The initiation knot is a signaling center required for molar tooth development

Abstract

Signaling centers, or organizers, regulate many aspects of embryonic morphogenesis. In the mammalian molar tooth, reiterative signaling in specialized centers called enamel knots (EKs) determines tooth patterning. Preceding the primary EK, transient epithelial thickening appears, the significance of which remains debated. Using tissue confocal fluorescence imaging with laser ablation experiments, we show that this transient thickening is an earlier signaling center, the molar initiation knot (IK), that is required for the progression of tooth development. IK cell dynamics demonstrate the hallmarks of a signaling center: cell cycle exit, condensation and eventual silencing through apoptosis. IK initiation and maturation are defined by the juxtaposition of cells with high Wnt activity to Shh-expressing non-proliferating cells, the combination of which drives the growth of the tooth bud, leading to the formation of the primary EK as an independent cell cluster. Overall, the whole development of the tooth, from initiation to patterning, is driven by the iterative use of signaling centers.

Keywords: Cell division; Embryonic development; Migration; Shh; Signaling center; Tooth; Wnt.

Fig. 1.

A molar initiation knot is established in the molar placode and early bud in G₁/G₀ cells positive for signaling center markers. (A,B) Confocal fluorescence images of mouse embryonic mandibles of the cell cycle indicator Fucci for G₁/G₀ phase nuclei (red) and the signaling center marker Shh^GFP (green). Images show immunofluorescence staining of the epithelium (EpCam, gray; the tooth epithelium perimeter is indicated by dotted lines), early G₁/G₀ focus (arrowheads) and presumptive pEK (asterisks). Images show a planar view from the mesenchyme toward the epithelium; i indicates the location of the areas shown at higher magnification on the right; z indicates sagittal optical sections. (A) G₁/G₀ cells were present throughout the dental lamina and the molar placode as the incisor and molar resolved into separate domains at E11.5. At E12.5, G₁/G₀ cells formed a focus mesially in the molar early bud. This focus remained close to epithelial surface mesiolingually. At E13.0, G₁/G₀ cells corresponding to the presumptive pEK emerged in the tip of the bud and condensed by E13.5. (B) Shh^GFP signaling center marker showing expression corresponding to G₁/G₀ foci throughout placode and bud morphogenesis, and in the emerging pEK.

Fig. 2.

The molar IK is a functional signaling center driving budding via proliferation. (A) Confocal fluorescence images of whole-mount explants showing Fucci G₁/G₀ nuclei (red), S/G₂/M nuclei (green) and epithelium/tooth perimeter (EpCam, white, dotted line). i indicates the areas shown at higher magnification on the right and z indicates sagittal optical sections. IK are indicated by arrowheads and pEK by asterisks. Initially, S/G₂/M cells were seen throughout the oral epithelium and, by E12.5, in the early bud posterior to the IK in both basal and suprabasal populations. IK and pEK cells remained in G₁/G₀. (B) Surface rendering of the nuclei in G₁/G₀ and S/G₂/M cell cycle phases in the developing molar placode/tooth bud epithelium. (C) Quantification of cell cycle phases in the IK and early bud with live imaging of E11.5+12 h molars. Data are mean±s.e.m. (n=5). (D) Quantification of cell cycle phases in the IK, emerging pEK and tooth bud with live imaging of E12.5+12 h molars Data are mean±s.e.m. (n=3).

Fig. 3.

Laser ablation of the IK arrests molar bud growth. Confocal fluorescence images of whole-mount explant cultures of K17-GFP epithelium, Fucci S/G₂/M nuclei (green), Fucci G₁/G₀ (red) and epithelium (EpCam, gray). Tooth placode/bud epithelium is indicated by dotted lines, i indicates the areas shown at higher magnification on the right and z indicates sagittal optical sections. The IK was laser ablated (position marked by a viewfinder symbol ¤ and by horizontal lines in z-plane views) at E11.5 or E12.5 followed by 24 h culturing. (A) Laser ablation of IK G₁/G₀ cells in early placode-stage epithelium (E11.5) abrogated epithelial invagination and growth. On the control side, tooth invagination proceeded normally. (B) Early bud stage (E12.5) ablation completely arrested bud invagination and elongation. (C) Bud dimensions of ablated and control molars at E11.5+24 h and E12.5+24 h. Data show the fold change over E11.5 and are mean±s.e.m.; n_E11.5+24h=9, n_E12.5+24h=8, Mann–Whitney U-test; *P≤0.05, **P≤0.01 and ***P≤0.001. (D) Laser ablation of the IK in the Fucci S/G₂/M model at E11.5 resulted in loss of bud cell proliferation. Physiological pattern of S/G₂/M cells in the adjacent oral epithelium confirmed good tissue health. (E) Quantification of proliferating cells in E11.5+24 h molars. Data are mean±s.e.m.; n=8, Mann–Whitney U-test, ***P≤0.001. (F) Bud cell proliferation and consequently bud growth were similarly abrogated in E12.5 ablated molars. (G) Quantification of proliferating cells in E12.5+24 h molars. Data show the fold change over E12.5 and are mean± s.e.m.; n=8, Mann–Whitney U-test, P≤0.01.

Fig. 4.

The IK remains an integral part of the tooth and does not contribute cells to the pEK, which arises independently. (A) Still images of live tissue time-lapse confocal fluorescence imaging from the placode stage at E11.5 (whole mandible) and molar close-up (i) at E11.5+12 h (E11.5, +3 h, +6 h, +9 h and +12 h) showing Fucci G₁/G₀ (red) and S/G₂/M (green) cells. Arrowheads indicate the IK position. Dotted lines indicate the tooth placode/bud epithelium. (B) Tracks and displacement vectors of individual Fucci G₁/G₀ and S/G₂/M cells in the E11.5+12 h molar culture. (C) Still images of Fucci G₁/G₀ (red) and S/G₂/M (green) reporter live imaging from the early bud stage at E12.5 to +12 h. i indicates the area shown at higher magnification in the molar volume rendering time lapse images. Arrowheads indicate the IK and asterisks indicate the pEK. (D) E12.5+12 h cell tracks and displacement of individual Fucci G₁/G₀ (red) and S/G₂/M (green) cells.

Fig. 5.

Cell condensation and active directional cell migration drive molar IK maturation. (A) Confocal fluorescence images of Fucci G₁/G₀ (red) molars, cell borders (EpCam, white, dotted line) and nuclei (Hoechst, blue). (B) Quantification of IK and oral epithelial cell density (plots represent minimum, 25th percentile, median, 75th percentile and maximum values; n_placode/bud=n_oral=10, Mann–Whitney U-test, ***P≤0.001). (C) Quantification of cell track length and net displacement in molar placode/bud and oral epithelium at E11.5+12 h (plots represent minimum, 25th percentile, median, 75th percentile and maximum values; n_{G1/G0 IK}=149, n_{G1/G0 oral}=45, n_S/G2/M=146, Mann–Whitney U-test, ***P≤0.001) and E12.5+12 h (data are mean±s.e.m., n_{G1/G0 IK}=90, n_{G1/G0 oral}=51, n_S/G2/M=317, Mann–Whitney U-test, *P≤0.05). (D) Quantification of molar IK and oral epithelial cell movement angles E11.5+12 h (data are mean±s.e.m., n_{IK cells}=n_{oral epithelial cells}=95, Rayleigh test: H₀=random, IK P≤0.001, oral P>0.05). (E) Pairwise comparison of molar IK G₁/G₀ and bud S/G₂M cell positions (plots represent minimum, 25th percentile, median, 75th percentile and maximum values; n_pairs=40, Mann–Whitney U-test, *P≤0.05). (F) Confocal fluorescence images of Fucci G₁/G₀ nuclei (red) and K17-GFP (epithelium, green) reporter. Tooth placode/bud epithelium is indicated by dotted lines, IK is indicated by arrowheads and pEK by an asterisk. i indicates the area shown at higher magnification on the right and z indicates sagittal optical sections. E11.75 cultures were treated at the time of most active IK G₁/G₀ cell movement with blebbistatin for 24 h to inhibit actomyosin-based cell motility. Blebbistatin treatment repressed IK G₁/G₀ cell condensation and arrested bud morphogenesis.

Fig. 6.

Dynamics between Wnt^Hi and Shh cell populations regulates IK maturation and maintenance. (A) Fucci G₁/G₀ fluorescence images overlaid with whole-mount DIG in situ hybridization for Wnt10b. IK is indicated by arrowheads and emerging pEK by an asterisk. i indicates the area shown at higher magnification underneath. (B) High-resolution analysis of sagittal optical sections of fixed samples of Fucci G₁/G₀ (red) and TCF/Lef:H2B-GFP (green) patterns. The tooth epithelium perimeter is indicated by white-dotted lines. At E11.5, TCF/Lef:H2B-GFP high intensity (Wnt^Hi, visualized with nuclear surface rendering) was present in the dental lamina and the molar IK G₁/G₀ condensate was located next to a border of Wnt^Hi cells. At E12.5, IK G₁/G₀-only cells were surrounded by Wnt^Hi cells. At E13.5, G₁/G₀ and Wnt^Hi cells were present in the presumptive pEK region (asterisk). (C) Quantification of cells in Wnt^Hi, G₁/G₀ and double-positive cell populations in fixed samples (data are mean±s.e.m., IK n_E11.5=413; n_E12.5=192; E13.5 n_IK=136, n_pEK=831, non-parametric Student's t-test, *P≤0.05, **P≤0.01, ***P≤0.001). (D) Quantification of cell number in Wnt^Hi, G₁/G₀ and double-positive cell populations from live tissue imaging at E11.5+12 h and E12.5+12 h (data are mean±s.e.m. n_E11.5+12h=n_E12.5+12h=3). (E) Surface-rendering still images of E11.5+12 h molar time-lapse, Fucci G₁/G₀, TCF/Lef:H2B-GFP and double-positive cells. At E11.5+3 h onwards, the G₁/G₀ cell population started differentiating closely juxtaposed to Wnt^Hi cells, with an increasing number of cells transitioning to G₁/G₀. (F) Track end point analysis (median is indicated by a red cross, n_{IK G1/G0}=128, n_{oral G1/G0}=63, n_WntHi=104). Molar IK G₁/G₀ cells showed preferential distribution toward the dental lamina Wnt^Hi region (forward). (G) Track straightness in molar IK G₁/G₀, oral epithelial G₁/G₀ and TCF/Lef:H2B-GFP+ cell populations (data are mean±s.e.m., n_{IK G1/G0}=128, n_{oral G1/G0}=42, n_WntHi =67, Mann–Whitney U-test, ***P≤0.001). (H) Decay in cell movement persistence (plots represent minimum, 25th percentile, median, 75th percentile and maximum values; n_{IK G1/G0}=80, n_{oral G1/G0}=50, n_WntHi=71).

Fig. 7.

Modulation of canonical Wnt signaling affects IK cell dynamics and molar bud shape. (A) Confocal fluorescence images of explant cultures showing K17-GFP (epithelium, green), Fucci nuclei G₁/G₀ (red) and the tooth placode/bud epithelium perimeter (dotted line). i indicates the area shown at higher magnification on the right and z the surface rendering side view. Canonical Wnt signaling levels were modulated from placode stage at E11.5+24 h by either stimulation with Wnt3a or inhibition with XAV939. Stimulation resulted in a flat bud with persistant G₁/G₀ IK cells throughout the invagination. Inhibition led to complete loss of the G₁/G₀ condensate and abrogated invagination. (B) Quantification of bud dimensions in Wnt3a-stimulated and control cultures (data show the fold change over E11.5 and are mean±s.e.m.; n_ctrl=12, n_Wnt3a=10, Mann–Whitney U-test, ***P≤0.001). (C) Quantification of IK cell number, density and area in Wnt3a-stimulated and control cultures (data show the fold change over E11.5 and are mean±s.e.m., n_ctrl=12, n_Wnt3a=10, Mann–Whitney U-test, ***P≤0.001). (D) Stimulation with Wnt3a in Fucci G₁/G₀ (red) and S/G₂/M (green) cultures resulted in lack of IK condensation followed by a drastic loss of cell proliferation in the bud. Inhibition with XAV939 resulted in the loss of the G₁/G₀ IK condensate and absence of proliferation and invagination. i indicates the area shown at higher magnification on the right and z the surface rendering side view. (E) Quantification of cell proliferation in Wnt3a-stimulated and control cultures (data show the fold change over E11.5 and are mean±s.e.m.; n_ctrl=7, n_Wnt3a=8, Mann–Whitney U-test, **P≤0.01). (F) A Wnt10b recombinant protein-releasing bead was placed next to the IK (visualized with a Fucci G₁/G₀ reporter) distally to the placode. Explants were imaged at E11.5, after 8 h and after 16 h. Morphogenesis and IK condensation proceeded normally in control buds without beads. Application of the Wnt10b-releasing bead resulted in a loss of condensation of G₁/G₀ IK cells. Instead, the G₁/G₀ IK cells were spread out toward the Wnt10b bead.