesenchyme.

16. 1.The enzymatic separation of ectomesenchyme from the overlying dental epithelium prevented tooth formation. Recombination of the separated elements resulted in tooth formation. 2. When the epithelium from a non dental site like skin combined with dental mesenchyme results in tooth formation.

17. 3. Ectomesenchyme from a molar area, when combined with epithelium of an incisor area forms a molar tooth and vice versa. Thus, the shape of a tooth is determined by dental mesenchyme. 4. When a molar mesenchyme combined with diastema (toothless region in the jaw) or a non oral epithelium, molar tooth was formed.

18. 5 Dental mesenchyme from a 10 day old embryo when combined with odontogenic epithelium of 11 day old embryo, fails to form a tooth, if vice versa, tooth formations results. Therefore it is essential for the ectomesenchyme epithelium to be in contact, interactions between the two occurs at an optimum time for a limited duration in odontogenesis . 6 Any change would result in incomplete or no differentiation.

19. 7. If dental papilla cells are grown in a monolayer culture, they loose their cytological characteristics and become primitive mesenchymal cells. When these cells are recombined with freshly dissociated epithelium, tooth germs are formed.

20. Role of ecm molecules in e-m interactions Structural components of ECM include:- Basement membrane components and their degrading enzymes affect cellular behavior in the embryo and involve in regulation of E-M interactions.

21. The ECM molecules exert their effect on the cells through binding to specific matrix receptor on the cell surface. The examined molecules include Syndecan-1, Tenascin, fibronectin, collagen III, laminins, integrins.

22. BM First ECM to appear during embryonic development Several functions including mediation of signals for orderly development of organs Manjunatha BS, Kumar BS, epithelial mesenchymal interactions in odontogenesis part-1, Oral and maxillofacial pathology, vol 9, issue 2, july-dec 2005 .

23. Earliest interaction between cells and ECM molecules Lead to budding of oral epithelium & condensation of neural crest cells around the bud Mesenchymal cells differentiate into odontoblasts Later by influence of IEE cells

24. Using various ECM molecules such as fibronectin, tenascin and syndecan Showed that these molecules may be involved in the cell matrix interaction which control differentiation of dental mesenchymal cells into odontoblasts Studies showed that integrin are involved in E-M interactions

25. Matrix degrading proteases like Matrix metalloproteases and their inhibitors, TIMP Influences E-M interactions and organ development by regulating connective tissue turnover as well as the functioning of signaling molecules.

26. The morphogenesis and cell differentiation changes in which remodeling of ECM plays important role. Remodeling of ECM requires regulation of both degradation and synthesis of various components of the ECM. It has been suggested that MMP’s and other proteases play a role in amelogenesis .

27. Odontoblast start secreting Enamel organ- dental papilla BM is gradually removed, allowing pre ameloblasts to interact directly with odontoblasts By the time enamel secretion begins, BM is completely removed.

28. Role of ecm molecules in e-m interactions Growth factors are an important group of signaling molecules which exert their growth factors locally and are either paracrine or autocrine in nature. Growth factors are grouped into many families TGF-beta EGF FGF IGF PDGF Neurotropin s

29. A study on growth factors in human tooth development using monoclonal antibodies against EGF and IGF-1 receptors showed that these factors act as signaling molecules in the modulation of cell proliferation and differentiation.

30. The expression of TGF 1 was associated with phenotypic properties of the odontoblastic cell lineage . They also suggested a role for TGF 1 in signaling between the epithelial and mesenchymal tissues that lead to the differentiation of odontoblasts . The study showed both paracrine and / or autocrine mode of action for TGF 2 in early (cap/bell stage) human tooth germs.

31. BMP-4 , a member of TGF super family was shown to mediate E-M interactions during early tooth development. Historically, it was the first signaling molecule in developing mammalian tooth germ. BMP-4 expressed in dental epithelium during initiation of tooth development and the expression shifts to the dental mesenchyme. This shift of BMP 4 expression pattern consistent with the known pattern of sequential and reciprocal E-M interactions .

32. The BMPs are homeodimeric proteins originally defined by their ability to induce bone formation in vitro. BMPs 2, 4, and 7 were frequently co distributed and showed marked association with E-M interactions.

33. Ketunen and Thesleff analyzed the expression of three FGFs 4, 8 and 9. They are in dental epithelium during E-M interactions, which regulate tooth morphogenesis. These FGFs act as signaling epithelial signals, mediating inductive interactions between dental epithelium and mesenchyme during several successive stages of tooth formation.

34. Kettunen petal studied the role of other FGFs like FGF 3, FGF 7 & 10. The results suggested that FGF 3 & FGF 10 function as mesenchymal signals regulating epithelial morphogenesis of the tooth.

35. Role of genes in e-m interactions Development of tooth is under strict genetic control which determines the number, position, size and shape of teeth. Presently about 350 genes have been identified that regulate tooth development.

36. Majority of the genes are central regulations of development that are associated with interactions between the cells. Majority of genes are associated with the signaling pathways between cells and tissues of various organs . The pathways include genes encoding the actual signals, their receptors, mediators of signaling pathways and transcription and transcription factors.

37. Genetic analysis of tooth development is done by means of mutations on known genes either by transgenic or knockout technique in mice. Transgenic- a gene is introduced into the mouse by direct pro nuclear injection Knockout technique- the function of a specific gene is inhibited and its importance is analyzed

38. Congenital absence of one or more permanent teeth is the most common developmental anomaly in human dentition Mutations in several genes in humans have been identified as causes of dental defects either in syndromic or non-syndromic cases. Mainly hypodontia, ectodermal dysplasia, cleidocranial dysplasia , reiger syndrome and others are associated with syndromic hypodontia or anodontia.

39. Msx genes First gene demonstrated which is essential for tooth development in mice. The Msx (muscle segment) gene family consists three members in the mammalian genome.

40. In vitro recombination experiments using dental and non dental epithelial and mesenchymal tissues in mouse embryos revealed that Msx genes expressed in tooth are dependent upon tissue interactions. Msx-1 and Msx-2 expression occurs at almost all sites of E-M interactions, including developing tooth germs during mid gestation.

41. The homeobox gene Msx-2 is the first gene found in the enamel knot, enamel septum and navel. Msx-2 and Msx-1 have a complex interactive pattern of expression during tooth development. Msx-1 is predominantly expressed at high levels in mesenchymal cells developing tooth germs whereas Msx 2 is expressed both in mesenchymal and epithelial cells.

42. The expression of Msx 1 and Msx 2 in dental mesenchyme requires the presence of epithelium until early bell stage. After cap stage, tooth becomes Msx 1 independent.

43. The dental epithelium receives Msx-1 dependent signals from the dental mesenchyme for tooth formation. Definitive support for a role of Msx genes in E-M interactions comes from genetic experiments.

44. Mice defects for Msx 1 or Msx 2 results in failure of E-M interactions and dental defects like anodontia or hypodontia and non functional cleft palate occurs. Msx 1 mutant mice die at birth and exhibit cleft of secondary palate, skull abnormalities and arrest in molar and incisor development at bud stage. Reduction in the degree of mesenchymal condensation around the tooth was also found.

45. Dlx Distaless gene Dlx 1 and 2 effects only the skeletal elements derived from the proximal ends of first and second arch but has no effect on tooth development. It was seen that when a mice was compounded with both Dlx 1 & 2 exhibited a selective absence of upper molars.

46. LIM-homeobox ( Lhx ) are a group of gene clusters which are dispersed outside the homeobox gene clusters and they are transcriptional regulators controlling pattern formation. Lhx 6 and 7 localizes during embryogenesis Fgf 8 have a role in regulating the expression of Lhx6 and 7 gene. Expression of lhx6 and 7 is restricted to neural crest ectomesenchyme of oral half of first branchial arch. AS Tucker, P T sharpe , Molecular genetics of tooth morphogenesis and patterning : the right shape in the right place, J Dent Resrch 78(4), 1999. LHX

47. pitx2 Protein coding gene Its epithelial expression prefigures the location of teeth. It is shown to be required for progression beyond epithelial thickening or bud stage

48. wnt signaling pathways The  Wnt signaling pathways  are a group of  signal transduction  pathways made of  proteins  that pass signals into a cell through cell surface receptors. Three Wnt signaling pathways have been characterized: The canonical Wnt pathway The noncanonical planar  cell polarity  pathway Noncanonical Wnt/ calcium  pathway.

49. All three pathways are activated by binding a Wnt-protein  ligand  to a  Frizzled  family  receptor , which passes the biological signal to the  Dishevelled protein  inside the cell. The canonical Wnt pathway leads to regulation of  gene   transcription , and is thought to be negatively regulated in part by the  SPATS1  gene.  The noncanonical planar cell polarity pathway regulates the  cytoskeleton  that is responsible for the shape of the cell.

50. The noncanonical Wnt/calcium pathway regulates  calcium  inside the cell. Wnt signaling pathways use either nearby cell-cell communication ( paracrine ) or same-cell communication ( autocrine ). They are highly evolutionarily  conserved  in animals, which means they are similar across animal species from fruit flies to humans

51. Wnt signaling was first identified for its role in  carcinogenesis , then for its function in  embryonic development . The embryonic processes it controls include  body axis  patterning,  cell fate  specification,  cell proliferation  and  cell migration . These processes are necessary for proper formation of important tissues including bone, heart and muscle.

52. Its role in  embryonic development  was discovered when genetic mutations in Wnt pathway proteins produced abnormal  fruit fly   embryos . Wnt signaling also controls  tissue regeneration  in adult bone marrow, skin and intestine.

53. Pax9 gene A 9 member gene family which plays a key role during embryogenesis. One of the first mesenchymal responding genes which define presumptive odontogenic mesenchyme, before the morphogenetic stage of odontogenesis .

54. Pax-9 knockout mice showed an arrest in molar tooth development at bud stages similar to Msx-1 mutants. The animals showed complete anodontia and secondary cleft palates along with craniofacial bone abnormalities.

55. runx2 Earlier called as Cbfa-1 is a gene encoding a transcription factor for osteoblasts and odontoblasts diffrentiation . Mutations in this gene results in cleidocranial dysplasia, a syndrome affecting bone and tooth development.

56. It is a master gene of bone and tooth development and is needed for osteoblast and odontoblasts differentiation. Syndrome includes hypoplasia of bone, supernumerary tooth and sometimes a third dentition develops, indicating that all humans have the potential to develop a third dentition which is normally inhibited by RUNX2.

57. shh gene Shh expression is observed in dental lamina in incisors but not in molar tooth germs from E-11- 12.5. Exclusively identified at the site of epithelial thickenings and functions as a mitogen. Crucial for odontogenesis to occur.

58. The shh expression correlates and spread along the inner enamel epithelium during bell stage . Expression of these genes in enamel knot, a signaling centre and role in patterning of tooth.

59. Oral mucosa It is assumed that the development and maintaince of oral epithelium similar to that of epidermis. They are controlled by means of E-M interactions . Mtalova et al, Tooth agenesis: from molecular genetics to molecular dentistry, J Dent Res (7), 2008

60. SHH Gsc GLi MSX DLX Lhx Fgf 8 BMP PAX 9 Pitx 2 PRX-1,2 Mfz - 6 Wnt-7 HOX Activin – ß A Lef-1 Pattern formation Role in development during embryogenesis Produce incisor tooth germ Specify the site of tooth initiation Position of prospective tooth mesenchyme Barx - 1 Regulates fgf 8 Marking the sites of dev of teeth Activate Wnt responsive target genes PRX-1,2

61. Mfz - 6 HOX Barx - 1 PRX-1,2 Regulates Bmp -4 & Fgf -8 Regulated by fgf-8 & Bmp -4 Control body plan of an embryo along head tail axis Expressed throughout the oral ectoderm, frizzled in shape

62. conclusion Odontogenesis is a highly co-ordinated and complex process which relies upon cell to cell interactions that results in generation of the tooth. Dental epithelium and mesenchyme can be separated and recombined with tissues of different origins, developmental stages.

63. The developing tooth provides one of the most useful experimental models for the study of induction and patterning mechanisms that are involved during organ morphogenesis. Tooth development is under genetic control , majority of them associated with signaling pathways transmitting interactions between cells and tissues.

64. The complex nature of these interactions deals with numerous growth factors and gene involved. E-M interactions in development of many other organs like salivary glands, lungs, kidneys, mammary glands, hair follicles, limb bud etc , depend upon such interactions for their development and differentiation.

65. Earlier because of experimental limitations little information was available for these interactions prior to the cap stage of tooth development. Presently recombinations have also been done prior to the bud stage of tooth development.

66. It has been clearly evidenced using experiments that ectomesenchyme has the necessary instructions so that the epithelium will give rise to enamel organ and later secretes both enamel and dentin matrices.