Changes In Morphology,Proliferation And Apoptosis Of Epithelial Root Sheath During Tooth Root Development In Vps4b Knockout Mice

Background and ObjectiveHertwig’s epithelial root sheath（HERS）,as the sign of the root’s initial development,is highly relevant to the normal physiological process of roots,with its integrity,timely cleavage and cell proliferation and apoptosis.Dentin dysplasia type I（DD-I）is an autosomal dominant trait with short,deformed roots resulting from severe root dysplasia.Vacuolar protein sorting 4B（VPS4B）is a multifunctional protein,which plays an important role in regulating cell proliferation,apoptosis,lysosomes degradation and intracellular protein trafficking.The whole genome sequencing of peripheral blood in a DD-I pedigree in our previous studies have found that the pathogenic gene in this family was VPS4B.Our research also have indicated that VPS4B may be a critical gene affecting tooth development,but its precise function in the development of dental root remains unclear.In this experiment,the Vps4b gene knockout mice were used to observe the morphological changes of HERS during root development,and to analyze the effects of mutant gene on the proliferation and apoptosis of HERS cells.Furthermore,the possible role of the mutant gene in tooth root teratogenesis was explored.Materials and MethodsThe male and female Vps4b gene knockout mice were mated and the postnatal（P）time was recorded.The neonatal mice were collected on 5,9,11,15 and 19days after birth,and then the mice were sacrificed to dissect the jaws and tails.Genotypes of newborn mice were identified by PCR technique.The mandibles of young mice were fixed,decalcified,and then embedded in paraffin after dehydration in order to prepare 5μm serial sections.Hematoxylin-eosin（HE）staining was used to define the developmental stages of root in each group of mice.The expressions of Cytokeratin 14（CK14）,proliferating cell nuclear antigen（PCNA）and B-cell lymphoma-2（Bcl-2）in HERS cells of normal and knockout mice at different developmental stages were localized and compared by immunohistochemical method.ResultsThe results of PCR showed that the genotypes of the offspring mice were Vps4b^+/+and Vps4b^+/-.The HE staining in normal mice manifested that HERS which induces root to grow was formed on P5 day.The structure of HERS was continuous,double layer with root furcation forming on P9 day,nevertheless,it began to disintegrate after P11 day.When it came to P15 day,one third of root has been formed.On P19 day,when the root achieved its physiological length we observed that HERS continued breaking while it remains intact in the apical end.The results of immunohistochemical staining in normal mice revealed that when the two-layer cell structure of HERS was formed on P5 day,the expression of PCNA was positive while Bcl-2 was negative in HERS cells.On P9 day,HERS continued to prolong,and PCNA was strong positive expression while Bcl-2 was positive expression in HERS cells.When HERS began to fragment on P11 day,the expression of PCNA in the terminal part was stronger than that in the neck loop while the Bcl-2 was weaken.On P15 day,few PCNA-positive HERS cells were found on the surface of root,while PCNA was still strongly positive and Bcl-2 was weakly positive at the end of HERS.When the root is about to be completed on 19day,we detected that there was only the positive expression of PCNA at the end of HERS,however,Bcl-2 was negative.After Vps4b gene was knocked out,anti-CK14protein showed that HERS began to interrupt on P9 day,but it was discontinuous and the number of HERS cells decreased after P15 day,and only cell clusters attached to the root surface.The expression intensity of PCNA in HERS cells was weaker than that in normal mice（P<0.05）.HERS cells showed positive expression of Bcl-2 only on P9 day and P11 day,and the intensity of expression decreased（P<0.05）.ConclusionThe continuity of HERS was disintegrated prematurely during the root development of Vps4b^+/-mice,which demonstrated that the deletion of Vps4b gene affected the formation of normal shape of HERS.The expression intensity of PCNA,Bcl-2 in HERS cells decreased,inferring that Vps4b might affect the development of tooth roots via regulating the proliferation and apoptosis of HERS cells.