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. 2003 Feb;23(3):1075-84.
doi: 10.1128/MCB.23.3.1075-1084.2003.

Essential role for NFI-C/CTF transcription-replication factor in tooth root development

George Steele-Perkins  1 , Kenneth G Butz, Gary E Lyons, Margarita Zeichner-David, Heung-Joong Kim, Moon-Il Cho, Richard M Gronostajski
Affiliations

Essential role for NFI-C/CTF transcription-replication factor in tooth root development

George Steele-Perkins et al. Mol Cell Biol. 2003 Feb.

Abstract

The mammalian tooth forms by a series of reciprocal epithelial-mesenchymal interactions. Although several signaling pathways and transcription factors have been implicated in regulating molar crown development, relatively little is known about the regulation of root development. Four genes encoding nuclear factor I (NFI) transcription-replication proteins are present in the mouse genome: Nfia, Nfib, Nfic, and NFIX: In order to elucidate its physiological role(s), we disrupted the Nfic gene in mice. Heterozygous animals appear normal, whereas Nfic(-/-) mice have unique tooth pathologies: molars lacking roots, thin and brittle mandibular incisors, and weakened abnormal maxillary incisors. Feeding in Nfic(-/-) mice is impaired, resulting in severe runting and premature death of mice reared on standard laboratory chow. However, a soft-dough diet mitigates the feeding impairment and maintains viability. Although Nfic is expressed in many organ systems, including the developing tooth, the tooth root development defects were the prominent phenotype. Indeed, molar crown development is normal, and well-nourished Nfic(-/-) animals are fertile and can live as long as their wild-type littermates. The Nfic mutation is the first mutation described that affects primarily tooth root formation and should greatly aid our understanding of postnatal tooth development.

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Figures

FIG. 1.
FIG. 1.
Targeted mutation of the mouse Nfic gene. (A) Targeting vector (top), the Nfic region encompassing exons 1 and 2 (middle), and their homologous recombination product (bottom). The termini of homologous sequences and relevant restriction sites are noted. The positive and negative selection gene expression cassettes are boxes labeled Neo and TK, respectively, and their transcription orientations are indicated by arrows. Exons are indicated by open boxes labeled Exon. The 3′-flanking probe (EcoRV-HindIII; short bar) and the wild-type and mutant EcoRI restriction fragments it identifies in Southern analyses (thick bars) are shown. Plasmid backbone sequences are denoted by a thin line. Locations of the three primers (a, b, and c) used for PCR genotyping are indicated by small arrows. (B) Data from Southern blot screen of G418- and ganciclovir-resistant ES clones. Each of six clones is numbered above, and the hybridizing wild-type (WT 9.5kb) and mutant (KO 3.7kb) EcoRI fragments are indicated on the left. Clones 3 and 6 have a targeted mutation, whereas clones 1, 2, 4, and 5 do not. (C) PCR genotyping of one litter from an Nfic heterozygous cross. PCRs employing the three primers indicated in panel A plus two Y-chromosome-specific primers were used to screen tail biopsies of a litter of nine (lanes 1 to 9). Labels on the left indicate the 545-bp targeted-gene product (KO), the 358-bp wild-type product (WT), and the male-specific products (Ychr). Lanes (lane/progeny, Nfic genotype, sex): 1, +/+, female; 2, +/−, male; 3, +/−, female; 4, −/−, male; 5, −/−, male; 6, −/−, female; 7, +/+, female; 8, +/−, female; 9, +/−, female. Lane Mr is a 100-bp DNA ladder. (D) RT-PCR analysis of Nfic transcripts in tissues of each Nfic genotype. The total RNA isolated from brains (lanes B) or livers (lanes L) of Nfic wild-type (+/+), heterozygote (+/−), and homozygote (−/−) male P193 littermates was reverse transcribed, and the cDNA was then analyzed by PCR with exon 1- and exon 3-specific primers. The products corresponding to wild-type (WT) and aberrant (Exon 2−) transcripts are indicated. Lane Mr is a 100-bp DNA ladder.
FIG. 2.
FIG. 2.
Growth retardation and increased mortality of Nfic−/− mice. (A) Female P54 Nfic+/− and Nfic−/− littermates. Note the severe runting of the −/− animal. A mirror image is shown in order to present the −/− sibling on the right. (B) Mortality of Nfic mice reared on standard chow. The percent survival of Nfic−/− (n = 34), Nfic+/− (n = 84), and Nfic+/+ (n = 33) mice are plotted versus time. The points of the +/− and +/+ curves indicate the time of sacrifice of littermates of Nfic−/− animals that had died. These studies were performed prior to observing tooth defects in Nfic−/− animals. Also, the increased mortality of Nfic−/− animals was not observed if they are reared on a soft-dough diet (see Results).
FIG. 3.
FIG. 3.
Abnormal mandibular incisors of Nfic−/− mice. (A) View of mouths of two P40 female littermates (Nfic+/− and Nfic−/−) reared on a soft-dough diet. Lower labia and tongues were excised to help expose incisors. Note the abnormally thin mandibular incisors and the extended gingiva of the −/− animal. (B) Mandible portion of an H&E-stained coronal section of the jaw of an Nfic+/+ P135 male. The tongue is at the top. An arrowhead and an arrow indicate the enamel and dentin, respectively. The majority of the enamel was lost during decalcification. (C) Same as panel B except the individual is a Nfic−/− littermate. Arrowheads indicate disorganized tissue where incisor structures are expected.
FIG. 4.
FIG. 4.
Abnormal maxillary incisors of Nfic−/− mice. (A) Close view of maxillary incisors in situ of a P135 Nfic+/− male reared on standard chow. The black bar indicates thickness of the right maxillary incisor. (B) Same as panel A except the individual is an Nfic−/− littermate. Note that the incisor is thin compared to panel A. (C) Upper portion of an H&E-stained coronal section of the head of a P135 Nfic+/+ male. The tongue is at the bottom. The box locates the right maxillary incisor. (D) Same as panel C except the individual is an Nfic−/− littermate. (E) Magnified view of boxed area in panel C. An arrowhead and an arrow indicate the enamel and dentin, respectively. Note the uniform layer of dentin around the circumference of the incisor. (F) Magnified view of boxed area in panel D. An arrowhead indicates the enamel deposition at the labial side, whereas the arrow indicates the absence of dentin at the lingual side of the incisor.
FIG. 5.
FIG. 5.
Failure of molar root development in Nfic−/− mice. The panels compare the molars of P190 male (A to D), P15 male (E and F), and P14 male (G and H) littermates of the indicated Nfic genotype. (A and C) Lateral views of first (left) and second maxillary (A) and mandibular (C) molars of a +/− mouse. (B and D) Lateral views of first (left) and second maxillary (B) and mandibular (D) molars of a −/− mouse. (E and F) Lateral views of first (left) and second (right) mandibular molars of +/+ (E) and −/− (F) littermates. Bar, 1 mm. (G and H) H&E-stained sagittal sections of the developing left mandibles of +/+ (G) and −/− (H) littermates. The perimeter of the immature first and second molars in each is demarcated by dark-staining dentin. Arrows denote the immature roots present in panel G but absent in panel H.
FIG. 6.
FIG. 6.
Abnormal alveolar bone formation in Nfic−/− mice. (A) View of maxillae of skulls prepared from P97 male +/− and −/− littermates. (B) Closeup of molar root areas in center of panel A. Note that the +/− mouse maxilla's left molars were extracted for the closeup, whereas the −/− mouse molars dislodged during skull preparation. (C) Lingual view of left mandible of the same +/− animal as in panel A. (D) Lingual view of left mandible of the same −/− animal as in panel A. (E) Lingual view of left mandibles isolated from P15 male +/+ and −/− littermates. Bar, 1 mm. (F) Closeup view of molar root sockets of mandibles shown in panel E. Both the +/+ and −/− mouse mandibles are forming bone between the three molars (arrowheads). However, the −/− mouse mandible is not forming bone at the regions between the missing roots of the first and second molars (arrows). The third molar of the +/+ mouse mandible did not dislodge during skull preparation (bottom).
FIG. 7.
FIG. 7.
Nfic expression during normal tooth development. Nfic expression was detected in wild-type animals by in situ hybridization using 35S-labeled (A and B) and DIG-labeled (C to H) antisense (B, C, E, and G) and control sense (D, F, and H) Nfic probes. Panels A and B show phase-contrast and dark-field images, respectively, of an E15.5 mouse first maxillary molar hybridized with a 35S-labeled Nfic antisense probe. Note the high Nfic expression in the molar mesenchymal cells (arrowhead), with weaker expression in the epithelium (asterisk). Panels C and D show staining by Nfic antisense and sense probes, respectively, of the mandible of a P8 mouse. Note the expression of Nfic in ameloblasts (arrow), odontoblasts (arrowheads), and surrounding connective tissue (top asterisk, stellate reticulum; bottom asterisk, mesenchymal tissues) and in the pulp of the molars and incisor (i) of panel C. No staining is seen with the control sense probe (D). Panels E and F show staining by Nfic antisense and control sense probes, respectively, in mouse P14 first maxillary molars. Sections were counterstained lightly with methyl green to identify cell types. Nfic is expressed in crown odontoblasts (arrow) and connective tissue (asterisks). Panels G and H are higher magnifications of panels E and F, respectively, showing the expression of Nfic in root odontoblasts (arrowhead), preodontoblasts (arrow), periodontal ligament (p), and bone (b) in panel G. Similar expression of Nfic was seen in mandibular molars at this stage (not shown).
FIG. 8.
FIG. 8.
Reduced tooth-specific gene expression in the mandibles of Nfic−/− mice. Nfix, Nfia, Nfib, α1 type I collagen (col), amelogenin (Amg), ameloblastin (Amb), and DSPP transcript levels were quantified by real-time quantitative PCR and normalized to β2-microglobulin levels in the same samples. The bars represent the average of three animals of each genotype. Values are expressed as the transcript levels found in heterozygous (white bars, +/−) and Nfic−/− animals (black bars, −/−) relative to the levels seen in wild-type littermates.
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References

    1. Bei, M., K. Kratochwil, and R. L. Maas. 2000. BMP4 rescues a non-cell-autonomous function of Msx1 in tooth development. Development 127:4711-4718. - PubMed
    1. Chai, Y., X. Jiang, Y. Ito, P. Bringas, Jr., J. Han, D. H. Rowitch, P. Soriano, A. P. McMahon, and H. M. Sucov. 2000. Fate of the mammalian cranial neural crest during tooth and mandibular morphogenesis. Development 127:1671-1679. - PubMed
    1. Chaudhry, A. Z., C. E. Lyons, and R. M. Gronostajski. 1997. Expression patterns of the four nuclear factor I genes during mouse embryogenesis indicate a potential role in development. Dev. Dyn. 208:313-325. - PubMed
    1. das Neves, L., C. Duchala, F. Godinho, M. Haxhiu, C. Colmenares, W. Macklin, C. Campbell, K. Butz, and R. Gronostajski. 1999. Disruption of the murine nuclear factor I-A gene (Nfia) results in perinatal lethality, hydrocephalus and agenesis of the corpus callosum. Proc. Natl. Acad. Sci. USA 96:11946-11951. - PMC - PubMed
    1. Dassule, H. R., P. Lewis, M. Bei, R. Maas, and A. P. McMahon. 2000. Sonic hedgehog regulates growth and morphogenesis of the tooth. Development 127:4775-4785. - PubMed

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