An integrated gene regulatory network controls stem cell proliferation in teeth

Abstract

Epithelial stem cells reside in specific niches that regulate their self-renewal and differentiation, and are responsible for the continuous regeneration of tissues such as hair, skin, and gut. Although the regenerative potential of mammalian teeth is limited, mouse incisors grow continuously throughout life and contain stem cells at their proximal ends in the cervical loops. In the labial cervical loop, the epithelial stem cells proliferate and migrate along the labial surface, differentiating into enamel-forming ameloblasts. In contrast, the lingual cervical loop contains fewer proliferating stem cells, and the lingual incisor surface lacks ameloblasts and enamel. Here we have used a combination of mouse mutant analyses, organ culture experiments, and expression studies to identify the key signaling molecules that regulate stem cell proliferation in the rodent incisor stem cell niche, and to elucidate their role in the generation of the intrinsic asymmetry of the incisors. We show that epithelial stem cell proliferation in the cervical loops is controlled by an integrated gene regulatory network consisting of Activin, bone morphogenetic protein (BMP), fibroblast growth factor (FGF), and Follistatin within the incisor stem cell niche. Mesenchymal FGF3 stimulates epithelial stem cell proliferation, and BMP4 represses Fgf3 expression. In turn, Activin, which is strongly expressed in labial mesenchyme, inhibits the repressive effect of BMP4 and restricts Fgf3 expression to labial dental mesenchyme, resulting in increased stem cell proliferation and a large, labial stem cell niche. Follistatin limits the number of lingual stem cells, further contributing to the characteristic asymmetry of mouse incisors, and on the basis of our findings, we suggest a model in which Follistatin antagonizes the activity of Activin. These results show how the spatially restricted and balanced effects of specific components of a signaling network can regulate stem cell proliferation in the niche and account for asymmetric organogenesis. Subtle variations in this or related regulatory networks may explain the different regenerative capacities of various organs and animal species.

Figure 1. Schematics of the Continuously Growing Mouse Incisor

(A) Basic overall organization of the incisor tooth. Growth occurs from proximal to apical (incisal) end. At the proximal end lie the lingual and labial cervical loops, each containing epithelial stem cells. (B) Enlargement of boxed region in (A) showing the individual cell types that comprise the proximal end. Stem cells reside within stellate reticulum, core of the cervical loop. See text for details. Color coding is as follows: enamel (red), dentin (blue), epithelium (orange), and follicular mesenchyme (green). DE, dental epithelium, FM, follicle mesenchyme, IDE, inner dental epithelium, ODE, outer dental epithelium, PM, papilla mesenchyme, SR, stellate reticulum, TA, transit amplifying.

Figure 2. Follistatin Inhibits the Proliferation of Epithelial Stem Cells and TA Cells and the Expression of Fgf3 in the Mouse Incisor

(A and B) At P17, the growth of K14-Follistatin mouse incisors is retarded relative to wild type. (C) At the newborn stage, wild-type incisors exhibit a large, labial (Lab) cervical loop (CL; arrow) and a much smaller lingual (Lin) cervical loop. (D) In newborn K14-Follistatin mice, the labial cervical loop is severely hypoplastic (red arrow). (E) In newborn Follistatin^−/− incisors, the lingual cervical loop is hyperplastic (red arrow). (F–H) BrdU incorporation experiments at E18 reveal a marked reduction in cell proliferation in K14-Follistatin incisors, especially in the TA cell region of the labial dental epithelium (DE). In Follistatin^−/− incisors, there is an increase in cell proliferation in the lingual dental epithelium. Dashed red lines indicate the epithelium. (I–K) In situ hybridization analysis of Fgf3 expression (red color represents signal). (I) In newborn wild-type mice, Fgf3 is expressed asymmetrically in dental papilla mesenchyme adjacent to the labial cervical loop. (J) Fgf3 expression is markedly down-regulated in P1 K14-Follistatin incisors. (K) Follistatin^−/− incisors exhibit ectopic Fgf3 (arrow) in lingual dental mesenchyme adjacent to proliferating epithelial cells. Yellow lines indicate the epithelium (C–E, I–K). Scale bar represents 100 μm (C–K).

Figure 3. Fgf Genetic Interactions in Incisor and Molar Development

(A–I) Lower incisor phenotypes in wild type and in Fgf3^−/− and Fgf3^−/−; Fgf10^+/− mutants. (A–C) Histology at P1 shows wild-type and Fgf3^−/− incisor development are indistinguishable, whereas Fgf3^−/−; Fgf10^+/− incisors are smaller and exhibit hypoplastic labial cervical loops (arrows). (D–F) Gross photographs of 5-wk-old incisors. Note the absence of pigmentation in Fgf3 mutants, and the thin, broken incisors in a Fgf3^−/−; Fgf10^+/− mutant. (G–I) Ground sections of incisors in D-F, respectively, showing equivalent enamel layer thickness (dashed red lines) in wild-type and Fgf3^−/− mutants, and enamel layer absence in a Fgf3^−/−; Fgf10^+/− mutant (arrows). (J–L) Five-week-old Fgf3^−/− and Fgf3^−/−; Fgf10^+/− lower molars are smaller than normal, and enamel is prematurely worn on the occlusal surface. (M–O) At P1, folding of Fgf3^−/− molar epithelium is aberrant and shallower than normal; this phenotype is more severe in Fgf3^−/−; Fgf10^+/− molars. (P and Q) Histological sections of E13 molar region in wild-type and Fgf3^−/−; Fgf10^−/− double mutants show that the development of both maxillary and mandibular molars is arrested prior to bud stage in the mutant. One out of 90 double-mutant embryos survived until E13. Scale bar represents 100 μm (A–C and M–Q).

Figure 4. Signaling Molecule and Receptor Gene Expression in Incisor Dental Mesenchyme

In situ hybridization for the indicated genes in incisor tooth germs from E14 to E18. (A) Note the symmetric mesenchymal expression of Bmp4 in the cervical loop regions and the onset of asymmetric mesenchymal expression of Activin βA and Fgf3 at E15 and E16, respectively (arrows). Follistatin is expressed throughout the thin layer of lingual dental epithelium as well as in the labial outer dental epithelium, but is down-regulated in the inner dental epithelium labially (arrow). (B) The BMP and Activin receptors Alk3 and Alk4, respectively, are expressed in the epithelium on both labial and lingual sides between E16 and E18. Scale bar represents 200 μm.

Figure 5. BMP4 Inhibits Fgf3 Expression in Dental Mesenchyme and Activin Counteracts This Effect and Stimulates Cell Proliferation

(A–F) E16 incisor explants containing the cervical loop region were cultured in vitro for 24 h with beads containing the indicated factors, and gene expression was analyzed by whole-mount in situ hybridization. (A) In BSA bead controls, Fgf3 is expressed only in labial (Lab) dental mesenchyme underlying the epithelium (arrow, 46/46). (B) BMP4 beads down-regulate Fgf3 expression (39/47). (C) Fgf10 expression is unaffected by BMP4 beads (14/14). (D) Noggin beads induce ectopic Fgf3 expression in lingual (Lin) mesenchyme directly adjacent to the inner dental epithelium (arrow, 8/18). (E) Activin A beads also induce ectopic Fgf3 expression in lingual mesenchyme (arrow, 21/35). (F) When both BMP4 and Activin A beads (a) are inserted, the repression of Fgf3 expression by BMP4 (b) is abolished (4/7). A, anterior, P, posterior. (G–O) When P1 incisor explants are cultured in the presence of Activin A for 4 d, cell proliferation is stimulated as assayed by BrdU incorporation, and both lingual (G and H) and labial (J and K) cervical loops are increased in size (21/25). Ectopic Fgf3 is induced in the lingual dental mesenchyme directly underneath the enlarged cervical loop (arrow, [M] and [N]). When the inhibitor of Activin receptor-like kinase (ALK) receptors (SB431542) is added to the culture medium of incisor explants, the labial cervical loop is thinner, containing fewer stellate reticulum cells, and epithelial cell proliferation is reduced in both labial and lingual cervical loops (11/12) (I and L). In these samples, Fgf3 is down-regulated in the dental mesenchyme (M and O). Dashed lines indicate the epithelium. Scale bar represents 100 μm (G–O).

Figure 6. Dental Epithelium Maintains Activin βA and Fgf3 Expression in Mesenchyme, and This Effect is Mimicked by FGF9

(A and B) When incisor mesenchyme (m) is isolated from dental epithelium (e) and cultured in vitro for 1 d, both Fgf3 (E14: 0/4; E15: 0/15) and Activin βA (E14: 0/3) expression are down-regulated to undetectable levels. (C and D) When isolated dental epithelium and mesenchyme are recombined, strong Fgf3 (E14: 10/11; E15: 13/15) and Activin βA (E14: 9/9) expression is induced in the mesenchyme at the epithelial-mesenchymal boundary. (E and F) FGF9 beads induce strong expression of Fgf3 (E14: 7/7; E15: 3/3) and Activin βA (E14: 6/6; E15: 2/2) in incisor mesenchyme around the bead. (G) Activin A beads do not induce Fgf3 expression in isolated dental mesenchyme (E15: 0/13). (H) FGF3 beads do not induce Activin βA in isolated dental mesenchyme (E15: 0/14). (I and J) No induction of Fgf3 (E14: 0/4; E15: 0/6) or Activin βA (E14: 0/3; E15: 0/6) occurs around BSA control beads.

Figure 7. Different Incisor Phenotypes of Several Mouse Mutants and the Signaling Network in the Stem Cell Niche

(A) Summary of the sizes of stem cell niches (cervical loops [CL]) and of the distribution of enamel (red) and dental epithelium (orange) in mouse incisors with the indicated genotypes. (B) Model showing the gene regulatory network that controls epithelial stem cell proliferation in the incisor stem cell niche. See text for details.