Molekula rnõâ podstata vyâ voje zubnõâchzaâ rodkuê Molecular basis of toothgerm development

Transcription

1 ORTODONCIE rocïnõâk16 Molekula rnõâ podstata vyâ voje zubnõâchzaâ rodkuê Molecular basis of toothgerm development *,**RNDr.Jana FleischmannovaÂ, Ph.D., ***MUDr. PrÏemysl KrejcÏ õâ, Ph.D., *,****RNDr. Eva MatalovaÂ, Ph.D., *,****prof.mvdr.ivan MõÂsÏ ek, CSc. *LaboratorÏ embryologie zï ivocï ichuê,uâ stav zï ivocïisïneâ fyziologie a genetiky, Akademie veïdcï R, v.v.i., Brno **Katedra fyziologie zï ivocï ichuê, PrÏõÂrodoveÏ deckaâ fakulta, JihocÏ eskaâ univerzita, CÏ eskeâ BudeÏ jovice ***Klinika zubnõâho leâ karïstvõâ, Le karïskaâ fakulta, Univerzita Palacke ho, Olomouc ****Veterina rnõâ a farmaceutickaâ univerzita Brno *Department of Animal Physiology, Institute of Animal Physiology and Genetics, AS CR, v.v.i., Brno **Department of Animal Physiology, Faculty of Natural Sciences, University of South Bohemia, CÏ eskeâ BudeÏ jovice ***Clinic of Dental Medicine, Medical Faculty, Palacky University, Olomouc ****University of Veterinary and Pharmaceutical Sciences Brno Souhrn Detailnõ znalosti o normaâ lnõâm vyâ voji zubuêjsou nezbytnyâ m prïedpokladem pro porozumeï nõâ zubnõâm defektuê m na molekulaâ rnõâ uâ rovni a pro jejich mozïnou terapii zalozïenou na tkaânï oveâ m inzïenyâ rstvõâ. Tento cïlaâ nek maâ za cõâl shrnout zaâ kladnõâ kroky odontogenetickyâ ch kaskaâ d. PrÏedevsÏ õâm bude diskutovaâ na role Shh (sonic hedgehog), Wnts (wingless-type MMTV integration site family), Fgf (fibroblast growth factors) a Bmp (bone morphogenetic proteins) proteinovyâ ch rodin v jednotlivyâchfaâ zõâch vyâ voje od iniciace po tvorbu tvrdyâ ch tkaâ nõâ zubu (Ortodoncie 2007, 16, cï. 4, s ). Abstract Detailed information about normal tooth development is an essential prerequisite for understanding dental defects at molecular level and their possible tissue engineering based therapy. This review aims to briefly summarise keystones of odontogenic molecular networks. Particularly, involvement of Shh (sonic hedgehog), Wnts (wingless-type MMTV integration site family), Fgfs (fibroblast growth factors) and Bmps (bone morphogenetic proteins) families in different stages of normal tooth development from initiation to hard tissues formation is discussed (Ortodoncie 2007, 16, No. 4, p ). Vy voj zubnõâho zaâ rodku ± morfologickeâ aspekty (Obr. 1) Zuby, podobneï jako dalsï õâ orgaâ ny ektodermaâ lnõâho puê vodu, se vyvõâjejõâ na zaâ kladeï epitelio-mesenchymaâlnõâch interakcõâ [1], iniciace, morfogeneze, mineralizace a erupce zubuê je tedy regulovaâ na vzaâ jemnyâmi homo a heterotypickyâ mi molekulaâ rnõâmi kaskaâ dami mezi stomodeaâ lnõâmektodermem(epitel) vystyâ lajõâcõâmdutinu uâ stnõâ a mezenchymem puê vodemz kraniaâ lnõâ neuraâ lnõâ lisï ty [2, 3]. Vy voj mysï õâ dentice je vhodnyâ mmodelempro studiumobecnyâ ch molekulaâ rnõâch mechanismuê vyâ voje Tooth germ development ± morphological aspects (Fig. 1) Teeth, like other organs of ectodermal origin, develop on basis of epithelio-mesenchymal interactions [1], indeed sequential and reciprocal homo and heterotypical molecular cascades between the stomodeal ectoderm(epithelium) that lines the inside of the oral cavity, and cranial neural crest-derived mesenchyme regulate tooth initiation, morphogenesis, mineralisation and eruption [2, 3]. Mouse tooth development has proved to be a powerful model to study the molecular mechanisms of redakce@orthodont-cz.cz 39

2 rocïnõâk16 ORTODONCIE Obra zek 1: VyÂvoj zubnõâho zaâ kladu. ED 12,5 epiteliaâ lnõâ ztlusïteï nõâ, ED 13,5 zubnõâ pupen, ED 14,5 zubnõâ pohaâ rek, ED 15,5 zubnõâ zvonek, ED 18,5 stadium histodiferenciace, ED 19,5 mineralizace. Figure 1: Tooth germ development. ED 12.5 epithelial thickening, ED 13.5 tooth bud, ED 14.5 tooth cap, ED 15.5 tooth cap, ED 18.5 tooth bell, ED 18.5 histodifferentiation stage, ED 19.5 mineralisation. savcï õâch zubuê, i kdyzï semysï õâ chrup znacïneï lisï õâ od lidskeâ ho. U mysï õâ se vyvõâjejõâ pouze dva odlisïneâ typy zubuê, trïi molaâ ry a jeden rïezaâ k v kazïdeâ mkvadrantu, a majõâ pouze jednu generaci zubuê, na rozdõâl od cï loveï ka, kteryâ maâ dveï generace zubuê, jednu docï asnou (docï asneâ zuby) a jednu staâ lou. Z hlediska morfologie zacï õânaâ vyâ voj zubuê jako ztlusïteï nõâ epitelu (11. embryonaâ lnõâ den- ED 11 u mysïi,6.tyâden embryonaâ lnõâho vyâvoje u cï loveï ka) zpuê sobeneâ zmeïnou orientace deï lõâcõâho vrïeteâ nka epiteliaâ lnõâch buneï k. Na sledneï se na zaâ kladeï koordinovaneâ interakce mezi epiteliaâ lnõâmi a mezenchymaâ lnõâmi bunï kami vytvaârïõâ zubnõâ pupen (ED 12): BunÏ ky epiteliaâ lnõâho ztlusï teï nõâ proliferujõâ a vnorïujõâ se hloubeï ji do okolnõâho mezenchymu, kteryâ na druheâ straneï kondenzuje kolemzubnõâho epitelu. BeÏ hemdalsï õâho vyâ voje zubnõâ epitel vruê staâ daâ le do mezenchymu, obklopuje kondenzujõâcõâ mezenchym a vytvaârïõâ tak zubnõâ pohaâ rek (ED 14) a pozdeï ji zubnõâ zvonek (ED 16). Proces tvorby zubnõâho pohaâ rku je kontrolovaâ n koncentricky usporïaâ danyâ mshlukembuneï k na vrcholu zubnõâho pupene, ktereâ se samy nedeï lõâ, takzvanyâmsklovinnyâmuzlem. U zubuê s võâce hrboly (mysï õâa lidskeâ molaâ ry, lidskeâ premolaâ ry) se tvar zubnõâho epitelu nadaâ le komplikuje a vznikajõâ epiteliaâ lnõâ invaginace, zubnõâ hrboly. Tento proces je kontrolovaâ n sekundaâ r- nõâmi sklovinnyâmi uzly vznikajõâcõâmi na vrcholu kazïdeâho budoucõâho hrbolu. Jakmile je urcï eno zaâ kladnõâ rozlozïenõâhrboluê, dochaâzõâ k diferenciaci buneï k produkujõâcõâch tvrdou zubnõâ hmotu. Mezenchyma lnõâ bunï ky prïileâ hajõâcõâ k vnitrïnõâmu mammalian tooth development. Mouse dentition, however, differs significantly fromhuman dentition. Mice only develop two different tooth types, three molars proximately and one incisor distally on each side on the jaws and have one set of teeth while humans have two, one deciduous (milk teeth) and one permanent. The first morphological sign of mammalian tooth development is a thickening of the oral epithelium (embryonic day ED 11 in the mouse, embryonic week 6 in the man) caused by a shift in orientation of mitotic spindles in epithelial cells. Subsequently (ED 12), tooth bud is formed by coordinated interaction between epithelial and mesenchymal cells: Cells of the epithelial thickening proliferate and invaginate further into the mesenchyme that on the other hand condenses around the epithelium. Later, the epithelium extends farther into the mesenchyme, and wraps around the condensing mesenchyme forming a tooth cap (ED 14), and later a bell (ED 16). The process of tooth cap morphogenesis is controlled by a signalling centre called the enamel knot, a concentrically arranged cluster of non-dividing cells at the tip of the late bud. Moreover, in multicuspid teeth (mouse and human molars, human premolars) the shape of the bell stage tooth germ complicates and gives rise to several epithelial invaginations, cusps. This process seems to be controlled by secondary enamel knots each at the future cusp tip. After the basic cusp pattern has been formed, differentiation of dental hard tissue producing cells proceeds. Mesenchymal cells closest to the dental epitheliumdifferentiate into odontoblasts and start to se redakce@orthodont-cz.cz

3 ORTODONCIE rocïnõâk16 sklovinneâ mu epitelu diferencujõâ v odontoblasty a zacï õânajõâ vylucï ovat organickou matrix, kteraâ slouzï õâ jako lesï enõâ pro uklaâ daâ nõâ anorganickeâ slozï ky dentinu, hydroxyapatitovyâ ch krystaluê. BezprostrÏedneÏ po pocï aâ tecï nõâ mineralizaci pre-dentinu diferencujõâ prïilehleâ epiteliaâ lnõâ bunï ky v ameloblasty produkujõâcõâ organickou matrix skloviny. Po ukoncï enõâ depozice sklovinneâ hmoty umõâraâ asi cï tvrtina ameloblastuê apoptoâ zou a zbyleâ bunï ky regulujõâ maturaci skloviny ve vysoce mineralizovanou tkaâ nï teâ m eï rï bez organickyâ ch slozï ek. Ameloblasty spolu s ostatnõâmi epiteliaâ lnõâmi bunï kami pokryâ vajõâ sklovinu azï do okamzïiku erupce. Histomorfogenezi v korunkoveâ oblasti naâ sleduje histomorfogeneze v oblasti korïene. BeÏ hemvyâ voje korïene dochaâ zõâ k apikaâ lnõâmu prodluzï ovaâ nõâ zubnõâho epitelu, vytvaârïõâ se takzvanaâ Hertwigova epitelovaâ pochva, dvouvrstva epiteliaâ lnõâch buneï k, kteraâ indukuje diferenciaci buneï k zubnõâ papily v odontoblasty. Po vytvorïenõâ korïenoveâ ho dentinu se Hertwigova pochva rozpadaâ, bunï ky vnitrïnõâ vrstvy dentaâ lnõâho vaku (mesenchymaâ lnõâ bunï ky obklopujõâcõâ zubnõâ zaâ klad) se dostaâ vajõâ do kontaktu s korïenovyâmdentinema diferencujõâ v cementoblasty. BunÏ ky vneï jsï õâ vrstvy zubnõâho vaku diferencujõâ ve fibroblasty a osteoblasty, ktereâ se spolu s cementoblasty uâ cï astnõâ vyâ voje periodoncia. Take periodontaâ lnõâ vlaâ kna, cement a vlastnõâ alveolaâ rnõâ kost jsou zcïaâ sti odvozeny ze zubnõâho vaku, kteryâ m aâ rozhodujõâcõâ vyâ znam pro erupci zubu [4]. VyÂvoj zubnõâho zaâ rodku - molekulaâ rnõâ aspekty Iniciace: Za kladnõâ zubnõâ vzorec (typ a pozice zubuê v cï elisti) je urcï en v cï asneâ faâ zi vyâ voje, jesï teï prïedtõâm nezï jsou zubnõâ zaâ rodky morfologicky patrneâ. Podle homeoboxoveâ teorie jsou oblasti vyâ voje molaâ ruê a rïezaâ kuê urcï eny signaâ ly z oraâ lnõâho epitelu. Na modelu dolnõâ cïelisti mysï i bylo popsaâ no, zï e rozdõâly v expresi Fgf8/Fgf9 a Bmp4 urcï ujõâ oblast vyâvoje stolicïek a rïezaâkuê. Fgf8 a Fgf9 jsou exprimovaâ ny lateraâ lneï v oblasti prïedpoklaâdaneâ ho vyâ voje molaâ ruê, zatõâmco Bmp4 je exprimovaâ n mediaâ lneï v oblasti budoucõâch rïezaâ kuê. Fgf8 indukuje v mezenchymu molaâ roveâ oblasti expresi Barx1 (BarH like homeobox gene 1) a Dlx2 (distal-less homeobox 2), Bmp4 indukuje expresi Msx1/2 (muscle-segment homeobox 1/2) v oblasti rïezaâ kuê a zaâ rovenï ve stejneâ oblasti inhibuje expresi Barx1 [5, 6]. Pokud je Bmp signalizace zablokovaâna puê sobenõâmantagonisty Noggin, je Barx1 exprimovaânivrïezaâ koveâ m mezenchymu a jeho puê sobenõâmdochaâ zõâ i zde k tvorbeï molaâ ruê [6]. Velikost a pocï et zubuê jednotlivyâ ch typuê je pravdeïpodobneï urcï en velmi zaâ hy velikostõâ oblasti cï elisti, ve ktereâ se pozdeï ji urcï ityâ typ zubuê vyvõâjõâ. Na molekulaâ rnõâ uâ rovni se v tomto procesu uplatnï uje ectodysplasin (Eda) protein z TNF (tumour necrosis factor) rodiny. Jak umeï laâ stimulace tak utlumenõâ Eda signaâ lnõâ draâ hy crete organic dentin matrix that serves as a scaffold for deposition of anorganic dentin compounds, hydroxyapatite crystals. Immediately after initial mineralisation of pre-dentin, the adjacent layer of epithelial cells differentiates into ameloblasts. Ameloblasts produce organic enamel matrix that then matures into highly mineralised tissue with almost no organic compound. After enamel matrix is deposited, about 25 % of ameloblasts die by apoptosis and remaining cells regulate enamel maturation. Ameloblasts together with other epithelial cells cover the enamel until eruption. Histomorphogenesis in the crown region is followed by histomorphogenesis in the root region. During root development, the odontogenic epithelium extends apically to formthe Hertwig's epithelial root sheath, a bilayer of epithelial cells that induces differentiation of dental papilla cells into odontoblasts. Once root dentin is formed, Hertwig's root sheath loses continuity and the cells of the inner layer of the dental follicle (mesenchymal cells surrounding the tooth germ) come in contact with the root dentin and differentiate into cementoblasts. Cells of the outer layer of the dental follicle differentiate into fibroblasts and osteoblasts that together with cementoblasts contribute to periodontium formation. The periodontal ligament, cementum, and alveolar bone proper are also partially derivated fromthe dental follicle that plays a decisive role in tooth eruption [4]. Tooth germ development - molecular aspects Initiation: The basic pattern of dentition (type and position of teeth in the jaw) is established early in mammalian development before any morphologically apparent sign of tooth development. According to the homeobox code theory the molar and incisor fields are established by signals from the oral epithelium. In the mouse lower jaw, different expressions of Fgf8/Fgf9 and Bmp4 determines the incisor and molar field. Fgf8 and Fgf9 are expressed laterally overlying the presumptive molar field; whereas, Bmp4 is expressed medially overlying the presumptive incisor field. In the molar field mesenchyme, Fgf8 positively regulates the expression of Barx1 (BarH like homeobox gene 1) and Dlx2 (distal-less homeobox 2), whereas Bmp4 positively regulates the expression of Msx1 and Msx2 (muscle-segment homeobox 1/2), and at the same time negatively regulates the expression of Barx1 in the incisor field [5, 6]. Inhibition of Bmp signalling by its antagonist Noggin leads to ectopic expression of Barx1 in the presumptive incisor mesenchyme and transformation of the tooth type fromincisors to molars [6]. The size of the tooth and the resultant number of teeth seems to be determined early in development by the size of the tooth field regulated probably by TNF (tumour necrosis factor) protein family member Ectodys- redakce@orthodont-cz.cz 41

4 rocïnõâk16 ORTODONCIE Obra zek 2: Schematicke znaâ zorneï nõâ vyâvoje zubuê.vyâvoj zubuê probõâhaâ na zaâ kladeï interakcõâ mezi epitelem a mezenchymem zprostrïedkovanyâch signaâ lnõâmi kaskaâ dami, ktereâ jsou opakovaneï vyuzïõâvaâ ny v ruê znyâch faâ zõâch vyâvoje. Figure 2: Schematic representation of the tooth development. Tooth development is based on epithelio-mesenchymal interactions repetitively exploiting the same reciprocal molecular cascades in different developmental stages. vede ke zmeï neï velikosti a pocï tu zubuê [7]. Pro velmi raneâ urcï enõâ velikosti i pocï tu zubuê sveïdcï õâ pokusy provedeneâ na Eda deficientnõâch mysï õâch, overexprese Eda v raneâ mstadiu vyâvoje vede ke kompenzaci pocï etnõâch defektuê zubuê, naopak nemaâ zïaâ dnyâ efekt, pokud je Eda overexprimovanyâ pozdeï ji, kdy uzï jsou formovaâ ny jednotliveâ zubnõâ zaâ rodky [8, 9]. Morfogeneze (Obr. 2): PoteÂ, co je urcïen zaâ kladnõâ zubnõâ vzorec, dochaâ zõâ v epitelu dutiny uâ stnõâ k prïesunu Fgf8 exprese do oblastõâ budoucõâch zubuê a Bmp4 do oblastõâ epitelu, kde k vyâ voji zubuê nedochaâ zõâ. Fgf8 indukuje v prïilehleâ m mezenchymu expresi genuê duê lezï ityâch pro dalsïõâvyâvoj zubuê (Pax9 - paired box gene 9). Bmp4 naopak puê sobõâ jako inhibitor vyâ voje zubuê a blokuje expresi Pax9 v oblastech, kde zuby vznikat nemajõâ [10]. V zubnõâmepitelu je exprimovaâ n Shh, kteryâ indukuje proliferaci epiteliaâ lnõâch buneï k a ruê st zubnõâho pupene [11]. Na rozdõâl od iniciace, kdy je vyâvoj zubuê rïõâzen epiteliaâ lnõâmi signaâ ly, ve stadiu zubnõâho pupene dochaâ zõâ kprïesunu odontogenetickeâ ho potenciaâ lu z epitelu do okolnõâho mezenchymu (ED 11,5) spolu s prïesunem exprese Bmp4 do mezenchymu. Bmp4 zpeï tneï puê sobõâ na epitel zubnõâho zaâ kladu a indukuje na vrcholu zubnõâho pupene expresi p21 a Msx2, kteraâ je spojena se zastavenõâmbuneï cï neâ ho cyklu a diferenciacõâ primaâ rnõâho sklovinneâ ho uzlu [12]. BunÏ ky sklovinneâ ho uzlu exprimujõâ celou sïkaâ lu signaâ lnõâch molekul, ktereâ jsou zodpoveï dneâ jednak za udrzï enõâ funkce sklovinneâ ho uzlu, proliferaci okolnõâch epiteliaâ lnõâch i mezenchymaâ lnõâch buneï k, morfogenezi plasin (Eda). Both up- and down-regulations in Eda signalling pathway lead to changes in tooth size and number [7] that seems to originate in a very early stage of tooth development. In Eda mutant mice, overexpression of Eda early in development can rescue the tooth number defect; whereas it has no effect when overexpressed after the tooth germs have started to form [8, 9]. Morphogenesis (Fig. 2): Once the basic dentition pattern has been established, the expression of Fgf8 in the epitheliumshifts to the sides of future tooth germs; whereas, Bmp4 is expressed in a mirror pattern. Fgf8 induces expression of odontogenic genes like Pax9 (paired box gene 9) in the adjacent dental mesenchyme. On the other hand, Bmp4 inhibits Pax9 expression [10]. Therefore, Bmp4 acts as an inhibitor of tooth development at presumptive toothless sides. Shh expressed in the dental epitheliuminduces it's proliferation and bud formation [11]. Contrary to the initiation stage when the epithelium directs tooth development, the odontogenic potential shifts from the epithelium to the mesenchyme at the bud stage (ED 11.5), coinciding with a shift of Bmp4 expression to the dental mesenchyme. Bmp4 induces the expression of p21 and Msx2 in the underlying epitheliumat the tip of the tooth bud, which is associated with cell cycle arrest and differentiation of the primary enamel knot signalling centre [12]. The enamel knot cells express in nested patterns several signalling molecules that are responsible for the maintenance of the enamel knot, proliferation of surrounding epithelial and mesenchymal cells, morphoge redakce@orthodont-cz.cz

5 ORTODONCIE rocïnõâk16 zubnõâho pohaâ rku i konecï neâ odstraneï nõâ sklovinneâ ho uzlu apoptoâ zou. RuÊ stoveâ faktory (Fgf4, Shh) exprimovaneâ bunï kami sklovinneâ ho uzlu stimulujõâ proliferaci ve sveâ mokolõâ, ovsï emne v primaâ rnõâmsklovinneâ muzlu sameâ m, kde pro neï chybeï jõâ receptory [13]. Vy slednaâ nerovnomeï rnaâ proliferacï nõâ aktivita v jednotlivyâch cï aâ stech zubnõâho zaâ kladu je hlavnõâmprincipemmorfogeneze zubnõâho pohaâ rku [14]. Spolu s molekulami indukujõâcõâmi proliferaci, produkuje sklovinnyâ uzel takeâ proapoptotickeâ molekuly (Bmp), ktereâ spousïteï jõâ jeho apoptotickou eliminaci poteâ, co vykonal svou funkci [15]. PrÏed prïedcï asnou apoptoâ zou je sklovinnyâ uzel zrïejmeï chraâneï n puê sobenõâmeda signaâ lnõâ draâ hy [16]. DalsÏ õâ tvarovaâ nõâ zubnõâho epitelu a tvorba hrboluê jsou regulovaâ ny sekundaâ rnõâmi sklovinnyâ mi uzly produkujõâcõâmi Slit1 a Fgf4. Slit1 inhibuje proliferaci uvnitrï sekundaâ rnõâch sklovinnyâ ch uzluê a Fgf4 indukuje proliferaci okolnõâch buneï k [17], cozï vede k vzniku dalsï õâch epiteliaâ lnõâch invaginacõâ. Po ukoncï enõâ sveâ funkce jsou sekundaâ rnõâ sklovinneâ uzly odstraneï ny apoptoâ zou [18]. PrÏi vyâvoji hrboluê se patrneï uplatnï uje EdaR protein. Mutace nesis of the tooth cap and terminal self-elimination by apoptosis. Growth factors (Fgfs, Shh) expressed by enamel knot cells stimulate proliferation in adjacent cell compartments but not in the enamel knot itself since it expresses no Fgf receptors [13]. Resulting unequal proliferation of different subpopulations of the tooth germis the main principle of the bud - cap morphogenesis [14]. Additionally to molecules inducing proliferation, enamel knot cells produce also proapoptotic proteins such as Bmp4 that control removal of enamel knot cells after their function has been already terminated [15]. Eda- EdaR signalling pathway is believed to protect enamel knot cells frompremature apoptosis [16]. Further involutions of the dental epitheliumand formation of cusps seems to be regulated by secondary enamel knots secreting Slit1 and Fgf4. Slit1 inhibits cell proliferation within the secondary enamel knot, whereas Fgf4 promotes proliferation of the adjacent dental epithelium and mesenchyme [17], leading to further epithelial invaginations. Secondary enamel knots are eliminated by apoptosis after the initiation of the cusp Obra zek 3: Schematicke znaâ zorneï nõâ morfologickyâch a molekulaâ rnõâch aspektuê procesu mineralizace. Dentinogeneze a amelogeneze zacï õânajõâ ve stadiu zubnõâho zvonku na zaâ kladeï reciprokyâch interakcõâ mezi bunï kami vnitrïnõâho sklovinneâ ho epitelu, sousednõâmi mezenchymaâ lnõâmi bunï kami a stratum intermedium. Vrstva mezenchymaâ lnõâch buneï k v kontaktu s bazaâ lnõâ membraâ nou diferencuje v odontoblasty produkujõâcõâ dentin, bunï ky vnitrïnõâho sklovinneâ ho epitelu daâ vajõâ vznik ameloblastuê m produkujõâcõâm sklovinu. Figure 3: Schematic representation of morphological and molecular aspects of the mineralisation proces. Dentinogenesis and amelogenesis starts at the bell stage by reciprocal interaction between inner enamel epithelium, adjacent mesenchymal cells and stratum intermedium. The layer of mesenchymal cells facing the basement membrane differentiates into dentin producing odontoblasts, inner enamel epithelium cells develop into ameloblasts producing enamel. redakce@orthodont-cz.cz 43

6 rocïnõâk16 ORTODONCIE v EdaR genu u mysï i vede ke vzniku stolicï ek s mensïõâm pocï temmeï lkyâ ch hrboluê, kdezï to konstitutivnõâ aktivace tohoto receptoru vede ke vzniku mnoha ostryâ ch hrboluê [9]. Tvorba tvrdyâ ch tkaâ nõâ zubu (Obr.3): Unika tnõâ strukturnõâ a funkcï nõâ vlastnosti zubu jsou prïedevsï õâm vyâ sledkem molekulaâ rnõâ regulace mineralizace dentinu a skloviny. Diferenciace odontoblastuê a ameloblastuê produkujõâcõâch tvrdeâ tkaâneï zubuê vychaâ zõâ ze vzaâ jemnyâch interakcõâ mezi stratum intermedium (neï kolik vrstev malyâ ch epiteliaâ lnõâch buneï k prïileâ hajõâcõâch k vnitrïnõâmu sklovinneâ mu epitelu), vnitrïnõâm sklovinnyâ m epitelem (budoucõâ ameloblasty) a mezenchymaâ lnõâmi bunï kami sousedõâcõâmi s vnitrïnõâm sklovinnyâ m epitelem (budoucõâ odontoblasty). Diferenciace odontoblastuê je pravdeïpodobneï regulovaâ na TGFOà (tumour growth factor Oà ) proteinovou super-rodinou, zvlaâsïteï TGFOà 3 produkovanyâ m pre-ameloblasty. PodobneÏ je terminaâ lnõâ diferenciace ameloblastuê kontrolovaâ na Bmp2 a TGFOà 1 produkovanyâ mi odontoblasty [20]. KromeÏ toho se diferenciace ameloblastuê uâcï astnõâ zrïejmeï i stratum intermedium, Msx2 deficientnõâ mysï i totizï trpõâ vaâ zï nyâ m posï kozenõâm skloviny zpuê sobenyâ m nedostatecï neï vyvinutyâm stratum intermedium [21]. Runx2 (runt related transcription factor 2) je potrïebnyâ pro zastavenõâ buneï cï neâ ho cyklu beï hemdiferenciace pre-odontoblastuê [22]. Diferencovane odontoblasty pak sekretujõâ komplexnõâ koktejl sekretovanyâch strukturnõâch proteinuê tvorïõâcõâch organickou matrix dentinu. Hlavnõ strukturnõâ slozï kou dentinoveâ matrix je kolagen typu I. Spolu s nõâmjsou za spraâ vnou mineralizaci dentinu zodpoveï dneâ takeâ nekolagennõâ proteiny, jednaâ se prïedevsïõâmo cï leny rodiny sibling (Small Integrin- Binding Ligand, N-linked Glycoprotein) proteinuê (osteopontin, bone sialoprotein, dentin matrix protein 1, dentin sialophosphoprotein a matrix extracellular phosphoglycoprotein), matrix metalloproteinaâ zy, ktereâ sï teï põâ a aktivujõâ sibling proteiny, a maleâ na leucin bohateâ proteoglykany. Mutace v Dspp (dentin sialophosphoprotein) genu zpuê sobujõâ vrozeneâ vady dentinu oznacï o- vaneâ jako dentinogenesis imperfecta nebo dentinovaâ dysplaâ zie [23]. Proces amelogeneze vytvaârïõâ nejvõâce mineralizovanou tkaânï celeâhoteï la. V prvnõâfaâ zi tvorby skloviny (sekretorickaâ faâ ze) produkujõâ ameloblasty organickou matrix a ionty nezbytneâ pro pocï aâ tecï nõâ uklaâ daâ nõâ hydroxyapatitovyâ ch krystaluê. KromeÏ toho vylucï ujõâ ameloblasty enzymy pro naâ sledneâ sï teï penõâ komponent organickeâ matrix a transportujõâ neï ktereâ slozï ky organickeâ matrix zpeï t mimo sklovinu (maturacï nõâ faâ ze) cozï je duê lezï iteâ pro dosazï enõâ vysokeâ ho stupneï mineralizace zraleâ skloviny. KlõÂcÏ oveâ proteiny sklovinneâ matrix jsou amelogeniny (Amel), enameliny (Enam), tuftelliny a ameloblastiny (Ambn). Ameloblastiny inhibujõâ proliferaci amelogrowth [18]. Cusp formation probably engages the EdaR protein. Mutation in EdaR gene leads to fewer shallow cusps, whereas its constitutive activation leads to molars with many sharp cusps [9]. Dental hard tissue formation (Fig.3): The unique structural and functional features of dentition are particularly a consequence of molecular regulation of dentin and enamel mineralisation. Differentiation of dental hard tissue producing odontoblasts and ameloblasts is directed by reciprocal interaction between stratum intermedium (small epithelial cells adjacent to the inner enamel epithelium), inner enamel epithelium (future ameloblasts) and the mesenchymal cells directly facing inner enamel epithelium (future odontoblasts). Differentiation of odontoblasts seems to be regulated by the TGFOà (tumour growth factor Oà ) super-family particularly TGFOà 3 produced by pre-ameloblasts. Reciprocally, terminal differentiation of ameloblasts is likely to be controlled by the dental Bmp2 and TGFOà 1 produced by odontoblasts [20]. Moreover, stratum intermedium seems to be involved in ameloblast differentiation as seen in Msx2 knockout mice that bear profound enamel defects resulting from poorly developed stratum intermedium [21]. Runx2 (runt related transcription factor 2) is necessary for differentiating pre-odontoblasts to undergo growth arrest [22]. Differentiated odontoblasts produce a complex cocktail of secreted structural proteins forming organic dentin matrix. Major structural compound of dentin organic matrix is type I collagen. However, non-collagenous dentin matrix components also contribute to proper mineralisation, these are mainly Sibling (Small Integrin-Binding Ligand, N-linked Glycoprotein) protein family members (osteopontin, bone sialoprotein, dentin matrix protein 1, dentin sialophosphoprotein, and matrix extracellular phosphoglycoprotein); matrix metalloproteinases that have the ability to bind and activate Sibling proteins; and small leucine-rich proteoglycans. Mutations in Dspp (dentin sialophosphoprotein) gene cause developmental dentin defects known as dentinogenesis imperfecta or dentin dysplasia [23]. Amelogenesis produces the most mineralised tissue in the body. In the first stage of enamel formation (secretory phase) ameloblasts provide organic matrix and ions necessary for initial hydroxyapatite crystal deposition. Moreover, ameloblasts secrete enzymes for subsequent digestion of organic matrix and transport some compounds of the organic matrix out of the enamel (maturation phase) that is necessary to achieve the high mineral density of mature enamel. Key enamel organic matrix molecules are amelogenins (Amel), enamelins (Enam), tuftellins and ameloblastins (Ambn). Ameloblastins inhibit proliferation of amelo redakce@orthodont-cz.cz

7 ORTODONCIE rocïnõâk16 blastuê a udrzïujõâ je v diferencovaneâ mstavu. Enameliny se uplatnï ujõâ v iniciacï nõâmstadiu amelogeneze prïi iniciaci tvorby hydroxyapatitovyâ ch krystaluê, amelogeniny jsou pak duê lezï iteâ pro ruê st krystaluê beï hemsekretorickeâ faâ ze. Amelogeniny a ameloblastiny jsou odstraneïny beï hem maturace, zatõâmco enameliny a tufteliny zuê staâ vajõâ soucï aâ stõâ skloviny. Enzymy sï teï põâcõâ komponenty proteinoveâ matrix skloviny jsou prïedevsï õâmenamelysin, matrix metaloproteinaâ za (koâ dovanaâ genem Mmp20), kteraâ je nejvõâce vylucï ovaâna beï hemsekrecï nõâ faâ ze vyâ voje skloviny a regulovaneï upravuje amelogenin [24], a kallikrein-4 serinovaâ proteaâ za (koâ dovanaâ genemklk4) vylucï ovanaâ hlavneï beï hemmaturacï nõâ faâze [25]. Defekty jak strukturnõâch molekul, tak proteinaâz sï teï põâcõâch sklovinnou matrix vedou k ruê znyâ mtypuê mposï kozenõâ skloviny souhrnneï oznacï ovanyâ mjako amelogenesis imperfecta. ZatõÂm byly u teï chto pacientuê identifikovaâ ny mutace ve cï tyrïech genech podõâlejõâcõâch se na vyâvoji skloviny (Amel, Enam, Klk4, Mmp20) [26]. DalsÏ õâ perspektivy Za kladnõâ principy vyâ voje zubuê jsou jizï v soucï asneâ dobeï znaâ my a mohou tedy prïispeï t k lepsï õâmu porozumeï nõâ zubnõâmdefektuê ma jejich diagnostice, prïõâpadneï kuâcï ineïjsï õâ terapii zalozïeneâ na vyuzï itõâ tkaânï oveâ ho inzïenyâ rstvõâ. VyuzÏ itõâ soucï asnyâ ch poznatkuê o molekulaâ rnõâ podstateï vyâ voje zubuê pro lepsï õâ pochopenõâ zubnõâch defektuê jako je hypodoncie bylo diskutovaâ no v nasï em prïedchozõâmcïlaâ nku [27]. I kdyzï jesïteï zbyâvaâ zodpoveïdeï t mnoho otaâzekaprïekonat mnoho obtõâzï õâ, otevõârajõâ soucï asneâ poznatky o molekulaâ rnõâ podstateï vyâ voje zubuê a morfogeneze zubnõâch zaâ rodkuê noveâ m ozï nosti dokonce azï k produkci zubuê ªde novoª za pouzïitõâ kmenovyâch buneï k zõâskanyâch jak ze zubuê samyâch, tak z jinyâch zdrojuê nebo liniõâ embryonaâ lnõâch kmenovyâch buneïk. Vy zkum v oblasti molekulaâ rnõâ odontologie je aktuaâ lneï podporovaâ n grantem GA AV CÏ R KJB , EU programem COST B23 (grant OC B23.001) a vyâ zkumnyâ m zaâ meï rem U ZÏ FG AVOZ blasts and maintain their differentiation stage. Enamelins contribute early in the initiation stage of amelogenesis to initial mineralisation of hydroxyapatite crystals, amelogenins are necessary for later crystal growth during secretory stage of amelogenesis. Amelogenins and ameloblastins are removed during maturation, whereas enamelins and tuftellins stay as an integral enamel part. Enzymes mediating cleavage of enamel matrix protein components are particularly enamelysin a matrix metalloproteinase (coded by the Mmp20 gene) that is secreted most abundantly during the secretory stage of enamel development and processes amelogenin in a highly controlled fashion [24] and kallikrein-4 a serine proteinase (coded by the Klk4 gene) secreted most abundantly during the maturation stage [25]. Defects in both structural matrix molecules as well as matrix processing proteinases result in weak enamel so called amelogenesis imperfecta (Stephanopoulos et al., 2005). To date, mutations have been identified in four genes (Amel, Enam, Klk4, Mmp20) known to be involved in enamel formation [26]. Future perspectives Basic principles of tooth development are now becoming clear and this knowledge could be exploited for better understanding and diagnostics of dental disorders, moreover, to more effective treatments based on tissue engineering approaches. Several applications of recent molecular knowledge about tooth germ development into understanding and possible treatment of dental disorders, such as hypodontia were discussed in our latest paper in Ortodoncie [27]. Recent understanding of molecular tooth development and tooth germ morphogenesis opens many challenges up to tooth production ªde novoª using adult stemcells fromdental and non-dental sources and embryonic stem cell lines. However, many difficulties are still to be overcome and questions to be answered. Molecular odontology research has been supported by the grants of the GA AS CR KJB , COST Programme B23 (grant OC B23.001) and IRP IAPG No. AVOZ Literature/References: 1. McCollumMA, Sharpe PT.: Developmental genetics and early hominid craniodental evolution. Bioessays. 2001; 23: Miletich I, Sharpe PT.: Neural crest contribution to mammalian tooth formation. Birth Defects Res C Embryo Today. 2004; 72: Tucker A, Sharpe P.: The cutting-edge of mammalian development; how the embryo makes teeth. Nat Rev Genet. 2004; 5: Marks SC Jr, Schroeder HE.: Tooth eruption: theories and facts. Anat Rec. 1996; 245: Bei M, Maas R.: FGFs and BMP4 induce both Msx1-independent and Msx1-dependent signaling pathways in early tooth development. Development. 1998; 125: Tucker AS, Matthews KL, Sharpe PT.: Transformation of tooth type induced by inhibition of BMP signaling. Science. 1998; 282: redakce@orthodont-cz.cz 45

8 rocïnõâk16 ORTODONCIE 7. Peterkova R, Lesot H, Peterka M.: Phylogenetic memory of developing mammalian dentition. J Exp Zoolog B Mol Dev Evol. 2006; 306: Mustonen T, Ilmonen M, Pummila M, Kangas AT, Laurikkala J, Jaatinen R, Pispa J, Gaide O, Schneider P, Thesleff I, Mikkola ML.: Ectodysplasin A1 promotes placodal cell fate during early morphogenesis of ectodermal appendages. Development. 2004; 131: Tucker AS, Headon DJ, Courtney JM, Overbeek P, Sharpe PT.: The activation level of the TNF family receptor, Edar, determines cusp number and tooth number during tooth development. Dev Biol. 2004; 268: Neubuser A, Peters H, Balling R, Martin GR.: Antagonistic interactions between FGF and BMP signaling pathways: a mechanism for positioning the sites of tooth formation. Cell. 1997; 90: Hardcastle Z, Mo R, Hui CC, Sharpe PT.: The Shh signalling pathway in tooth development: defects in Gli2 and Gli3 mutants. Development. 1998; 125: Jernvall J, Aberg T, Kettunen P, Keranen S, Thesleff I.: The life history of an embryonic signaling center: BMP- 4 induces p21 and is associated with apoptosis in the mouse tooth enamel knot. Development. 1998; 125: Dassule HR, Lewis P, Bei M, Maas R, McMahon AP.: Sonic hedgehog regulates growth and morphogenesis of the tooth. Development. 2000; 127: Jernvall J, Thesleff I.: Reiterative signaling and patterning during mammalian tooth morphogenesis. Mech Dev. 2000; 92: Jernvall J, Aberg T, Kettunen P, Keranen S, Thesleff I.: The life history of an embryonic signaling center: BMP- 4 induces p21 and is associated with apoptosis in the mouse tooth enamel knot. Development. 1998; 125: Laurikkala J, Mikkola M, Mustonen T, Aberg T, Koppinen P, Pispa J, Nieminen P, Galceran J, Grosschedl R, Thesleff I.: 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. 2001; 229: Jernvall J, Kettunen P, Karavanova I, Martin LB, Thesleff I.: Evidence for the role of the enamel knot as a control center in mammalian tooth cusp formation: non-dividing cells express growth stimulating Fgf-4 gene. Int J Dev Biol. 1994; 38: Vaahtokari A, Aberg T, Thesleff I.: Apoptosis in the developing tooth: association with an embryonic signaling center and suppression by EGF and FGF-4. Development. 1996; 122: Begue-Kirn C, Smith AJ, Loriot M, Kupferle C, Ruch JV, Lesot H.: Comparative analysis of TGF beta s, BMPs, IGF1, msxs, fibronectin, osteonectin and bone sialoprotein gene expression during normal and in vitro-induced odontoblast differentiation. Int J Dev Biol. 1994; 38: Camilleri S, McDonald F.: Runx2 and dental development. Eur J Oral Sci. 2006; 114: Bei M, Stowell S, Maas R.: Msx2 controls ameloblast terminal differentiation. Dev Dyn. 2004; 231: Camilleri S, McDonald F.: Runx2 and dental development. Eur J Oral Sci. 2006; 114: Kim JW, Simmer JP.: Hereditary dentin defects. J Dent Res. 2007; 86: Bartlett JD, Simmer JP.: Proteinases in developing dental enamel. Crit Rev Oral Biol Med. 1999; 10: Simmer JP, Hu JC. Expression, structure, and function of enamel proteinases. Connect Tissue Res. 2002; 43: Stephanopoulos G, Garefalaki ME, Lyroudia K. Genes and related proteins involved in amelogenesis imperfecta. J Dent Res. 2005; 84: KrejcÏ õâ P, Fleischmannova J, Matalova E, MõÂsÏ ek I.: Molecular basis of hypodontia. A review of the literature. Ortodoncie. 2007; 16: RNDr.Jana FleischmannovaÂ, Ph.D. Katedra fyziologie zï ivocï ichuê, PrÏõÂrodoveÏ deckaâ fakulta, JihocÏ eskaâ univerzita v CÏ eskyâ ch BudeÏ jovicõâch redakce@orthodont-cz.cz