TGF-beta mediated Dlx5 signaling plays a crucial role in osteo-chondroprogenitor cell lineage determination during mandible development.

Transforming growth factor-beta (TGF-beta) signaling is crucial for mandible development. During its development, the majority of the mandible is formed through intramembranous ossification whereas the proximal region of the mandible undergoes endochondral ossification. Our previous work has shown that TGF-beta signaling is required for the proliferation of cranial neural crest (CNC)-derived ectomesenchyme in the mandibular primordium where intramembranous ossification takes place. Here we show that conditional inactivation of Tgfbr2 in CNC cells results in accelerated osteoprogenitor differentiation and perturbed chondrogenesis in the proximal region of the mandible. Specifically, the appearance of chondrocytes in Tgfbr2(fl/fl);Wnt1-Cre mice is delayed and they are smaller in size in the condylar process and completely missing in the angular process. TGF-beta signaling controls Sox9 expression in the proximal region, because Sox9 expression is delayed in condylar processes and missing in angular process in Tgfbr2(fl/fl);Wnt1-Cre mice. Moreover, exogenous TGF-beta can induce Sox9 expression in the mandibular arch. In the angular processes of Tgfbr2(fl/fl);Wnt1-Cre mice, osteoblast differentiation is accelerated and Dlx5 expression is elevated. Significantly, deletion of Dlx5 in Tgfbr2(fl/fl);Wnt1-Cre mice results in the rescue of cartilage formation in the angular processes. Finally, TGF-beta signaling-mediated Scleraxis expression is required for tendonogenesis in the developing skeletal muscle. Thus, CNC-derived cells in the proximal region of mandible have a cell intrinsic requirement for TGF-beta signaling.

Smad4 is required to regulate the fate of cranial neural crest cells.

Smad4 is the central mediator for TGF-beta/BMP signals, which are involved in regulating cranial neural crest (CNC) cell formation, migration, proliferation and fate determination. It is unclear whether TGF-beta/BMP signals utilize Smad-dependent or -independent pathways to control the development of CNC cells. To investigate the functional significance of Smad4 in regulating CNC cells, we generated mice with neural crest specific inactivation of the Smad4 gene. Our study shows that Smad4 is not required for the migration of CNC cells, but is required in neural crest cells for the development of the cardiac outflow tract. Smad4 is essential in mediating BMP signaling in the CNC-derived ectomesenchyme during early stages of tooth development because conditional inactivation of Smad4 in neural crest derived cells results in incisor and molar development arrested at the dental lamina stage. Furthermore, Smad-mediated TGF-beta/BMP signaling controls the homeobox gene patterning of oral/aboral and proximal/distal domains within the first branchial arch. At the cellular level, a Smad4-mediated downstream target gene(s) is required for the survival of CNC cells in the proximal domain of the first branchial arch. Smad4 mutant mice show underdevelopment of the first branchial arch and midline fusion defects. Taken together, our data show that TGF-beta/BMP signals rely on Smad-dependent pathways in the ectomesenchyme to mediate epithelial-mesenchymal interactions that control craniofacial organogenesis.

The role of TGF-beta signaling in regulating chondrogenesis and osteogenesis during mandibular development.

During craniofacial development, Meckel's cartilage and the mandible bone derive from the first branchial arch, and their development depends upon the contribution of cranial neural crest (CNC) cells. We previously demonstrated that conditional inactivation of Tgfbr2 in the neural crest of mice (Tgfbr2(fl/fl);Wnt1-Cre) results in severe defects in mandibular development, although the specific cellular and molecular mechanisms by which TGF-beta signaling regulates the fate of CNC cells during mandibular development remain unknown. We show here that loss of Tgfbr2 does not affect the migration of CNC cells during mandibular development. TGF-beta signaling is specifically required for cell proliferation in Meckel's cartilage and the mandibular anlagen and for the formation of the coronoid, condyle and angular processes. TGF-beta-mediated connective tissue growth factor (CTGF) signaling is critical for CNC cell proliferation. Exogenous CTGF rescues the cell proliferation defect in Meckel's cartilage of Tgfbr2(fl/fl);Wnt1-Cre mutants, demonstrating the biological significance of this signaling cascade in chondrogenesis during mandibular development. Furthermore, TGF-beta signaling controls Msx1 expression to regulate mandibular osteogenesis as Msx1 expression is significantly reduced in Tgfbr2(fl/fl);Wnt1-Cre mutants. Collectively, our data suggest that there are differential signal cascades in response to TGF-beta to control chondrogenesis and osteogenesis during mandibular development.

Cell autonomous requirement for TGF-beta signaling during odontoblast differentiation and dentin matrix formation.

TGF-beta subtypes are expressed in tissues derived from cranial neural crest cells during early mouse craniofacial development. TGF-beta signaling is critical for mediating epithelial-mesenchymal interactions, including those vital for tooth morphogenesis. However, it remains unclear how TGF-beta signaling contributes to the terminal differentiation of odontoblast and dentin formation during tooth morphogenesis. Towards this end, we generated mice with conditional inactivation of the Tgfbr2 gene in cranial neural crest derived cells. Odontoblast differentiation was substantially delayed in the Tgfbr2(fl/fl);Wnt1-Cre mutant mice at E18.5. Following kidney capsule transplantation, Tgfbr2 mutant tooth germs expressed a reduced level of Col1a1 and Dspp and exhibited defects including decreased dentin thickness and absent dentinal tubules. In addition, the expression of the intermediate filament nestin was decreased in the Tgfbr2 mutant samples. Significantly, exogenous TGF-beta2 induced nestin and Dspp expression in dental pulp cells in the developing tooth organ. Our data suggest that TGF-beta signaling controls odontoblast maturation and dentin formation during tooth morphogenesis.

Identification of novel genes expressed during mouse tooth development by microarray gene expression analysis.

To identify genes heretofore undiscovered as critical players in the biogenesis of teeth, we have used microarray gene expression analysis of the developing mouse molar tooth (DMT) between postnatal day (P) 1 and P10 to identify genes differentially expressed when compared with 16 control tissues. Of the top 100 genes exhibiting increased expression in the DMT, 29 were found to have been previously associated with tooth development. Differential expression of the remaining 71 genes not previously associated with tooth development was confirmed by quantitative reverse transcription-polymerase chain reaction analysis. Further analysis of seven of the latter genes by mRNA in situ hybridization found that five were specific to the developing tooth in the craniofacial region (Rspo4, Papln, Amtn, Gja1, Maf). Of the remaining two, one was found to be more widely expressed (Sp7) and the other was found to be specific to the nasal serous gland, which is close to, but distinct from, the developing tooth (Vrm).