Roles of SATB2 in osteogenic differentiation and bone regeneration.

Expressed in branchial arches and osteoblast-lineage cells, special AT-rich sequence-binding protein (SATB2) is responsible for preventing craniofacial abnormalities and defects in osteoblast function. In this study, we transduced SATB2 into murine adult stem cells, and found that SATB2 significantly increased expression levels of bone matrix proteins, osteogenic transcription factors, and a potent angiogenic factor, vascular endothelial growth factor. Using an osterix (Osx) promoter-luciferase construct and calvarial cells isolated from runt-related transcription factor 2 (Runx2)-deficient mice, we found that SATB2 upregulates Osx expression independent of Runx2, but synergistically enhances the regulatory effect of Runx2 on Osx promoter. We then transplanted SATB2-overexpressing adult stem cells genetically double-labeled with bone sialoprotein (BSP) promoter-driven luciferase and ß-actin promoter-driven enhanced green fluorescent protein into mandibular bone defects. We identified increased luciferase-positive cells in SATB2-overexpressing groups, indicating more transplanted cells undergoing osteogenic differentiation. New bone formation was consequently accelerated in SATB2 groups. In conclusion, SATB2 acts as a potent transcription factor to enhance osteoblastogenesis and promote bone regeneration. The application of SATB2 in bone tissue engineering gives rise to a higher bone forming capacity as a result of multiple-level amplification of regulatory activity.

LEF1 is a critical epithelial survival factor during tooth morphogenesis.

LEF1 is a cell-type-specific transcription factor and mediates Wnt signaling pathway by association with its co-activator beta-catenin. Wnt signaling is known to be critical for the specification of cranial neural crest (CNC) cells and may regulate the fate diversity of the CNC during craniofacial morphogenesis. Loss of Lef1 results in arrested tooth development at the late bud stage and LEF1 is required for a relay of a Wnt signaling to a cascade of FGF signaling activities to mediate the epithelial-mesenchymal interaction during tooth morphogenesis. It remains unclear, however, what is the cellular mechanism of LEF1 signaling in regulating tooth morphogenesis. To test the hypothesis that LEF1 signaling regulates the fate of the dental epithelial and the CNC-derived mesenchymal cells during tooth morphogenesis, we investigated and compared the cellular migration, proliferation, and apoptotic activity within the tooth germ between the wild-type and Lef1 null mutant mice. Using the Wnt1-Cre/R26R transgenic system for indelibly marking the progenies of CNC cells, we show that there is no CNC migration defect in the Lef1 null mutant mice, indicating that the arrest in tooth development is not the result of shortage of the CNC contribution into the first branchial arch in the Lef1 mutant. Furthermore, there is no alteration in cell proliferation or condensation of the CNC-derived dental mesenchyme in the Lef1 null mutant, suggesting that LEF1 may not affect the cell cycle progression of the multipotential CNC cells during tooth morphogenesis. Importantly, apoptotic activity is significantly increased within the dental epithelium in the Lef1 null mutant mice. As the result of this increased cell death, the bud stage tooth germ fails to advance to the cap stage in the absence of Lef1. Inhibition of apoptotic activity by FGF4 rescues the tooth development in the Lef1 null mutant. Our studies suggest that LEF1 is a critical survival factor for the dental epithelial cells during tooth morphogenesis.

FGF4, a direct target of LEF1 and Wnt signaling, can rescue the arrest of tooth organogenesis in Lef1(-/-) mice.

Lymphoid enhancer factor (LEF1), a nuclear mediator of Wnt signaling, is required for the formation of organs that depend on inductive interactions between epithelial and mesenchymal tissues. In previous tissue recombination experiments with normal and Lef1(-/-) tooth germs, we found that the effect of LEF1 expression in the epithelium is tissue nonautonomous and transferred to the subjacent mesenchyme. Here we examine the molecular basis for LEF1 function and find that the epithelium of the developmentally arrested Lef1(-/-) tooth rudiments fails to express Fgf4, Shh, and Bmp4, but not Wnt10a. We identify the Fgf4 gene as a direct transcriptional target for LEF1 and show that beads soaked with recombinant FGF4 protein can fully overcome the developmental arrest of Lef1(-/-) tooth germs. In addition, we find that FGF4 beads induce rapidly the expression of Fgf3 in dental mesenchyme and that both epithelial and mesenchymal FGF proteins induce the delayed expression of Shh in the epithelium. Taken together, these data indicate that a single target of LEF1 can account for the function of LEF1 in tooth development and for a relay of a Wnt signal reception to a cascade of FGF signaling activities, allowing for a sequential and reciprocal communication between epithelium and mesenchyme.

Regulation of hair follicle development by the TNF signal ectodysplasin and its receptor Edar.

X-linked and autosomal forms of anhidrotic ectodermal dysplasia syndromes (HED) are characterized by deficient development of several ectodermal organs, including hair, teeth and exocrine glands. The recent cloning of the genes that underlie these syndromes, ectodysplasin (ED1) and the ectodysplasin A receptor (EDAR), and their identification as a novel TNF ligand-receptor pair suggested a role for TNF signaling in embryonic morphogenesis. In the mouse, the genes of the spontaneous mutations Tabby (Ta) and downless (dl) were identified as homologs of ED1 and EDAR, respectively. To gain insight into the function of this signaling pathway in development of skin and hair follicles, we analyzed the expression and regulation of Eda and Edar in wild type as well as Tabby and Lef1 mutant mouse embryos. We show that Eda and Edar expression is confined to the ectoderm and occurs in a pattern that suggests a role of ectodysplasin/Edar signaling in the interactions between the ectodermal compartments and the formation and function of hair placodes. By using skin explant cultures, we further show that this signaling pathway is intimately associated with interactions between the epithelial and mesenchymal tissues. We also find that Ta mutants lack completely the placodes of the first developing tylotrich hairs, and that they do not show patterned expression of placodal genes, including Bmp4, Lef1, Shh, Ptch and Edar, and the genes for beta-catenin and activin A. Finally, we identified activin as a mesenchymal signal that stimulates Edar expression and WNT as a signal that induces Eda expression, suggesting a hierarchy of distinct signaling pathways in the development of skin and hair follicles. In conclusion, we suggest that Eda and Edar are associated with the onset of ectodermal patterning and that ectodysplasin/edar signaling also regulates the morphogenesis of hair follicles.