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. 2016 Jul 13:7:12133.
doi: 10.1038/ncomms12133.

αE-catenin inhibits YAP/TAZ activity to regulate signalling centre formation during tooth development

Chun-Ying Li  1   2 , Jimmy Hu  2 , Hongbing Lu  2 , Jing Lan  2 , Wei Du  2 , Nicole Galicia  2 , Ophir D Klein  2   3
Affiliations

αE-catenin inhibits YAP/TAZ activity to regulate signalling centre formation during tooth development

Chun-Ying Li et al. Nat Commun. .

Abstract

Embryonic signalling centres are specialized clusters of non-proliferating cells that direct the development of many organs. However, the mechanisms that establish these essential structures in mammals are not well understood. Here we report, using the murine incisor as a model, that αE-catenin is essential for inhibiting nuclear YAP localization and cell proliferation. This function of αE-catenin is required for formation of the tooth signalling centre, the enamel knot (EK), which maintains dental mesenchymal condensation and epithelial invagination. EK formation depends primarily on the signalling function of αE-catenin through YAP and its homologue TAZ, as opposed to its adhesive function, and combined deletion of Yap and Taz rescues the EK defects caused by loss of αE-catenin. These findings point to a developmental mechanism by which αE-catenin restricts YAP/TAZ activity to establish a group of non-dividing and specialized cells that constitute a signalling centre.

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Figures

Figure 1
Figure 1. Deletion of Ctnna1 induces YAP nuclear localization as well as cell proliferation and inhibits EK formation.
(a) Schematic diagram of incisor morphogenesis. At E12.5, the dental epithelium (light purple) begins to invaginate into the underlying mesenchyme (MES, tan). By E13.5, the EK is formed. Subsequent epithelial-mesenchymal interactions result in mesenchymal condensation around the epithelium (yellow shaded area). At E14.5, the epithelium turns posteriorly, and the EK regresses by E16. (b,c) At E13.5, the EK consists of post-mitotic cells that are Ki67 negative (open yellow arrowhead). (d,e) Ablation of Ctnna1 (Ctnna1cKO) by K14Cre results in ectopic cell proliferation in this region (yellow arrowhead in c). (f,g) p21 is expressed in the control EK (yellow arrowhead) but not in Ctnna1cKO (compare yellow arrowhead in f to open yellow arrowhead in g). (h–j) EK markers, Shh, Bmp4 and Fgf3, are absent in the Ctnna1cKO (compare black arrowhead in h and white arrowheads in j to open black arrowhead in i and open white arrowheads in j). Mesenchymal Bmp4 and Fgf3 are also reduced. (k,l,o–q) At E13.5, the EK contains mostly cytoplasmic YAP (open yellow arrowheads in k,l), while strong nuclear YAP staining is detected in the surrounding epithelium (q). This is visualized through detection of overlapping signals (white) between YAP (green) and DAPI (red) (open yellow arrowheads in o,p). (m,n,r–t) Deletion of Ctnna1 results in YAP nuclear localization in the presumptive EK (yellow arrowheads in m,n,r,s) and the neighbouring epithelium (t). (u,v) Quantification of Ki67-negative (−) cells in the basal layer (u) and Ki67-positive (+) cells in the area surrounding the EK (v) at E13.5 (mean±s.e.m., n=3, P<0.01). (w) Quantification of p21-positive (+) cells in control and Ctnna1cKO (mean±s.e.m., n=3, P<0.01). (x,y) Quantification of nuclear YAP in the EK region (x) and in the protruding epithelium (y) in control and Ctnna1cKO (mean±s.e.m., n=4, P<0.01 and 0.05 respectively). All quantifications are analysed by using Student's t-test. *P<0.05, **P<0.01. Dotted lines outline the epithelium of the incisor tooth germ. VL, vestibular lamina. Scale bar, 25 μm (b,d,f,g,k,m,o,r); 12 μm (l,n,p,q,s,t); 8 μm (c,e) 50 μm (h,i,j).
Figure 2
Figure 2. αE-catenin is required for maintaining proper cell polarity in the tooth germ epithelium.
(a,b) PAR3 is apically localized in cells of the basal layer at E13.5. (c,d) Deletion of Ctnna1 causes basal mislocalization of PAR3. (e) Quantification of the cells with mislocalized PAR3 in basal layers of control and Ctnna1cKO tooth germs (mean±s.e.m., n=3, P<0.05). (f,g) Scribble is laterally localized in the basal layer at E13.5. (h,i) The level of Scribble protein is decreased after ablation of αE-catenin. (j–q) Inhibition of aPKC with Gö 6983 induces nuclear localization of YAP in the explant culture. (r) Quantification of nuclear YAP in vehicle and antagonist treated tissues (mean±s.e.m., n=3, P<0.01). (s,t) Knockdown of PAR3 with shRNA induces YAP nuclear localization in primary epithelial cells. (u) Quantification of nuclear YAP in control (shcon) and shRNA (shPar3)-treated cells (mean±s.e.m., n=3, P<0.01). All quantifications are analysed by using Student's t-test. *P<0.05, **P<0.01. Dotted lines outline the epithelium of the incisor tooth germ. Scale bar, 25 μm (a,c,f,h,j,l,n,p); 8 μm (b,d,g,i,k,m,o,q); 100 μm (s,t).
Figure 3
Figure 3. Proper YAP localization is essential for the formation of EK during tooth development.
(a) Schematic diagram of an E14.5 incisor tooth germ. Square box represents areas imaged in d–o. (b) In situ hybridization of Axin2 at E13.5 tooth germ (arrowhead indicates the expression of Axin2 in the EK). (c) Time course for treatment of Axin2CreER;Lats1/2fl/fl and ShhCreER;Lats1/2fl/fl embryos with tamoxifen to delete Lats1 and Lats2 in the EK. (d,g,j,m) In the EK, YAP is excluded from the nucleus with increased p21 and decreased Ki67 expression at E14.5. (e,f) Double deletion of Lats1 and Lats2 with EK-specific Axin2CreER and ShhCreER induces the nuclear localization of YAP. (h,i) Double deletion of Lats1 and Lats2 with EK-specific Axin2CreER and ShhCreER decreases p21 expression. (k,l) Overlay images of YAP and p21 staining. (n,o) Double deletion of Lats1 and Lats2 in the EK induces ectopic cell proliferation, as shown by Ki67 staining. (p) In situ hybridization of the EK marker Bmp4 at E14.5 (black arrowhead). (q,r) Double deletion of Lats1 and Lats2 inhibits Bmp4 expression (open white arrowheads). Dotted lines outline the tooth germ epithelium in b–r. Scale bar, 50 μm (b,c); 25 μm (d–o); 100 μm p–r).
Figure 4
Figure 4. αE-catenin is required for incisor epithelium invagination.
(a–d) αE-catenin is normally expressed throughout the dental epithelium at E12.5 and E13.5, but expression is absent in Ctnna1cKO. (e–l) Histological analysis and 3D reconstruction show smaller and abnormally shaped tooth germs at E13.5 and developmentally arrested epithelia at E14.5 in Ctnna1cKO (f,h,j,l) when compared with littermate controls (e,g,i,k). Dotted lines outline the tooth germ epithelium in a–h, and yellow arrowheads indicate the tooth germ epithelium in i–l. VL, vestibular lamina. Scale bar, 25 μm (a–d); 100 μm (e–h); 200 μm (i–l).
Figure 5
Figure 5. αE-catenin is required for the epithelium-induced mesenchymal condensation during incisor development.
(a–d) In situ hybridization for Pax9 and Msx1 in control and Ctnna1cKO. In control embryos, Pax9 and Msx1 are expressed in the mesenchyme of the tooth germ at E13.5. In Ctnna1cKO, the expression of these genes is reduced or absent. (e,f) 3D reconstruction of control and Ctnna1cKO tooth germs at E13.5 (epithelium is red and mesenchyme is green). The thickness of the mesenchyme is reduced in the absence of αE-catenin. (g) Quantification of thickness of the condensed mesenchyme around the incisor dental epithelium at E13.5 (mean±s.e.m., n=3, P<0.01). (h) Schematic illustration of the in vitro condensation assay. ED, embryonic day; PA, pharyngeal arch. (i,j) H2B-GFP labelled wild-type mesenchymal cells robustly condense around the dissected control epithelium in vitro (i) but fail to condense around the Ctnna1cKO epithelium (j). White circles mark the position of GFP-negative epithelium. (k) Quantification of GFP-positive mesenchymal cells moving towards (IN) or away from (OUT) the epithelium shows Ctnna1cKO epithelium is unable to retain mesenchymal cells in a condensed state (mean±s.e.m., n=4 in control, n=3 in Ctnna1cKO, P<0.05). (l) The efficiency of mesenchymal cell movement (displacement over distance travelled) is not affected by αE-catenin deletion (mean±s.e.m., n=4 in control, n=3 in Ctnna1cKO, P>0.05). All quantifications are analysed by using Student's t-test. *P<0.05, **P<0.01. Dotted lines outline the epithelium of the incisor tooth germ. Scale bar, 100 μm (a–d,i,j).
Figure 6
Figure 6. αE-catenin regulates enamel knot formation and tooth germ invagination by controlling YAP/TAZ activity during tooth development.
(a,b) H&E staining of E13.5 tooth germ shows that double deletion of Yap/Taz in the Ctnna1cKO dental epithelium is able to rescue the invagination defect seen in Ctnna1cKO. (c) Quantification of the depth of invaginated tooth germ in control, triple knockouts and Ctnna1cKO at E13.5 (mean±s.e.m., n=3, P<0.01). (d–g) Deletion of Yap/Taz in Ctnna1cKO restores the non-proliferating zone at the posterior region of the tooth germ, as assessed by Ki67 staining, indicating the formation of the EK (open yellow arrowheads). (h–k) p21 staining confirms that deletion of Yap/Taz in Ctnna1cKO reestablishes a non-proliferating zone, indicating the formation of the EK in the tooth germ (yellow arrowheads). (l) Quantification of the number of p21-positive cells per section in control and triple knockout tooth germs (mean±s.e.m., n=3, P>0.05). (m,n) Shh expression at the posterior region of the tooth germ epithelium is rescued on Yap/Taz deletion in the Ctnna1cKO (dark arrowheads). (o,p) Expression of the mesenchymal gene, Msx1, is rescued in the dental mesenchyme at E13.5 after deletion of Yap/Taz in Ctnna1cKO in the tooth germ. All quantifications are analysed by using Student's t-test. *P<0.05, **P<0.01. Dotted lines outline the epithelium of the incisor tooth germ. Scale bar, 100 μm (a,b); 50 μm (d,f,h,j,m–p); 8 μm (e,g,i,k).
Figure 7
Figure 7. The αE-catenin-YAP/TAZ signalling axis is required for further tooth germ invagination and development.
(a,b) H&E staining of E14.5 tooth germ shows that the Ctnna1cKO tooth germs passes the bud stage and progress to the cap stage after deletion of Yap/Taz. Black arrowheads mark tears within the tissue and yellow arrowheads mark EK. (c–f) p21-expressing cells (yellow arrowheads) are maintained at E14.5 in the Ctnna1cKO tooth germ after deletion of Yap/Taz. Scale bar, 100 μm (a,b,c,e); 25 μm (d,f).
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