n Xu

Ectodermal Smad4 and p38 MAPK are functionally redundant in mediating TGF-β/BMP signaling during tooth and palate development

Smad4 is a central intracellular effector of TGF-beta signaling. Smad-independent TGF-beta pathways, such as those mediated by p38 MAPK, have been identified in cell culture systems, but their in vivo functional mechanisms remain unclear. In this study, we investigated the role of TGF-beta signaling in tooth and palate development and noted that conditional inactivation of Smad4 in oral epithelium results in much milder phenotypes than those seen with the corresponding receptor mutants, Bmpr1a and Tgfbr2, respectively. Perturbed p38 function in these tissues likewise has no effect by itself; however, when both Smad4 and p38 functions are compromised, dramatic recapitulation of the receptor mutant phenotypes results. Thus, our study demonstrates that p38 and Smad4 are functionally redundant in mediating TGF-beta signaling in diverse contexts during embryonic organogenesis. The ability of epithelium to utilize both pathways illustrates the complicated nature of TGF-beta signaling mechanisms in development and disease.

Smad4‐Shh‐Nfic signaling cascade–mediated epithelial‐mesenchymal interaction is crucial in regulating tooth root development

Transforming growth factor β (TGF‐β)/bone morphogenetic protein (BMP) signaling is crucial for regulating epithelial‐mesenchymal interaction during organogenesis, and the canonical Smad pathway–mediated TGF‐β/BMP signaling plays important roles during development and disease. During tooth development, dental epithelial cells, known as Hertwigs epithelial root sheath (HERS), participate in root formation following crown development. However, the functional significance of HERS in regulating root development remains unknown. In this study we investigated the signaling mechanism of Smad4, the common Smad for TGF‐β/BMP signaling, in HERS in regulating root development. Tissue‐specific inactivation of Smad4 in HERS results in abnormal enamel and dentin formation in K14‐Cre;Smad4fl/fl mice. HERS enlarges but cannot elongate to guide root development without Smad4. At the molecular level, Smad4‐mediated TGF‐β/BMP signaling is required for Shh expression in HERS and Nfic (nuclear factor Ic) expression in the cranial neural crest (CNC)‐derived dental mesenchyme. Nfic is crucial for root development, and loss of Nfic results in a CNC‐derived dentin defect similar to the one of K14‐Cre;Smad4fl/fl mice. Significantly, we show that ectopic Shh induces Nfic expression in dental mesenchyme and partially rescues root development in K14‐Cre;Smad4fl/fl mice. Taken together, our study has revealed an important signaling mechanism in which TGF‐β/BMP signaling relies on a Smad‐dependent mechanism in regulating Nfic expression via Shh signaling to control root development. The interaction between HERS and the CNC‐derived dental mesenchyme may guide the size, shape, and number of tooth roots.

SMAD4-mediated WNT signaling controls the fate of cranial neural crest cells during tooth morphogenesis

TGFβ/BMP signaling regulates the fate of multipotential cranial neural crest (CNC) cells during tooth and jawbone formation as these cells differentiate into odontoblasts and osteoblasts, respectively. The functional significance of SMAD4, the common mediator of TGFβ/BMP signaling, in regulating the fate of CNC cells remains unclear. In this study, we investigated the mechanism of SMAD4 in regulating the fate of CNC-derived dental mesenchymal cells through tissue-specific inactivation of Smad4. Ablation of Smad4 results in defects in odontoblast differentiation and dentin formation. Moreover, ectopic bone-like structures replaced normal dentin in the teeth of Osr2-IresCre;Smad4fl/fl mice. Despite the lack of dentin, enamel formation appeared unaffected in Osr2-IresCre;Smad4fl/fl mice, challenging the paradigm that the initiation of enamel development depends on normal dentin formation. At the molecular level, loss of Smad4 results in downregulation of the WNT pathway inhibitors Dkk1 and Sfrp1 and in the upregulation of canonical WNT signaling, including increased β-catenin activity. More importantly, inhibition of the upregulated canonical WNT pathway in Osr2-IresCre;Smad4fl/fl dental mesenchyme in vitro partially rescued the CNC cell fate change. Taken together, our study demonstrates that SMAD4 plays a crucial role in regulating the interplay between TGFβ/BMP and WNT signaling to ensure the proper CNC cell fate decision during organogenesis.

PDGFR‐α signaling is critical for tooth cusp and palate morphogenesis

Platelet‐derived growth factor receptor alpha (PDGFR‐α) and PDGF ligands are key regulators for embryonic development. Although Pdgfrα is spatially expressed in the cranial neural crest (CNC)‐derived odontogenic mesenchyme, mice deficient for Pdgfrα are embryonic lethal, making it impossible to investigate the functional significance of PDGF signaling in regulating the fate of CNC cells during tooth morphogenesis. Taking advantage of the kidney capsule assay, we investigated the biological function of PDGF signaling in regulating tooth morphogenesis. Pdgfrα and Pdgfa are specifically and consistently expressed in the CNC‐derived odontogenic mesenchyme and the dental epithelium, respectively, throughout all stages of tooth development, suggesting a paracrine function of PDGF signaling in regulating tooth morphogenesis. Highly concentrated expression patterns of Pdgfrα and Pdgfa are associated with the developing dental cusp, suggesting possible functional importance of PDGF signaling in regulating cusp formation. Loss of the Pdgfrα gene does not affect proper odontoblasts proliferation and differentiation in the CNC‐derived odontogenic mesenchyme but perturbs the formation of extracellular matrix and the organization of odontoblast cells at the forming cusp area, resulting in dental cusp growth defect. Pdgfrα−/− mice have complete cleft palate. We show that the cleft palate in Pdgfrα mutant mice results from an extracellular matrix defect within the CNC‐derived palatal mesenchyme. The midline epithelium of the mutant palatal shelf remains functionally competent to mediate palatal fusion once the palatal shelves are placed in close contact in vitro. Collectively, our data suggests that PDGFRα and PDGFA are critical regulators for the continued epithelial–mesenchymal interaction during tooth and palate morphogenesis. Disruption of PDGFRα signaling disturbs the growth of dental cusp and interferes with the critical extension of palatal shelf during craniofacial development. Developmental Dynamics 232:75–84, 2005.

Cell autonomous requirement for TGF-β 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.

Indirect modulation of Shh signaling by Dlx5 affects the oral-nasal patterning of palate and rescues cleft palate in Msx1-null mice

Cleft palate represents one of the most common congenital birth defects in human. During embryonic development, palatal shelves display oronasal (O-N) and anteroposterior polarity before the onset of fusion, but how the O-N pattern is established and how it relates to the expansion and fusion of the palatal shelves are unknown. Here we address these questions and show that O-N patterning is associated with the expansion and fusion of the palatal shelves and that Dlx5 is required for the O-N patterning of palatal mesenchyme. Loss of Dlx5 results in downregulation of Fgf7 and expanded Shh expression from the oral to the nasal side of the palatal shelf. This expanded Shh signaling is sufficient to restore palatal expansion and fusion in mice with compromised palatal mesenchymal cell proliferation, such as Msx1-null mutants. Exogenous Fgf7 inhibits Shh signaling and reverses the cranial neural crest (CNC) cell proliferation rescue in the Msx1/Dlx5 double knockout palatal mesenchyme. Thus, Dlx5-regulated Fgf7 signaling inhibits the expression of Shh, which in turn controls the fate of CNC cells through tissue-tissue interaction and plays a crucial role during palatogenesis. Our study shows that modulation of Shh signaling may be useful as a potential therapeutic approach for rescuing cleft palate.

TGF-β mediated FGF10 signaling in cranial neural crest cells controls development of myogenic progenitor cells through tissue-tissue interactions during tongue morphogenesis

Skeletal muscles are formed from two cell lineages, myogenic and fibroblastic. Mesoderm-derived myogenic progenitors form muscle cells whereas fibroblastic cells give rise to the supportive connective tissue of skeletal muscles, such as the tendons and perimysium. It remains unknown how myogenic and fibroblastic cell-cell interactions affect cell fate determination and the organization of skeletal muscle. In the present study, we investigated the functional significance of cell-cell interactions in regulating skeletal muscle development. Our study shows that cranial neural crest (CNC) cells give rise to the fibroblastic cells of the tongue skeletal muscle in mice. Loss of Tgfbr2 in CNC cells (Wnt1-Cre;Tgfbr2(flox/flox)) results in microglossia with reduced Scleraxis and Fgf10 expression as well as decreased myogenic cell proliferation, reduced cell number and disorganized tongue muscles. Furthermore, TGF-beta2 beads induced the expression of Scleraxis in tongue explant cultures. The addition of FGF10 rescued the muscle cell number in Wnt1-Cre;Tgfbr2(flox/flox) mice. Thus, TGF-beta induced FGF10 signaling has a critical function in regulating tissue-tissue interaction during tongue skeletal muscle development.

Epithelial-Specific Requirement of FGFR2 Signaling During Tooth and Palate Development

Reciprocal interactions between epithelium and mesenchyme are crucial for embryonic development. Fibroblast growth factors (FGFs) are a growth factor family that play an important role in epithelial-mesenchymal tissue interaction. We have generated epithelial-specific conditional knockout mice targeting Fibroblast growth factor receptor 2 (Fgfr2) to investigate the function of FGF signaling during craniofacial development. K14-Cre;Fgfr2(fl/fl) mice have skin defects, retarded tooth formation, and cleft palate. During the formation of the tooth primordium and palatal processes, cell proliferation in the epithelial cells of K14-Cre;Fgfr2(fl/fl) mice is reduced. Thus, FGF signaling via FGFR2 in the epithelium is crucial for cell proliferation activity during tooth and palate development.