The Regulatory Effect Of Bmp4/P38 Signaling In Cranial Neural Crest Cells On The Patterning Of Maxillo-mandibular Molars

The early initiation,positioning and morphogenesis of the mouse tooth are regulated by epithelial-mesenchymal signaling which involves the signaling molecules including FGF,hedgehog(Hh),Wnt and BMP families.As a member of BMP signaling pathyway,Bmp4 is the first signal molecule expressing in mammalian tooth and plays a decisive role in the determination of type and position where tooth occurres.Previously,our laboratory has constructed the mouse with forced expression of Bmp4 in cranial neural crest cells:Wnt1-cre;pMes-Bmp4,which has a molar germ forming dorsally toward to the tongue at the maxillo-mandibular junction.Most of the fused molar germs are degenerate,while a small part of them develop normally and have the same morphology as wild type embryos.Taking advantage of the exceptional phenotype of the genetically modified mouse,this research explores the causes of fused tooth germs and analyzes the segregation mechanism of the maxillo-mandibular molars,which discusses the regulatory effect of Bmp4 gene on tooth development from gene gain-of-function.Firstly,the expression patterns of Fgf8,Pax9 and Pitx2 in early first branchial arches by whole-mount in situ hybridization of wild type embryos were examined.We found that at E9.0,the expressed region of Fgf8,which can be regarded as the start site of odontogenesis,was restricted to the epithelium of oral-surface tooth aera of the first branchial arches in wild type embryos.Along with the migration and growth of the first branchial arches,the Fgf8 expression locus split in half followed by gradual extension.And there was one Fgf8 concentrated expression in the epithelium of oral-surface tooth aera of the maxillary and mandibulary processes at E10.5,respectively.Pax9 began at E9.75 with a weak expression in the mesenchyme of oral surface-tooth aera of the maxillary processes and the expression also existed in E10.0 mandibulary processes,until E10.5,Pax9 further streched to the distal end.From E9.0 to E10.5,Pitx2 expressed in the ectoderm covering the oral-surface of the first branchial arches.Moreover,the subrenal culture of the first branchial arches discovered that the majority of the grafts,which contained two molar germs after E9.5,had single molar germ prior to E9.5.The above results suggest that the maxilla-mandibular molars of mouse are emerged from the same tooth germ and experience a procedure of "one divides into two" during early positioning.On account of the fused molar germs caused by enhanced Bmp4 activity in neural crest cells,we similarly detected the expression patterns of Fgf8,Pax9 and Pitx2 in early first branchial arches by whole-mount in s:itu hybridization of Wnt1-cre,pMes-Bmp4 embryos to investigated the effect of elevated Bmp4 activity in cranial neural crest cells on the location of the early tooth germs.It found that the epithelium of oral-surface tooth aera of the first branchial arches always existed single expression part of Fgf8 in mutant embryos and the locus cannot split.Besides formation of the minimal maxillary processes,Fgf8 expression concentrated on the maxillo-mandibular junction at E10.5.Additionally,mutant embryos and littermates had no difference in Pax9 expression that the mutants were absent in E10.0 mandibular processes,and till E10.5,Pax9 extended around the maxillo-mandibular junction.Pitx2 also expressed in the ectoderm covering the oral-surface of the mutants first branchial arches from E9.0 to E10.5.Then,we conducted mouse kidney capsule culture of the early first branchial arche.While a small portion of the implants had only one molar germ,no tooth formation was d:iscovered in most of them.Both of these indicate that the Bmp4-overexpression in cranial neural crest cells has impact on the normal development of the early first branchial arches and gives rise to nondividing molars germs locus,consequently,the molars aberrantly positioned at the maxillo-mandibular junction.Further explored the mechanism that Bmp4 gene regulates the separation of the maxillo-mandibular from cellular and molecular levels,we found the significantly augmented of Bmp4/P38 signaling in the early first branchial arches which had no abnormalities in cell proliferation and apoptosis.It implicates that the destruction of BMP homeostasis leads to abnormal development of the early first branchial arches as well as ectopic patterning of the maxillo-mandibular molars.Furthermore,we monitored the expression level of BMP intracellular signal transduction in these three kinds of tooth germs,including BMP/Smad4,P38,Erk1/2 and JNK,our findings revealed distinct increase of the Bmp4/P38 activity in mutants tooth germs,and pronounced decrease of the level of pSmad1/5/8 in degenerate tooth germs.The small-molecule inhibitor SB203580 of P38 signaling pathway was applied to the rescue-experiment and we discovered partial rescue of the aberrant phenotypes such as cleft palate and the ectopic segregation of maxillo-mandibular in mutant embryos.These consequences confirm the functional role of the BMP homeostasis in the patterning of the maxillo-mandibular molars and tooth development.To sum up,this research clarifies the separation method of the maxillo-mandibular molars in mice is "one split into two",and verifies the significance of the Bmp4/P38 signaling to the patterning of maxillo-mandibular molars and dental development.The experiment further perfects the molecular mechanism of mouse tooth development,and in the meanwhile,it contributes to deepen appreciation of the tooth developmental patterning and themechanism in mammal.