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First published online 1 March 2006
doi: 10.1242/dev.02307

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Cessation of Fgf10 signaling, resulting in a defective dental epithelial stem cell compartment, leads to the transition from crown to root formation

¹ Department of Oral Anatomy and Developmental Biology, Osaka University Graduate School of Dentistry, Osaka, Japan.
² Division of Anatomy and Cell Biology of the Hard Tissue, Department of Tissue Regeneration and Reconstruction, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan.
³ Department of Oral Anatomy II, School of Dentistry, Iwate Medical University, Morioka, Japan.
⁴ Department of Biological Science and Technology, Faculty of Engineering, University of Tokushima, Tokushima, Japan.

View larger version (35K): [in a new window]   Fig. 1. Root development in mouse molar, mouse incisor and vole molar. (A) Differential gene expression of Fgf10 during the development of mouse incisors and molars and sibling vole molars. In mouse molar development, Fgf10 is expressed in the neighboring mesenchyme adjacent to the proliferating epithelium during crown morphogenesis, but disappears at the root formation stage. In the vole molar and the mouse incisor, Fgf10 is expressed continuously only in the mesenchyme of the crown analog, but not in that of the root analog. However, slight expression of Fgf10 is detectable around the tip of the lingual epithelium of mouse incisors. (B) Higher magnification of the boxed area in A during the root development of mouse molars. The broken red line indicates the border between the crown and root. (C) Higher magnification of the boxed area during root analog (lingual side) formation in mouse incisors. The histological features at the lingual side (C) mimic the root development of mouse molars (B). The HERS-like epithelial sheath and its fragmented epithelium, like Malassez epithelial rests, are visible at the apical end. Periodontal ligaments are formed between the fragmented epithelium. d, dentin; e, enamel.

View larger version (28K): [in a new window]   Fig. 2. Incisor germ transplantation. To observe the growth of the mutant incisors, we implanted the apical regions of mutant incisors under kidney capsules. Because a tooth germ is too large to implant under a kidney capsule, we separated the region surrounding the apical end from the tooth germ and implanted only the apical end. After 3 weeks of incubation, we removed the implants.

View larger version (79K): [in a new window]   Fig. 3. Incisor growth under kidney capsules. Wild-type and mutant incisors growing under kidney capsules. (C,D) Higher magnification of wild-type (A) and mutant (B) incisors, respectively. (E-H) Translucent wild-type (E,G) and mutant (F,H) specimens. (G,H) Higher magnification of the apical region at the labial side of wild-type (E) and mutant (F) incisors, respectively. (I,J) HE staining of wild-type (I) and mutant (J) incisors. The apical regions of wild-type mouse incisors grew under the kidney capsules similar to naturally growing incisors in the mandible. The apical region is covered by alveolar bone (C, asterisk), and incisors erupt from the sockets of the bones. In the wild type, the enamel, the apical bud, the inner enamel epithelium, differentiated ameloblasts and odontoblasts are clearly recognizable at the labial side. The lingual side is root analog consisting of dentin, cementum and periodontal tissues. These features are consistent with incisors growing in the mandible. However, at the labial side of the mutant incisor, thin enamel is visible, along with enamel formation over half the developing tooth. Red arrows indicate the border between the crown and root (F,H,J). Neither the apical bud nor the dental epithelium at the surface of the dentin is recognizable. ab, apical bud; d, dentin; da, differentiated ameloblast; e, enamel; iee, inner enamel epithelium; od, odontoblasts; p, periodontal ligament. Scale bars: 2 mm in A; 1 mm in B,C; 500 µm in D,E; 300 µm in F; 100 µm in G-I; 150 µm in J.

View larger version (62K): [in a new window]   Fig. 4. Immunohistochemical analysis. Double immunostaining of CK14 (A,D,G) and Notch2 (B,E,H) in the epithelial sheaths of mutant incisors grown under kidney capsules (A-I), and staining for Notch2 in lower incisors of wild-type mice at PN d1 (J). (A-I) Both labial and lingual HERS are strongly labeled by anti-CK14 and Notch2 antibodies. Fragmentation of the dental epithelium (arrowheads), similar to Malassez epithelial rests, is also recognizable at the dentin surface. Arrows indicate non-specific staining against calcified dentin. (D-F,G-I) Higher magnifications of the labial HERS and of the lingual HERS,, respectively, in A-C. (C,F,I) Phase pictures of A,B, D,E and G,H, respectively. (J) Immunostaining for Notch2 at the apical end of PN d1 mouse lower incisors. Notch2 is expressed in the OEE and the stellate reticulum at the labial side (J, smaller arrows), and in the epithelial sheath at the lingual side (J, larger arrow). Scale bars: 25 µm in A-C; 10 µm in D-I; 200 µm in J.

View larger version (77K): [in a new window]   Fig. 5. Localization of proliferating cells at the initiation of HERS formation. BrdU labeling analysis of mutant incisors grown under kidney capsules (A-C) and of the formation of HERS in in vitro cultures (D,E). (A-C) Small amounts of labels are detected in the dental epithelium and in the surrounding mesenchyme at the apical end. (B,C) Higher magnification of BrdU immunostaining at the labial and lingual sides. BrdU-positive cells are visible in the outer layer (arrowheads) and in the inner layer (red arrows) of the epithelial sheath. Black arrows indicate BrdU-positive papilla cells. An asterisk indicates artificial space separating the inner and outer layers of the epithelial sheath formed when making sections. (D,E) BrdU staining of the mouse molar germ cultures at PN d3 and d5. The number of BrdU-positive cells (red arrows) in the IEE and the inner layer of HERS is much smaller than the number of positive cells in the OEE and the outer layer (arrowheads). (F,G) BrdU-labeled cells in the inner and outer layers and dental papilla cells in mutant incisors (F), and in vitro cultures of molar germs of PN d3 and d5 (G). More BrdU-positive cells are visible in the outer layer than in the inner layer on either side (F). Comparing the OEE/stellate reticulum (sr) and the IEE in molar germ at PN d3, or comparing the outer and inner layers at PN d5, showed similar results as those found in the mutant incisors. The total number of BrdU-positive cells increased at PN d5. Data are presented as mean±s.d. Significant differences in the mitotic indices between two groups (with error bars) were calculated using a Student's t-test (asterisks indicate P<0.01). The broken red line indicates the border between the epithelium and the mesenchyme, which is identified by the CK14 immunostaining of serial sections. Scale bars: 100 µm in A; 50 µm in B-E.

View larger version (62K): [in a new window]   Fig. 6. Analysis of semi-thin cross-sections and electron microscopy. Transverse sections of the apical regions of mutant incisors grown under kidney capsules (A-C), and electron microscopic observation of a labial region (crown analog side) at an apical end (D). (E,F) Transverse section of upper second molar mesio-buccal root of PN w3 mice. (B,C,F) Higher magnifications of boxes in A and E. (A) This transverse section is very similar to that showing root (E,F) in a mouse molar. The ring is made of dentin, and the inside is dental pulp composed of odontoblasts and dental pulp cells. At the outer surface of dentin, the epithelial cell rests (B,C, arrowheads) and the invading dental follicle cells were apparent. (D) Electron microscopic analysis of the labial region of a mutant incisor. Cementum matrix is deposited at all dentin surfaces. Cementoblast-like cells (asterisk) are observed at all outer dentin surfaces. These cells have well-developed organelles and retain thick bundles of collagen fibers for periodontal ligament (arrows). A fragmented epithelial cell rest is also recognizable (D, arrowhead). cm, cementum matrix; d, dentin; od, odontoblasts; df, dental follicle; p, pulp; pl, periodontal ligament. Scale bars: 100 µm in A; 25 µm in B,C; 5 µm in D; 50 µm in E; 10 µm in F.

View larger version (48K): [in a new window]   Fig. 7. Overexpression of Fgf10 leads to the formation of apical bud in molar germs. Stereomicroscopic observation (A-C) and frozen sections (D-F) of cultured molar germs transfected with Fgf10 cDNA. (A) The molar germ was cultured for 2 days after the transfection of Fgf10 cDNA expression vector. Fgf10 expression was identified by expression of green fluorescence protein using a fluorescence stereomicroscope and was restricted to the proximal dental papilla cells (arrow). (B) The molar germ was cultured for 5 days. The expression expands along with the growth of the dental epithelium. (C) Stereomicroscopic observation of the cultured molar germ. The proximal dental epithelium (arrow) elongates and extends to a greater extent than the distal dental epithelium (control side). (D) CK14 immunostaining (red) of a frozen section clearly shows the formation of an apical bud (larger arrow) and an expanded inter cuspal epithelium (arrowheads) following Fgf10 overexpression. However, at the control (distal) side, the epithelium forming the HERS consists of two or three epithelial cell layers (smaller arrow). (E) The expression of Fgf10 (green) is seen in the proximal dental papilla cells between the proximal dental epithelium and inter-cuspal epithelium. (F) The section was counterstained with DAPI (blue) and the three colors were merged. (G) Schematic illustration of the effects of Fgf10 overexpression. After the morphogenesis of crown, Fgf10 mRNA disappears and the formation of HERS starts (Fig. 1A). However, at the proximal side, ectopic overexpression of Fgf10 protein leads to apical bud formation. Conversely, at the distal side (control), HERS forms as Fgf10 disappeared. Scale bars: 20 µm.

View larger version (41K): [in a new window]   Fig. 8. Hypothesis of HERS formation. Schematic illustration of our hypothesis of the process of HERS formation (A-D) and of mutant incisors grown under kidney capsules (E). The more active proliferation of OEE and stellate reticulum elongates beyond the IEE after cessation of crown formation, leading to the formation of HERS. OEE, stellate reticulum and HERS expressed both Notch2 and CK14 with strong intensity (A-C, dark-yellow cells). The epithelial sheaths proliferate, and fragmentation occurs at the surface of the dentin. Dental follicle cells migrate among the fragmented epithelium and make up the periodontal tissues (D). (E) In mutant incisors, the histological features at the labial side mimic the development of mouse molar germ. A broken red line indicates the border between the crown analog and the root analog at the labial side. d, dentin; e, enamel; iee, inner enamel epithelium; oee, outer enamel epithelium; sr, stellate reticulum.

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