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The Dentin Matrix Protein 1 (Dmp1) is Specifically Expressed in Mineralized, but not Soft, Tissues during Development
J.Q. Feng1,2,*,#,
H. Huang1,2,
Y. Lu1,*,
L. Ye1,
Y. Xie1,
T.W. Tsutsui3,
T. Kunieda3,4,
T. Castranio3,
G. Scott3,
L.B. Bonewald1 and
Y. Mishina3
1 Department of Oral Biology, School of Dentistry, University of Missouri-Kansas City, 650 E. 25th Street, Kansas City, MO 64108, USA;
2 Department of Orthopaedic Surgery, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing, China;
3 Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences/NIH Research Triangle Park, NC;
4 Graduate School of Natural Science and Technology, Okayama University;
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Figure 1. Generation of Dmp1 null mice by gene targeting. (A) Schematics of mouse Dmp1 wild-type locus with locations of key restriction enzyme sites, and the NLSLacZpA vector (NLS, nuclear localization signal peptide) with the neo cassette inserted into exon 6 at the ApaI and EcoRV sites. Southern blot screening strategy is also displayed. Note that the coding region for 384 out of 503 amino acids, including RGD, has been removed. (B) Representative Southern blot analysis of DNA from a wild-type and a heterozygous ES clone. DNA was digested with BamHI, and hybridized with 5' and 3' probes separately, resulting in a 15-Kb band that corresponds to the wild type. There are two additional BamHI cut sites on the lacZ neo cassette, which gives two bands for the heterozygous ES cell clone: 9.1 Kb for the 5' side and 6.8Kb for the 3' side, respectively. Analysis of the data demonstrates that one of the Dmp1 alleles is replaced by the inserted lacZ cDNA, whose expression will be used to reflect the endogenous Dmp1 expression pattern (see Fig. 2 ). (C) Northern blot and RT-PCR analyses. The mRNAs were extracted from wild-type (+/+), heterozygous (+/-), and homozygous knock-in (-/-) calvarial cells. PCR amplification was then performed with 2-µL aliquots of the target cDNA. A 5' primer (5'-CGGCTGGTGGACTCTCTAAG-3', corresponding to an oligonucleotide of 374 to 393 of Dmp1 cDNA) and a 3' primer (5'-CGGGGTCGTCGCTCTGCATC-3', corresponding to an oligonucleotide of 750 to 769 of Dmp1 cDNA) were used for Dmp1 RT-PCR. No wild-type Dmp1 mRNA was detected in the homozygous knock-in samples by both Northern and RT-PCR analysis, while a 5.8-Kb fusion transcript containing exons 1-5 of Dmp1 and the lacZ reporter gene was detected in both heterozygous and homozygous knock-in samples. (D) Dmp1 immunostaining. The femurs from neonatal Day 19 wild-type (left) and homozygous knock-in (right) mice were stained by an antibody to DMP1 peptide (105 to 125). There is no detectable signal (brown) in Dmp1 lacZ-knock-in homozygous mice, while a strong signal is obvious in osteocytes in control mice.
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Figure 2. Dmp1 lacZ expression pattern during early development. (A) Dmp1 expression in skeletogenesis. Dmp1 lacZ heterozygous embryos (E15.5), newborn, and day 7 pups were selected for the tracking of Dmp1 expression during early skeletal development. (B) Dmp1 expression pattern in tooth. Sections of incisor (left, newborn) and an adjacent area between the dentin and the cement (middle, newborn), and a 2nd molar subjected to in situ hybridization (right, lacZ probe) are shown. Note that lacZ staining, which reflects endogenous Dmp1 expression, is detected in all mineralized tissues mainly in the nucleus, due to the addition of a nuclear localization signal peptide. LacZ staining (green) is seen in hypertrophic chondrocytes (HC), osteoblasts (Ob), and osteocytes (Ocy), as well as in cementoblasts (Cb), pulp cells, and odontoblasts (Od). Dmp1 lacZ signals are also detected in ameloblasts (Am) by in situ hybridization. There is no X-Gal staining in brain tissue. AB, alveolar bone.
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Figure 3. Dmp1 null embryos and newborns appear grossly "normal". (A) Representative radiographs of the wild-type (+/+) and the knockout (-/-) embryos of day 15 are shown. (B,C) Alizarin red/Alcian blue staining of the wild-type (+/+) and the knockout (-/-) embryos of day 18 (B) and newborns (C) is shown.
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Figure 4. Histological analysis (H&E) of incisors and distal femurs from 18-day-old control and Dmp1 null embryos. (A) Representative tooth germs (upper incisors) of the wild-type (+/+, left) and the homozygous null (-/-, right) embryos of day 18 are shown. There are no apparent morphological changes in dentin (red) and odontoblast and ameloblast layers (purple). (B) Representative long bone (femur) of the wild-type (+/+, left) and the homozygous null (-/-, right) embryos of day 18 are shown. A slightly expanded hypertrophic zone and a widened bone shaft are shown. Am, ameloblast; HC, hypertrophic chondrocytes; Od, odontoblasts; d, dentin.
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Journal of Dental Research, Vol. 82, No. 10,
776-780 (2003)
DOI: 10.1177/154405910308201003
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