Role Of Growth Factor Signaling In The Development Of Tooth And Palate

1. Exogenous FGF8 rescues development of mouse diastemal vestigial tooth ex vivoPURPOSE:Regression of vestigial tooth buds results in the formation of the toothless diastema, a unique feature of mouse dentition. Revitalization of the diastemal vestigial tooth bud provides an excellent model for studying tooth regeneration and replacement. It has been previously shown that suppression of FGF signaling in the diastema results in vestigial tooth bud regression. In this study, we report that application of exogenous FGF8 to mouse embryonic diastemal region rescues the development of diastemal vestigial tooth.METHODS:①Embryonic day 13.5 (E13.5) embryos from timed pregnant CD-1 females were collected. Mandibular quadrants were carefully dissected out, containing only the diastema, incisor and molar germs. It was further dissected into the diastema, the incisor, and molar tissues. Affi-gel blue agarose beads (100-200μm in diameter) were prepared and soaked with growth factor proteins.3-4 protein soaked beads were inserted into the diastemal mesenchyme. After 24 hours in culture, samples were subjected to subrenal culture using adult male CD1 mice as hosts. Grafts were cultured underneath the kidney capsule for 4 weeks prior to being harvested for further analyses.②Mandibular quadrants were carefully dissected out. Each quadrant was either used as a tissue transplant. For whole quadrant transplant,3-4 protein soaked beads were inserted into the diastemal mesenchyme from the aboral side, and then beads-containing quadrants were transferred to a semisolid culture. BSA beads as control. Grafts were cultured underneath the kidney capsule for 4 weeks prior to being harvested for further analyses.③Affi-gel blue agarose beads (100-200μm in diameter) were prepared and soaked with growth factor proteins.3-4 protein soaked beads were inserted into the diastemal mesenchyme. After 24 hours or 48 hours in culture, samples were futher used for histological analysis, in situ hybridization, BrdU labeling and TUNEL assay.RESULTS:Isolated diastemal vestigial buds appeared to escape the suppressing effects of factors from the adjacent developing tooth germs, and in the presence of FGF8, became revitalized and continued to development. FGF8 promotes cell proliferation and inhibits apoptosis in diastemal tooth epithelium, and revitalizes the tooth developmental program, evidenced by the expression of genes critical for tooth development.CONCLUSION:FGF8 promotes cell proliferation and inhibits apoptosis in diastemal tooth epithelium, and revitalizes the tooth developmental program, evidenced by the expression of genes critical for tooth development. Our results also support the idea that adjacent tooth germs contribute to the suppression of diastemal vestigial tooth buds via multiple signals. 2. The role of BMP signaling in tooth and palate developmentPURPOSE:The family of BMPs comprises over 20 multi-functional cytokines that belong to the TFG-βsuperfamily. The BMP signaling plays a pivotal role in the development of craniofacial organs, including the tooth and palate. BmprⅠa and BmprⅠb encode two typeⅠBMP receptors that are primarily responsible for BMP signaling transduction. Despite the essential role for BmprⅠa in the epithelial component for tooth and palate development, the requirement of BmprⅠa in the mesenchymal component remains unknown. In this study, we investigated mesenchymal tissue-specific requirement of BmprⅠa and its functional redundancy with BmprⅠb during the development of mouse tooth and palate.METHODS:①Embryos containing inactivated BmprⅠa in their neural crest cells (Wnt1Cre; BmprⅠaF/-) were obtained by crossing Wnt1Cre;BmprⅠa+/- mice with BmprⅠaF/F line. To prevent the embryonic lethality, drinking water was supplemented with 200μg/ml isoproterenol and 2.5 mg/ml ascorbic acid from7.5 post-coitum (dpc). Embryos were collected from timed-mate pregnant females in ice-cold PBS. Embryonic head samples were dissected and fixed individually in 4% paraformaldehyde (PFA) overnight at 4℃, and processed for paraffin section for histological and in situ hybridization analyses or for frozen section for immunostaining.②To obtain embryos carrying Wnt1Cre; BmprⅠaF/- alleles and a pMes-caBmprⅠb transgenic allele, Wnt1Cre; BmprⅠa+/- mice were crossed with BmprⅠaF/+; pMes- caBmprⅠb mice. Mice containing such compounded alleles are referred as Wnt1Cre; BmprⅠaF/-; caⅠb. Samples were processed as previous described.③To determine if the Wnt1Cre; BmprⅠaF/-; caⅠb mouse exhibited delay of tooth development. Mandibular molar germs were isolated from Wnt1Cre; BmprⅠaF/-; caⅠb embryos and wild type controls, and were subjected to subrenal culture. Adult CD-1 male mice were used as hosts for subrenal culture.RESULTS:BmprⅠa and BmprⅠb exhibit partially overlapping and distinct expression patterns in the developing tooth and palatal shelf. Neural crest-specific inactivation of BmprⅠa leads to formation of an unusual type of anterior clefting of the secondary palate, an arrest of tooth development at the bud/early cap stages, and severe hypoplasia of the mandible. Defective tooth and palate development is accompanied by the down-regulation of BMP-responsive genes and reduced cell proliferation levels in the palatal and dental mesenchyme. To determine if BmprⅠb could substitute for BmprⅠa during tooth and palate development, we expressed a constitutively active form of BmprⅠb (caBmprⅠb) in the neural crest cells in which BmprⅠa was simultaneously inactivated. We found that substitution of BmprⅠa by caBmprⅠb in neural rest cells rescues the development of molars and maxillary incisor, but the rescued teeth exhibit a delayed odontoblast and ameloblast differentiation. In contrast, caBmprⅠb failed to rescue the palatal and mandibular defects including the lack of lower incisors. CONCLUSION:BmprⅠa is essential in the mesenchymal compartment for palate and tooth development. BmprⅠb has a restricted redundant function with BmprⅠa in a tissue specific manner in craniofacial development.3. The role of BMP signaling homeostasis in tooth and palate developmentPURPOSE:In PartⅠ, we have shown that neural crest-specific inactivation of BmprⅠa leads to formation of an unusual type of anterior clefting of the secondary palate, an arrest of tooth development at the bud/early cap stages, and severe hypoplasia of the mandible. Substitution of BmprⅠa by an constitutively active form of BMP receptor IB (caBmprⅠb) in the cranial neural crest cells rescues the development of molars and maxillary incisor, but the rescued teeth exhibit a delayed odontoblast and ameloblast differentiation (Li et al.,2011). In parallel to the caBmprⅠb rescue study, we used a conditional transgenic allele that expresses a constitutively active form of BMP receptor IA (caBmprⅠa) as a positive control. Using transgenic mous mouse model, we set to investigate the role of BMP signaling homeostasis in tooth and palate development.METHODS:①Embryos with different combination of BmprⅠa allele in the neural crest-derived tissues were obtained by crossing Wnt1Cre;BmprⅠa+/- mice with BmprⅠaF/+;pMes-caBmprⅠa mice. The genotypes of the embryos are:Wnt1Cre; BmprIaF/-, Wnt1Cre; BmprⅠaF/-pMes-caBmprⅠa, Wnt1Cre; BmprⅠaF/+; pMes-caBmprⅠa and Wnt1Cre; pMes-caBmprⅠa. To prevent the embryonic lethality, drinking water was supplemented with 200μg/ml isoproterenol and 2.5 mg/ml ascorbic acid from7.5 post-coitum (dpc).③Embryos were collected from timed-mate pregnant females in ice-cold PBS. Embryonic head samples were dissected and fixed individually in 4% PFA overnight at 4℃, and processed for paraffin section for histological and in situ hybridization analyses or for frozen section for immunostaining.③To determine if the Wnt1Cre; pMes-caBmprⅠa mouse exhibited delay of tooth development. Mandibular molar germs were isolated from Wnt1Cre; BmprⅠaF/-; caⅠb embryos and wild type controls, and were subjected to subrenal culture. Adult CD-1 male mice were used as hosts for subrenal culture.RESULTS:①The changes in BmprⅠa-mediated BMP signaling lead to different craniofacial abnormalities. BMP signaling homeostasis is essential for the development of tooth and palate.②Over expression of caBmprⅠa in neural crest cells resulted in a delay in tooth differentiation and complete clefting of the secondary palate, accompanied with a reduced cell proliferation in anterior palatal mesenchyme and ectopic cartilage formation in the posterior palate.CONCLUSION:The BMP signaling homeostasis is required in proper development of tooth and palate. Tissue specific activation of BMP signaling leads to reduced cell proliferation rate in the anterior palate and ectopic cartilage formation in the posterior palate. Enhanced BMP signaling activity in the dental mesenchyme results in a delayed differentiation of odontoblasts and ameloblasts.