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Identification of Cis-DNA Regions Controlling Bmp4 Expression during Tooth Morphogenesis in vivo

School of Dentistry, Dept. of Oral Biology, University of Missouri-Kansas City, 650 E. 25th St., Kansas City, MO 64108;

View larger version (69K): [in this window] [in a new window]   Figure 1. LacZ is strongly expressed during early tooth morphogenesis in Bmp4lacZneo knock-in mice, while LacZ expression in transgenic mice harboring the 2.4-kb Bmp4 promoter LacZ (Bmp4 LacZ 2.4p) construct is absent when evaluated at the same developmental stages. (A) Stage E11.5 whole-mount embryos from heterozygous Bmp4lacZneo knock-in (left panel) and Bmp4LacZ2.4p (right panel) mice were stained for β-galactosidase activity overnight. Note that expression signals in the somites are similar in Bmp4lacZneo knock-in mice and Bmp4LacZ2.4p mice, although the expression levels are different. A series of frozen sections from early incisor tooth buds from E12.5 (B), E13.5 (C), and E15.5 (D) embryos was stained with x-gal. A schematic construct map is displayed in the lower panels. Expression of LacZ in mice harboring the Bmp4lacZneo knock-in construct, in which the NLS-LacZ-neo cassette replaces part of Exon3 in the mouse Bmp4 gene, is a sensitive indicator of endogenous Bmp4 expression. Abbreviations: E, epithelium; M, mesenchyme; DF, dental follicle; DO, dental organ; DP, dental papilla. The data shown are representative of three separate experiments in the same developmental stages.

View larger version (71K): [in this window] [in a new window]   Figure 2. Bmp4 LacZ expression in mesenchyme-derived pulp cells and odontoblasts is absent in Bmp4LacZ2.4p mice. Sagittal sections from the head region of newborns were stained for β-galactosidase activity. The upper panels are from Bmp4LacZ2.4p mice, while the lower panels are from Bmp4lacZneo knock-in mice. Schematic construct maps are displayed in the right panels. LacZ expression patterns in Bmp4lacZneo knock-in and Bmp4LacZ2.4p mice are the same in all incisors and molars. Abbreviations: Am, ameloblast; Od, odontoblast; P, pulp cells. Note that the construct used in Bmp4LacZknock-in mice contains nuclear localization signal peptide; thus, the expressed β-galactosidase is mainly in the nucleus. The data shown are representative of four separate experiments.

View larger version (60K): [in this window] [in a new window]   Figure 3. The 1.1-kb Bmp4 gene region contains the control domains for Bmp4 expression in the epithelium-derived ameloblast. Sections of molars from newborn Bmp4lacZneo knock-in (A, labeled Bmp4LacZknock-in) mice, Bmp4LacZ2.4p (B), and Bmp4LacZ1.1p (C) mice were stained by the β-galactosidase antibody (green). A white arrow indicates the odontoblast layer in each panel, while a red one designates the ameloblast layer. Schematic presentation of the Bmp4 LacZ constructs is shown in the lower panels. Bmp4 promoter fragments –2372 to +258 (2.4 kb) and the -1140 to +212 (1.1 kb) are used to generate Bmp4LacZ2.4p and Bmp4LacZ1.1p mice. Abbreviations: Am, ameloblast; Od, odontoblast. The data shown are representative of three separate experiments in the same developmental stages.

View larger version (39K): [in this window] [in a new window]   Figure 4. Temporal and spatial expression of Bmp4 during tooth development is controlled by different domains of the Bmp4 gene. The upper panel depicts a schematic of the working hypothesis for control of Bmp4 expression during tooth development. Supporting data show that LacZ staining in Bmp4lacZneo knock-in mice, representative of endogenous Bmp4 expression, first appears in the epithelium (A, 200x), followed by the mesenchyme and enamel knot (B, 200x), and then in the mature tooth cells such as pulp cells (C, 200x), odontoblast, and ameloblast cells (D, 40x). By contrast, panel E shows that LacZ stains from the Bmp4LacZ2.4p mice are present only in the epithelium-derived ameloblasts. We propose that the Bmp4 gene region from –260 bp to –1.1 kb controls Bmp4 expression in the epithelium-derived ameloblasts, while Bmp4 expression in the mesenchyme-derived pulp cells and odontoblast cells is controlled by other regions. The control domains for Bmp4 expression in primordial tooth cells (PTC, see Fig 1 for more support data) are located in other regions of the Bmp4 gene, outside of the 2.4-kb Bmp4 gene area. Abbreviations: Am, ameloblast; Od, odontoblast; P, pulp cells.

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