Morphological changes in molar spatial patterning in mouse after maternal transfer of 5 a-j

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Fig. 1. Morphological changes in molar spatial patterning in mouse after maternal transfer of 5E1. (A-J) Five types of molar patterns obtained in maxilla (Mx) and mandible (Mn): Type-I, wild-type-like with first (M1), second (M2) and third (M3) molar; Type-II, M1-M2 fusion; Type-III, M1-M2 fusion with extra lingual cusps (arrows); Type-IV, M2-M3 fusion; Type-V, M1-M2-M3 fusion. (K-N) Three-dimensional micro-computed-tomography images showing buccal aspect of type-I and type-II patterns at embryonic day (E) 14 after injection of phosphate-buffered saline (E14-PBS) and after 5E1 injection (E14-5E1) in maxilla and mandible. (O-T) Dental lamina (arrow) is evident in the frontal section of M1 in both E14-PBS and E14-5E1 at one day after injection (O,R). After two days, dental lamina and lingual epithelial bud (arrowhead) are observed only in an E14-PBS (compare P with S), and M1-M2 separation (red arrow) is evident in E14-PBS (Q). M1-M2 fusion is evident in E14-5E1 (T). (U,V) Three-dimensional images of dental epithelium from bottom view show the larger buccolingual diameter of M2 (between arrows) in E14-5E1 (V) than in E14-PBS (U) after one day. The boundary (red arrowheads) between M1 and M2 is clearly shown in E14-PBS after two days both from bottom and buccal view (U), whereas M1-M2 fusion is evident in E14-5E1 (V). Scale bars: 500 μm.

Fig. 2.

Morphological changes in mouse molars after 5E1 treatment in vivo. (A,B) Supernumerary incisors (red arrow) develop in embryonic day (E) 14 mice treated with 5E1 (B, E14-5E1; 2/82 in mandibular quadrants) but not in control mice treated with PBS (A, E14-PBS). (C-E) Supernumerary molars (arrowheads) develop in E12-5E1 [1/40 in the maxillary quadrants (C), 1/32 in the mandibular quadrants], E14-5E1 (4/78 in the maxillary quadrants, D) and E16-5E1 (1/54 in the mandibular quadrants, E). (F,G) Transparent enamel (arrows) is seen covering the dentine in sagittal sections of fused molars in Mx and Mn of E14-5E1. (H-K) Three-dimensional images from occlusal view show pattern of molars in 4-week-old E14-PBS and E14-5E1. E14-PBS mice have three molars in Mx (H) and Mn (J). M1-M2 fusion is observed in Mx (I) and Mn (K) of E14-5E1. Extra cusps are evident on lingual side of a fused molar (red arrowheads in K). Scale bars: 1 mm. Mx, maxilla; Mn, mandible.

Fig. 3.

Transcriptional changes in molars after maternal transfer of 5E1 into mouse embryos at E14. RT-qPCR analysis shows up- or downregulation of Ptch1, Hhip, Gli1, Sostdc1, Sp5, Fgf4 and Krt73. The amount of each of the RT-qPCR products was normalized using β-2-microglobulin (B2m) as an internal control. Student's t-test was performed for statistical analysis with level of statistical significance set at P<0.05. Error bars indicate s.d. on the normalized ratio.

Fig. 4.

Alterations in gene expression pattern in 5E1-treated mouse molars. (A-D) Whereas the pattern of Shh expression is the same in E14-PBS and E14-5E1 mice after one day in vivo (A,B), Shh expression in M2 (black arrowhead) is evident only in E14-5E1 in vitro, showing accelerated M2 development (compare D with C). (E-H) Ptch1 is strongly expressed in E14-PBS in vivo (E) and in vitro (G), but disappeared in E14-5E1 (F,H). (I-L) Sostdc1-expressing areas are markedly reduced in E14-5E1 (J,L) compared with E14-PBS (I,K). (M-P) Sp5 expression in M1 and M2 is separated in E14-PBS in vivo (red arrowhead in M). Sp5 is observed in M1 of E14-PBS after one day (+1D) culture in vitro (O) and expressed in M1 and M2 separately after two days (+2D) (red arrowhead in O′), but connected in E14-5E1 in vivo and from one day in culture (white arrowheads in N and P). (Q,R) Gli3 expression pattern of E14-PBS is the same as that of E14-5E1 after one day in vitro. (S,T) Lef1 expression is observed in M1 after one day in vitro in both E14-PBS and E14-5E1, but its domain of expression in M2 (red arrows) is larger in E14-5E1 (T) than in E14-PBS (S). (U,V) krt73 is expressed in a line in E14-PBS (U), but is widely expressed throughout M1 and M2 in E14-5E1 (V). (W,X) Fgf4 expression in M1 (black arrow) is evident in both E14-PBS (W) and E14-5E1 (X), but its expression in M2 (red arrow) is only present in E14-5E1 (X). Scale bar: 200 μm

Fig. 5.

Effects of Shh on Sostdc1 expression in mouse. (A-D) Apart from endogenous Ptch1 (red arrows in A and B), exogenous Ptch1 is expressed around the Shh protein bead in both dental epithelium and mesenchyme (black arrows in B,D), but not around PBS beads (A,C). (E-H) Sostdc1 is not expressed around the control PBS beads (E,G). Sostdc1 is induced by exogenous Shh protein in mesenchyme (arrow in H), but not in epithelium (F). (I-L) Sostdc1 is expressed in dental epithelium and mesenchyme around wild-type tooth germs at E14 but is not induced in either epithelium or mesenchyme around beads soaked in Shh protein. Occlusal views are shown in I and K. Frontal section is shown in J and L at the level of the dashed lines in I and K, respectively. Yellow dashed lines indicate the boundary of the dental epithelium and mesenchyme. Scale bars: 200 μm.

Fig. 6.

Wnt-Shh-Sostdc1 negative feedback loop for the regulation of the tooth spatial patterning. (A,B) Frontal section of M1 at E14 shows that Lef1 is expressed in the enamel knot (inside the red dashed circles) and dental mesenchyme surrounding the enamel knot, whereas Sostdc1 is expressed mainly outside the Lef1-expressing area. (C) Schematic of Wnt-Shh-Sostdc1 negative feedback loop in M1 at E14 from frontal view. Wnt signals in the enamel knot induce Shh in the enamel knot and Wnt moves laterally to induce Lef1 and Sp5. Secreted Shh in the enamel knot also moves laterally to induce Sostdc1. The Sostdc1-expressing area (Sostdc1 area) is non-overlapping with the Lef1-expressing area (Lef1 area). (D) Wild-type mouse has three molars, each of which has an activation zone (Lef1 area) and an inhibition zone (Sostdc1 area). Loss of the inhibition zone in K14-Cre;Shhflox/flox, K14-Cre;Smoflox/flox, Sostdc1−/−, Lrp4−/− and 5E1-transferred mice enhances molar fusion. Sustained Wnt/β-catenin signals in K14-Cre;Ctnnb1(Ex3)fl/+ and K14-Cre;APCcko/cko induce multiple activation and inhibition zones to form multiple molars. (E) Schematic of the proposed reaction-diffusion model showing interactions between activator (A), mediator (M) and inhibitor (I). (F) Simulated patterns of activator, mediator and inhibitor show three molars in wild-type mice. The black dotted lines in A-C indicate the boundary of the dental epithelium and mesenchyme. Scale bar: 100 μm.

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