Depolarization induces calcium-dependent BMP4 release from mouse embryonic palate mesenchyme
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ABSTRACT Ion channels are essential for proper morphogenesis of the craniofacial skeleton. However, the molecular mechanisms underlying this phenomenon are unknown. Loss of the Kcnj2 potassium channel disrupts Bone Morphogenetic Protein (BMP) signaling within the developing palate. BMP signaling is essential for the correct development of several skeletal structures, including the palate, though little is known about the mechanisms that govern BMP secretion. We introduce a tool to image the release of bone morphogenetic protein 4 (BMP4) from mammalian cells. Using this tool, we show that depolarization induces BMP4 release from mouse embryonic palate mesenchyme cells in a calcium-dependent manner. We show native transient changes in intracellular calcium occur in cranial neural crest cells, the cells from which embryonic palate mesenchyme derives. Waves of transient changes in intracellular calcium suggest that these cells are electrically coupled and may temporally coordinate BMP release. These transient changes in intracellular calcium persist in palate mesenchyme cells from embryonic day (E) 9.5 to 13.5 mice. Disruption of Kcnj2 significantly decreases the amplitude of calcium transients and the ability of cells to secrete BMP. Together, these data suggest that temporal control of developmental cues is regulated by ion channels, depolarization, and changes in intracellular calcium for mammalian craniofacial morphogenesis. SUMMARY We show that embryonic palate mesenchyme cells undergo transient changes in intracellular calcium. Depolarization of these cells induces BMP4 release suggesting that ion channels are a node in BMP4 signaling.Keywords:
Mesenchyme
Calcium in biology
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Going in circles: conserved mechanisms control radial patterning in the urinary and digestive tracts
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Previous studies have suggested that Bmp4 is a key Msx1-dependent mesenchymal odontogenic signal for driving tooth morphogenesis through the bud-to-cap transition. Whereas all tooth germs were arrested at the bud stage in Msx1(-/-) mice, we show that depleting functional Bmp4 mRNAs in the tooth mesenchyme, through neural crest-specific gene inactivation in Bmp4(f/f);Wnt1Cre mice, caused mandibular molar developmental arrest at the bud stage but allowed maxillary molars and incisors to develop to mineralized teeth. We found that expression of Osr2, which encodes a zinc finger protein that antagonizes Msx1-mediated activation of odontogenic mesenchyme, was significantly upregulated in the molar tooth mesenchyme in Bmp4(f/f);Wnt1Cre embryos. Msx1 heterozygosity enhanced maxillary molar developmental defects whereas Osr2 heterozygosity partially rescued mandibular first molar morphogenesis in Bmp4(f/f);Wnt1Cre mice. Moreover, in contrast to complete lack of supernumerary tooth initiation in Msx1(-/-)Osr2(-/-) mice, Osr2(-/-)Bmp4(f/f);Wnt1Cre compound mutant mice exhibited formation and subsequent arrest of supernumerary tooth germs that correlated with downregulation of Msx1 expression in the tooth mesenchyme. In addition, we found that the Wnt inhibitors Dkk2 and Wif1 were much more abundantly expressed in the mandibular than maxillary molar mesenchyme in wild-type embryos and that Dkk2 expression was significantly upregulated in the molar mesenchyme in Bmp4(f/f);Wnt1Cre embryos, which correlated with the dramatic differences in maxillary and mandibular molar phenotypes in Bmp4(f/f);Wnt1Cre mice. Together, these data indicate that Bmp4 signaling suppresses tooth developmental inhibitors in the tooth mesenchyme, including Dkk2 and Osr2, and synergizes with Msx1 to activate mesenchymal odontogenic potential for tooth morphogenesis and sequential tooth formation.
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The signalling peptide encoded by the sonic hedgehog gene is restricted to localised thickenings of oral epithelium, which mark the first morphological evidence of tooth development, and is known to play a crucial role during the initiation of odontogenesis. We show that at these stages in the murine mandibular arch in the absence of epithelium, the Shh targets Ptc1 and Gli1 are upregulated in diastema mesenchyme, an edentulous region between the sites of molar and incisor tooth formation. This ectopic expression is not associated with Shh transcription but with Shh protein, undetectable in the presence of epithelium. These findings suggest that, in diastema mesenchyme, restriction of Shh activity is dependent upon the overlying epithelium. This inhibitory activity was demonstrated by the ability of transplanted diastema epithelium to downregulate Ptc1 in tooth explants, and for isolated diastema mesenchyme to express Ptc1. A candidate inhibitor in diastema mesenchyme is the glycosylphosphatidylinositol-linked membrane glycoprotein Gas1. Gas1 is normally expressed throughout mandibular arch mesenchyme; however, in the absence of epithelium this expression was downregulated specifically in the diastema where ectopic Shh protein was identified. Although Shh signalling has no effect upon Gas1 expression in mandibular arch mesenchyme, overexpression of Gas1 results in downregulation of ectopic Ptc1. Therefore, control of the position of tooth initiation in the mandibular arch involves a combination of Shh signalling at sites where teeth are required and antagonism in regions destined to remain edentulous.
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