| Cleft lip and/or cleft palate (CL/P) as the representative of maxillofacialmalformations ranks the first in the congenital deformities. CL/P is caused byinteraction of multiple genetic factors with environmental factors. It is so complicatedthat until now little is known about its causation. For a long time, most researchers havebeen focused on the development of palate itself, while the cause of cleft may beintrinsic to the palatal shelves or secondary to deficiencies in other regions of thecraniofacial complex, such as developmental perturbations on maxilla or mandibularretrusion resulting in a physical obstruction to palatal shelf elevation by the displacedtongue. Nevertheless, until now defects in other craniofacial structures, especially themechanisms of tongue development which is closely related to the organizationalstructure, physiological function and the growth and development of palate as well asthe correlation of cleft palate with tongue malformation have not been explored.Retinoic acid (RA), derivatives of vitamin A, as cosmetic additives and skindisease and anticancer drugs, becomes a kind of common teratogenic environmentalfactor. RA pathway, interacted with WNT and SHH signal pathways, participates in thedevelopmental regulation of the maxillofacial region. In this study, we detectedmechanisms and correlation of cleft palate with tongue malformation by the in vivo andin vitro study using retinoic acid-induced and Wnt5a knockout maxillofacialabnormalities models. This research will provide a further realization to the mechanismsof tongue development, and a theoretical basis and experimental foundation for theprevention and treatment of clinical cleft palate with tongue abnormality.Objective: To study the mechanisms and correlation of cleft palate and tonguemalformation using retinoic acid-induced and Wnt5a knockout maxillofacialabnormalities models Methods: We established and applied the retinoic acid-induced and Wnt5aknockout maxillofacial abnormalities models. Then the histological changes wereobserved by stereomicroscope, cells proliferation was detected by BrdU incorporation,and the apoptosis was examined by TUNEL staining, the fibrosis was tested by Massonstaining, Genechip array was used to screen the different genes in cleft palate andtongue malformation, and then validated the results of genechip by real-time PCR.Further detection was done for the expression and distribution of GSK3β protein in Wntsignal pathway in palate and the expression of Myf5and MyoD in tougue byimmunohistochemical assay. The Trowell organ culture system was utilized for thefusion test of the palatal shelves with the tongue excision, to investigate the correlationof development of palate and tongue.C2C12cell line was stably transfected by Wnt5a gene to detect the effect ofWnt5a in skeletal muscle development. Cell proliferation was assessed by CellCounting kit-8(CCK8) assay, and cell apoptosis was detected by flow cytometryAnnexin V-FITC/PI double staining and the cell viability was assessed by CCK8assayafter serum starved-induced apoptosis; detected the expression of Myf5, MyoG andMRF4during C2C12myoblasts differentiation by real-time PCR, and the expression ofMyosin was detected by immunocytochemical staining, and the functional implicationof Wnt5a in myogenesis was explored.Results:1. Development of palate, tongue, maxilla and mandible in RA inducedcleft palate.(1) The analysis of genechip results showed that gene functional categories andpathways, particularly involved in transcription regulator activity, regulation of growth,direction of cell movement, cytoskeleton. Wnt and Shh families were also identified,furthermore, some of these genes such as GSK3β and β-TrCP were involved in both ofWnt and Shh signaling pathway suggesting the crosstalk in signal pathway. Real-timePCR validated that the expression of GSK3β and β-TrCP were down-regulated,expression of Wnt pathway receptor FZD3was not changed. Immunohistochemicalassay showed, at E14of the vigrous proliferation stage, GSK3β expressed in both ofepithelium and mesenchyme, while at E15.5, the delayed skeletal development inmaxilla was obvious, and GSK3β expressed in a great deal of undifferentiatedmesenchymal cells in palatal shelves of cleft palate group. From E14to E14.5, the keystage for elevation and horizontal growth, the palatal shelves still grew vertically at theboth side of tongue, and there were multiple TUNEL positive apoptosis cells in the margin of meckel cartilage.(2) At E13.5, palatal shelves with the tongue excision were able to fuse and themesenchymal cells went through each other after organ culture for3days in the Trowellculture system. In the process of palate development, there was no difference in thenumber of nucleus in genioglossus, while the cross section area of genioglossus wasevidently smaller in cleft palate group compared with control group. There was nodifference in the proliferation and apoptosis and no abnormal fibrosis was detected intongue musculature. The analysis of genechip result of E14tongue showed that RArelated signaling molecules network was identified and some genes such as DTNA andCAMK2D were up-regulated in tongue musculature of RA-treated group.2. Role of Wnt5a in the development of tongue and skeletal muscle(1) Wnt5a showed dynamic expression pattern in development of tongue, it washighly expressed at E13.5in control group and RA-treated group, while the expressionlevel was still at high level at E15.5in RA-treated group compared with the declinedexpression in control group.(2) In Wnt5a knockout mouse, there was no difference in BrdU positive rate eitherin tongue body or in genioglossus compared with control group, and there was regionaldense in mesenchymal cells in control group, while it was not obvious in Wnt5aknockout mouse. Either the expression of Myf5or MyoD protein were down-regulatedin tongue musculature.(3) In the C2C12cell line stably transfected by Wnt5a, there is no change in cellproliferation and migeration, and no change in cell apoptosis and cell viability afterserum starved-induced apoptosis compared with control group. The expression of Myf5was down-regulated at D2(Differentiation2day) and D3, the expression of MyoG wasdeclined at D2, and the expression of MRF4was down-regulated at D2and D4duringC2C12myoblasts differentiation. The expression of Myosin was detected byimmunocytochemical staining, the fuse of myoblasts resulted in formation ofmultincleatedmyotubes in both group.Conclusion: Overdose of retinoic acid induced multiple maxillofacialmalformation represented by cleft palate, while the fusion of MEE was not affected.Retinoic acid induced cleft palate associated with delayed tongue development as wellas delayed skeletal development in maxilla and abnormal apoptosis in meckel cartilage,suggested that overdose of retinoic acid may disturb collaborative development ofmaxillofacial system. The malformation in palate was related with WNT and SHH signaling pathway inRA-induced model, RA-related molecular network was involved in tonguemalformation. Knockout in vivo or overexpression in vitro of Wnt5a delayeddifferentiation of skeletal muscle and suggested that Wnt5a may play roles inmyogenesis through multi-regulatory routes. |
PDF Full Text Request