Fisp12/CTGF is a Target of BMP Signaling during Early Odontogenesis

Objective: Bone morphogenetic protein-4 (BMP-4) is an important signaling molecule involved in epithelial-mesenchymal interactions during odontogenesis. In recent work, we showed that another signaling molecule, Fisp12/Connective Tissue Growth Factor (CTGF), is expressed by dental lamina and dental mesenchyme at bud stage, and then reverts to dental epithelium at cap/bell stage. Because such expression pattern is similar to that of BMP-4, we asked whether BMP-4 regulates Fisp12/CTGF expression and what roles Fisp12/CTGF has during odontogenesis. Methods: Mandibular explants containing bud stage tooth germs from Day 12-13 mouse embryos were reared in organ culture using serum free-BGJb medium. Epithelium was enzymatically removed from the explants prior to culture. Beads pre-coated with recombinant BMP-4 were placed onto the mesenchyme, and explants were re-incubated for various time and processed for whole mount in situ hybridization. Results: BMP-4-coated beads led to effective induction of Fisp12/CTGF expression. Doses of 100 to 250 ng/ml BMP-4 were needed to induce Fisp12/CTGF expression in dental and non-dental mesenchyme. Fisp12/CTGF expression was induced within 1 hr, lasted for 24 hrs and decreased by 40 hrs. To determine whether BMP-4 is an endogenous regulator of Fisp12/CTGF expression, beads pre-coated with the BMP antagonist Noggin were used. Indeed, endogenous Fisp12/CTGF expression as well as that induced by BMP-4-coated beads was significantly inhibited. In contrast, recombinant CTGF (rCTGF) was unable to induce BMP-4 expression. To analyze the roles of Fisp12/CTGF, primary cultures of mandibular mesenchymal cells from E11-12 were treated with rCTGF; this resulted in appreciable stimulation of proliferation. To confirm this finding, rCTGF-coated beads placed onto bud stage dental mesenchyme led local stimulation of cell proliferation. Conclusion: Our data demonstrate that Fisp12/CTGF is a down-stream target of BMP-4 signaling and appears to act as a mitogen for dental mesenchyme during early stages of odontogenesis. NIDR grant RO1DE13206 to E.K.