The Role And Mechanism Of Alx1 During Upper Incisor Development In Mice

As a transcription factor,ALX1 has a critical role in the development of neural tube closure,limb and craniofacial region in vertebrates.Mutations in the ALX1 gene cause severe craniofacial deformities in humans.In our previous studies,we found that deletion of Alx1 gene in mouse cranial neural crest cells results in cleft palate and absence of upper incisor.However,the role of Alx1 in incisor development has not been reported.In this study,loss-of function mouse genetic model was used to analyzed the function of Alx1 in the development of incisor.Firstly,we examined the expression pattern of Alx1 in wildtype mouse incisor using in situ hybridization.The results showed that Alx1 was only expressed in the mesenchyme of incisor at E12.5and E14.5.At E16.5,the expression of Alx1 is concentrated in the odontoblast of incisor.These results indicate that Alx1 may play an important role in the development and differentiation of incisors.Secondly,we generated Wnt1-Cre;Alx1^f/fmice carrying Alx1 specific deletion in cranial crest cells using Alx1^f/f and Wnt1-cre alleles.To further study the role of Alx1 in teeth,we collected mouse embryos for paraffin section.The molar is normal in Wnt1-Cre;Alx1^f/fmice compared to their wild-type littermates.However,the Wnt1-Cre;Alx1^f/f mice exhibited enamel knot missing,bud stage tooth arrest and the degenerated incisor epithelium The development of mandibular incisors were slightly delayed in Wnt1-Cre;Alx1^f/f mice.In addition,the size of incisor germreduced,and dentin secretion increased.Our data indicated that Alx1 played an important role in secretion of dentin and bud-to-cap stage transition of incisor.Finally,we further performed transcriptome differential analysis to uncover the molecular events involved.Gene ontology enrichment analysis demonstrated that differential genes in upper incisor of Alx1 knockout mice were functionally enriched in terms associated with WNT signaling pathway,BMP signaling pathway and dentin formation.We identified downregulation of several genes associated with development of incisor,such as Bmp4,Shh,Msx1,β-catenin and Lef1.We verified the expression of these factors by in situ hybridization,immunofluorescence,immunohistochemistry,and q PCR.the results indicated that Alx1 may regulate the development of incisor in mice through Bmp4,Shh,Msx1,β-catenin and Lef1.In summary,mandibular incisors delayed and maxillary incisor bud stage tooth arrested in Wnt1-Cre;Alx1^f/f mice.In addition,the size of tooth germ was reduced and dentin secretion was increased in the mandibular incisor.Our results suggest that the condition knockout of Alx1 inhibit the development and differentation of incisors by regulating Bmp4,Msx1,β-catenin,Lef1 and Shh.These results furter deepen our understanding of incisor development.