Análise da expressão dos fatores de crescimento fibroblásticos 2, 3 e 4 durante a odontogênese de molares do gambá Didelphis albiventris, um modelo promissor para estudos em biologia do desenvolvimento

Abstract

Odontogenesis is guided by a complex signaling cascade in which several molecules, including the fibroblastic growth factors 2, 3 and 4 (FGF2, FGF3 and FGF4), ensure dental groups development and specificity. Most of the data on odontogenesis derives from rodents, which does not have all dental groups. Didelphis albiventris is a marsupial mammal with the closest dentition to humans. Besides, odontogenesis in this species occurs when the newborns are already in the pouch, favouring samples harvesting for the study of tooth development. This study aimed to determinate the expression pattern of FGF2, FGF3 and FGF4 during the D. albiventris odontogenesis. For this, D. albiventris newborns were harvested and processed for the following analysis: (1) histological, to allow the morphological characterization of the D. albiventris molars odontogenesis; (2) immunolozalization, to establish FGF2, FGF3, and FGF4 expression pattern during odontogenesis; and (3) of gene expression, to allow the cloning and sequencing of these molecules in this opossum species, providing tools for the localtemporal characterization of the FGFs mRNA expression patterns, by in situ hybridization. Histological analysis was performed using HE; and FGFs immunolocalization was performed by indirect immunoperoxidase. For gene expression analysis, oligos were obtained based on sequences available for Monodelphis domestica, the closest marsupial with genome sequenced. Oligos were obtained to amplify D. albiventris FGF3 and FGF4 (as FGF2 was cloned and sequenced before). Antisense RNA probe were in vitro synthesized, using digoxigenin for labelling. Histological analysis of the odontogenesis stages showed similarity of dental structures between D. albiventris and rodents, at the same odontogenesis stages, in addition to the presence of a vestigial secondary dental lamina in opossum. Immunolocalization allowed the observation of FGF2, FGF3 and FGF4 in a larger number of dental structures than related data for rodents, suggesting broader functions for these molecules in this opossum species. It was not possible to amplify D. albiventris FGF3, suggesting differences in the sequence level between these two opossum species. A FGF4 fragment was cloned and sequenced. This clone was also used to allow the synthesis of an antisense RNA probe, for in situ hybridization. FGF4 trancripts were detected in the dental germ, connective tissue, skeletal muscle, cartilage, epithelium, liver, and nervous tissue. The knowledge of the signaling that determinates odontogenesis in an animal model with complete dentition may contribute to the development of therapies for the replacement of lost teeth. The present study represents one step further in knowing this opossum species and in its implementation for Developmental Biology studies.