INVOLVEMENT OF MSX PROTEINS IN EARLY HUMAN TOOTH DEVELOPMENT

Cilj istraživanja: Ciljevi ovog istraživanja bili su prikazati izražajne domene i obrasce izražaja po staničnim odjeljcima za homeoproteine MSX1 i MSX2 u zametku ljudskog zuba tijekom ranih stadija odontogeneze (pupoljak, kapa i zvono) i usporediti dobivene rezultate sa podatcima iz literature o izražaju pripadajućih gena tijekom ranog razvoja zuba u čovjeka i eksperimentalnih životinja (miš), te funkcijskoj povezanosti MSX1 i MSX2 sa regulatorima staničnog ciklusa kako bismo dobili uvid o važnosti ovih čimbenika za normalan razvoj zuba u čovjeka. Materijali i metode: U svrhu ovog istraživanja korišteni su uzorci tkiva deset ljudskih fetusa starih 7, 8/9, 10 i 14 tjedana trudnoće, prikupljeni nakon spontanih pobačaja i ektopičnih trudnoća s Odjela patologije KBC-a Split. Za imunohistokemijsku analizu korištena su fetalna tkiva u području glave i/ili dijelovi čeljusti koji su sadržavali zametke zubi (mliječni sjekutići). Rezovi su bojani primarnim monoklonskim protutijelima za MSX1 i MSX2. Preparati su fotografirani digitalnom kamerom postavljenom na fluorescencijski mikroskop, a dobivene slike su obrađene u AdobePhotoshop® CS6. Superponiranjem slika (merge) te obrađivanjem istih u ImageJ softveru analizirali su se izražajna domena i izražajni obrasci ispitivanih čimbenika. Rezultati: MSX1 i MSX2 bili su izraženi u tkivima zametka ljudskog zuba s proliferativnom aktivnosti tijekom svih promatranih razvojnih stadija. Preklapanje izražajih domena obaju biljega zabilježeno je u području epitelno-mezenhimnog sučelja, s tim da je MSX1 bio jače izražen u području mezenhimnog dijela zametka ljudskog zuba (dentalna papila), a MSX2 u epitelnom dijelu (pupoljak, caklinski organ). Oba čimbenika su prikazala dominantno citoplazmatski izražajni obrazac tijekom svih promatranih stadija razvoja. Zaključak: MSX1 i MSX2 imaju specifične uloge tijekom ranih stadija odontogeneze koje se mogu povezati s regulacijom proliferacijske aktivnosti u tkivima zametka zuba u čovjeka.Objectives: The objectives of this study were to investigate expression domains and patterns for homeodomain proteins MSX1 and MSX2 in the human tooth germ during the early stages of odontogenesis (bud, cap and bell) and compare the results with the data from previous studies on activity of the corresponding genes and cell cycle regulatory mechanisms during early tooth development in humans and experimental animals (mouse). Material and Methods: For the purpose of this study, tissue samples of ten human fetuses were aged 7, 8/9, 10 and 14 gestational weeks, collected after spontaneous abortions and ectopic pregnancies at the Department of Pathology KBC Split. For the immunohistochemical analysis, fetal tissues in the head area and / or jaw parts containing tooth germs (primary incisors) were used. Tissue sections were stained with primary monoclonal antibodies against MSX1 and MSX2, photographed by digital camera mounted on a fluorescence microscope, and the microphotographs were processed in AdobePhotoshop® CS6. Merged image doublets were then assembled and processed by using ImageJ software, to evaluate the expression domains and expression patterns of MSX1 and MSX2. Results: MSX1 and MSX2 were expressed in proliferative tissues of human tooth germs throughout the investiagted stages of development. The expression domains of MSX1 and MSX2 overlapped in epithelial-mesenchymal interface area. MSX1 was predominantly expressed in mesenchymal parts of the human tooth germ (dental papilla), whereas MSX2 expression was mostly observed in the epithelial part (tooth bud, enamel organ). Both markers displayed cytoplasmic expression pattern in human tooth germ tissues throughout the investigated period. Conclusion: MSX1 and MSX2 have specific roles during the early stages of odontogenesis which may be associated with the regulation of proliferative activity in the tissues of human tooth germs