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FGF10 maintains stem cell compartment in developing mouse incisors

Hidemitsu Harada1,*, Takashi Toyono1, Kuniaki Toyoshima1, Masahiro Yamasaki2, Nobuyuki Itoh2, Shigeaki Kato3, Keisuke Sekine3 and Hideyo Ohuchi4

1 Second Department of Oral Anatomy and Cell Biology, Kyushu Dental College, 2-6-1, Manazuru, Kokurakita-ku, Kitakyushu, 803-8580, Japan
2 Department of Genetic Biochemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida-shimo-Adachi-cho, Sakyo-ku, Kyoto City, Kyoto, 606-8501, Japan
3 Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo 113-0032, Japan
4 Department of Biological Science and Technology, Faculty of Engineering, University of Tokushima, 2-1 Minami-Jyosanjima-cho, Tokushima City, Tokushima 770-8506, Japan



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  		Fig. 1. The developing mouse incisor during the initiation (E11), bud (E13), cap (E14), early bell (E16) and late bell (E19.5) stages. The initial stages of morphogenesis are very similar in all teeth. The first morphological sign of incisor development is a thickening of the oral epithelium (E11), which subsequently buds into the underlying mesenchyme (E13, bud stage). At later stages, the developing incisor rotates anteroposteriorly (E14, cap stage) and becomes parallel to the long axis of the incisors (E16, early bell stage). At early bell stage (E16), the cervical loop is seen at the apical end of the labial epithelium. Only the labial epithelium gives rise to the enamel-forming ameloblasts (E19.5, late bell stage). Epithelium in dark blue, dental mesenchyme in light blue dots, enamel in red and dentin in green. cl, cervical loop; cm, condensed mesenchyme; d, dentine; dp, dental papilla; e, enamel; eo, enamel organ; m, molar; oe, oral epithelium; p, pulp; vl, vestibular lamina.

 


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  		Fig. 2. Absence of cervical loop in the developing incisor germ of FGF null mice. These pictures show Hematoxylin and Eosin stained sections of lower incisor germs at cap (A,C), early bell (B,D) and late bell (E,G) stages in Fgf10+/+ embryos (A,B,E,F) and Fgf10–/– embryos (B,D,G,H). At cap stage (E14), FGF10 null mice did not show clear morphological abnormalities in incisor development (C). Arrows show the end of labial epithelium. At early bell stage (E16), the epithelial bulge of FGF null mice was smaller than that of wild type (B,D, arrows). At late bell stage (E19), the cervical loop was missing in FGF10 null mice (G,H). F and H show higher magnification of E and G at the region of the apical end. (I-L) Immunostaining of cytokeratin (CK) at late bell stage (E19). At the apical end of wild type, the center region of the stellate reticulum in the cervical loop was strongly CK positive (J). The peripheral region of the stellate reticulum, found in close proximity to basal epithelium, was CK negative (J, smaller asterisk). In mutant incisor, CK-negative stellate reticulum cells were missing around strongly CK-positive cells shown by arrowheads (L). Arrows show the apical end of labial epithelium. The staining of erythrocytes was due to nonspecific binding of antibody (L, larger asterisk). Hematoxylin and Eosin staining of the lower first molar germ in Fgf10+/+ (M) and Fgf10–/– (N) embryos. be, basal epithelium; cl, cervical loop; ie, inner enamel epithelium; oe, outer enamel epithelium; sr, stellate reticulum. Scale bars: 200 µm in A-D,E,G,I,K,M,N; 100 µm in F,H,J,L.

 


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  		Fig. 3. Gene expression patterns of Fgf3 and Fgf10 in the developing lower incisor by in situ hybridization. Both Fgf3 and Fgf10 mRNA was co-expressed in the dental papilla at the cap stage (E14) (A,B). At early bell stage (E16), Fgf3 mRNA was restricted to the mesenchyme underlying the inner enamel epithelium (D). Fgf3 mRNA was not seen around the cervical loop (D, arrowheads) and lingual epithelium (arrow). Cells expressing Fgf10 mRNA extended to the mesenchyme neighboring the inner enamel epithelium and covered the cervical loop (C, arrowheads). Scale bar: 200 µm. cl, cervical loop.

 


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  		Fig. 4. Development of the apical end of incisors in Fgf10+/+ and Fgf10–/– mice by organ culture and estimation of growing dental epithelium. (A) The dissected incisors from the mandible of Fgf10+/+ or Fgf10+/– (n=8) and Fgf10–/– mice (n=7) at E19 were cultured for 20 days as described. (B) All pictures were digitized, and the growth length of dental epithelium was estimated using NIH image 1.62. The graph of average growth rate was created using Microsoft Excel 98. red line, Fgf10+/+ or Fgf10+/–; blue line, Fgf10–/–. (C) These explants were cultured for 30 days. Arrows show disruption of the dental epithelium in the mutant. Arrowheads indicate apical end of the dental epithelium in the explants. Scale bars: 200 µm. *P<0.05.

 


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  		Fig. 5. Distribution of dividing cells in apical end of dental epithelium. (A) Representative longitudinal section of lower incisors and first molars in Fgf10+/+ and Fgf10–/– embryos at E18 as shown by BrdU labeling. In lower incisor of Fgf10+/+ embryos (a,b), BrdU-incorporating cells are sparsely distributed in the cervical loop (b) and densely distributed in inner enamel epithelium. In lower incisor of Fgf10–/– embryos (c,d), the labeling cells were seen densely at the edge of dental epithelium. The sparse pattern seen in the cervical loop of the Fgf10+/+ embryo was not observed (arrows in c,d). b and d show higher magnification of a and c at the apical end of the dental epithelium. Black dots outline the dental epithelium. In molar germs of Fgf10+/+ and Fgf10–/– embryos (e,f), BrdU is densely incorporated into the inner dental epithelium. Arrows show the apical end of dental epithelium. (B) Quantification of percentage of BrdU-positive cells at the apical region of dental epithelium in incisors and molars of wild type and mutant. The percentage of BrdU-posisitve cells in the cervical loop in wild type is significantly lower than the percentage in the inner enamel epithelium. Each bar presents data from three samples. Error bars represent standard deviation. ie, inner enamel epithelium; dp, dental papilla; I, incisor; M, molar. Scale bars: 200 µm in a,c,e,f; 100 µm in b,d.

 


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  		Fig. 6. Effect of anti-FGF10 neutralizing antibody on cultured incisor explants. The explants were cultured for 24 hours in the presence of anti-FGF10 antibody (A-C), anti-FGF3 antibody (D-F), anti-FGF10 antibody and human recombinant FGF10 (G-I), or normal goat IgG (J-L). Apoptotic cells were detected by Annexin V staining. Anti-FGF10 antibody disrupted the cervical loop epithelium (A,B) and caused apoptosis in the cervical loop cells (C). In the presence of anti-FGF3 antibody (D,E) or anti-FGF10 antibody and human recombinant FGF10 (G,H), the cervical loop was recognized. Anti-FGF3 antibody caused partly apoptosis in the inner enamel epithelium and the neighboring mesenchyme, but apoptotic cells were rarely seen in the cervical loop and the surrounding mesenchyme. In the presence of control IgG, few apoptotic cells were detected (K,L). Asterisks show the cervical loop. Scale bar: 200 µm.

 


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  		Fig. 7. FGF10 rescued the isolated cervical loop epithelium from apoptosis. The cervical loop epithelium was separated from the mesenchyme (A) and cultured for 48 hours (B,D,F-J) in the medium supplemented with recombinant human FGF10 at a concentration of 0.1 ng/ml (F,F'), 1.0 ng/ml (D,D',E,E'), 10 ng/ml (G,G'), with bovine serum albumin (B,B',C,C'), with FGF10-releasing beads (H,H'), with bovine serum albumin (BSA) -soaked beads (I,I'), or with FGF10 (1 ng/ml) and FGF10 neutralizing antibody (50 µg/ml) (J,J'). Apoptotic cells were detected using Annexin V staining (B'-J',L',N',O'). In the presence of FGF10 (1 ng/ml and 10 ng/ml), the outline of the isolated epithelium was clearly maintained (D,G) and apoptotic cells bound to Annexin V were sparse (D',E',G'). Higher magnification (E) shows that the cell morphologies of the basal epithelium and the peripheral stellate reticulum (putative stem cells) in the explant were clearly maintained. The explants cultured with BSA, BSA beads or FGF10 (1 ng/ml) and FGF10 neutralizing antibody (50 µg/ml) showed disrupted epithelium (B,C,I,J) and many apoptotic cells (B',C',I',J'). The shape of cells in the disrupted cervical loop epithelium was not clearly recognized (C) and a large number of cells were bound to Annexin V (C'). The explant in 0.1 ng/ml FGF10 showed the outline clearly (F), but many of these cells underwent apoptosis (F'). (K,P) The isolated mesenchyme (M) was placed in contact with the isolated cervical loop epithelium (A) and cultured for 2 days. In the mesenchyme adherent to the epithelium, apoptosis was rarely seen (L,L'). Dots outline the border between the epithelium and the mesenchyme (L,L'). In the isolated mesenchyme, several apoptotic cells were seen in the presence (O,O') and absence (N,N') of FGF10 beads. Scale bars: 200 µm in A,B,D,F-O; 40 µm in C,E.

 

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© The Company of Biologists Ltd 2002