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doi: 10.1242/10.1242/dev.00100

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Shh signaling within the dental epithelium is necessary for cell proliferation, growth and polarization

Amel Gritli-Linde1,*, Marianna Bei2, Richard Maas2, Xiaoyan M. Zhang3, Anders Linde1 and Andrew P. McMahon4,*

1 Department of Oral Biochemistry, Sahlgrenska Academy at Göteborg University, SE-405 30 Göteborg, Sweden
2 Division of Genetics, Brigham and Women's Hospital, Harvard Medical School, 20 Shattuck Street, Boston, MA 02115, USA
3 Curis Inc., 45 Moulton Street, Cambridge, MA 02138, USA
4 Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02135, USA



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  		Fig. 1. Histological and ultrastructural analysis of the Smo mutant tooth phenotype at 1 dpp. Parasagittal sections through control (A) and mutant (B) molars showing fusion between the first (m1) and second (m2) molars in the mutant. Frontal sections through molars from control (C) and mutant (D) pups. Note the absence of the dental cord (dc) and the cellular paucity and lack of vascularization in the mutant stellate reticulum (sr). The mis-positioned red blood cells in the SR in C is an artefact and therefore does not indicate the exact location of blood vessels. High magnifications of boxed areas in C and D are included as insets. Parasagittal sections at the anterior segment of incisors from control (E) and mutant (F) pups. Note the severe alterations in the ameloblast (a) and stratum intermedium (si) layers in the mutant. TEM micrographs from control (G) and mutant (H) incisors. The white double-headed arrows indicate ameloblast length in G and H. dp, dental papilla mesenchyme; G, Golgi; o, odontoblasts; Tp, Tomes' process; tw, terminal web. Scale bars: 500 µm (A,B); 200 µm (C,D); 50 µm (E,F); 5 µm (G,H).

 


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  		Fig. 2. Immunohistochemical localization of cytoskeletal and junctional complex proteins in Smo mutant molars and incisors at 1 dpp. Frontal sections of control (A,C,E,G,I,K,M,O) and Smo mutant (B,D,F,H,J,L,N,P) teeth. Parasagittal sections of control (Q) and mutant (R) incisors. ZO-1 (A-H), E-cadherin (I-P) and ß-tubulin (Q, R). Sections at the middle segment of incisors (E,F,M,N) and at their anterior-most segment (G,H,O,P). The boxed areas in A and B are shown in C and D, respectively. In the control molars (A,C) and incisors (E), ZO-1 accumulates between polarizing ameloblasts (a) and the stratum intermedium (si) as well as at the apicolateral membrane domain of polarizing ameloblasts (arrowhead in C) facing predentin matrix. The outer dental epithelium (ode) shows gaps (arrow in C) with no ZO-1 staining. At this time, ZO-1 is absent from the apical and basal aspect of Smo mutant ameloblasts (a) in molars (arrowhead in D) and incisors (F), whereas it accumulates in the ode (arrow in D), which remains as a continuous layer (B,D). At the anterior-most aspect of the control incisors, ZO-1 accumulation is polarized in the apicolateral and basolateral poles of secretory ameloblasts (G) but is absent in mutant ameloblasts (H). Note that by this stage mutant ameloblasts are severely shrunken, and the strong staining visible is associated with cells of the stratum intermedium and ode (H). E-cadherin staining highlights the gaps in the ode (arrow in K) of control molars (I,K) which are absent (arrow in L) in the mutant molars (J,L). Boxed areas in I and J are shown in K and L, respectively. In some cusps of mutant molars, ameloblasts show some E-cadherin staining at their apicolateral pole (red arrow in L). However, these cells do not show as strong a basolateral E-cadherin accumulation (arrowhead in L) as in controls (arrowhead in K). In the middle segment of control incisors, accumulation of E-cadherin is polarized at the apicolateral and basolateral membrane domains of polarizing ameloblasts (M) but is barely detectable in the mutant ameloblats (N). E-cadherin staining decorates the papillary layer (arrow in O) and accumulates at the apicolateral pole of secretory ameloblasts (O). In mutant incisors, E-cadherin is barely detectable in ameloblasts, and the papillary layer (arrow in P) is unrecognizable. ß-tubulin is virtually absent in mutant ameloblasts (a), whereas it is present in odontoblasts (od) (R). Immuno-positive sites are purple and immuno-negative sites are stained in blue. Scale bars: 200 µm (A,B,I,J); 50 µm (C-H,K-R).

 


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  		Fig. 5. Shh expression and Shh responsiveness during late tooth development in Smo mutants. Section through control (A,C,E,G,I,K,M,O,Q,S,U,W,Y) and Smo mutant (B,D,F,H,J,L,N,P,R,T,V,X,Z) teeth at 1 dpp. In situ hybridizations for Shh (A,B,M,N,Q,R); Ptc1 (C,D,S,T); Gli1 (E,F,O,P,W,X); Hip1 (G,H); Ptc2 (I,J,U,V); Gli2 (Y,Z). Immunohistochemistry with Ab80 showing SHH protein distribution (K,L). Parasagittal (A-H,K,L) and frontal (I,J) sections through the molar region. Frontal sections at the middle (M-P) and anterior (Q-Z) segments of incisors. Shh expression is dramatically decreased in mutant ameloblasts facing predentin (arrow in B). The stratum intermedium continues to express Shh for only a short period before expression is severely decreased (asterisk in N). The signal in the stellate reticulum (SR, arrowhead in G) is artefactual and is due to refractile properties of displaced red blood cells and not to silver grains in SR cells. In I, the arrow points to the inner dental epithelium at the cervical loop and the arrowhead shows preameloblasts. In L, the arrowhead indicates preameloblasts adjacent to polarized odontoblasts and the arrow, preameloblasts adjacent to predentin matrix. In the mutant molar (L), Shh protein has accumulated in large amounts in the predentin facing preameloblasts, which show low amounts of Shh staining. This is probably due to minute amounts of Shh protein, emanating from preameloblasts and possibly also from the SI, which become trapped within predentin matrix, as these mutant cells express extremely low levels of Ptc2 and Ptc1 and thus may be unable to sequester Shh protein. The insets in K and L show high magnifications of the boxed areas. In Q, the arrowhead points to polarizing ameloblasts and the arrow to presecretory ameloblasts. a, ameloblasts; ode, outer dental epithelium; si, stratum intermedium; sr, stellate reticulum. Scale bars: 200 µm.

 


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  		Fig. 3. Ptc2 and Gli1 transcripts are polarized in ameloblasts. In situ hybridization on control incisors at 1 dpp at the level of presecretory (A,D) and young secretory ameloblasts (B,C,E,F). Oblique sections (A,D). Frontal sections (B,C,E,F). The dotted lines indicate the ameloblast apical plasma membrane. Ptc2 (A,C) and Gli1 (B) mRNAs are enriched at the basal and perinuclear compartment (green arrow) of ameloblasts, causing the signal (red) to mask the blue staining of ameloblast nuclei, whereas the apical pole (red arrow) displays considerably less signal. The extent of the stratum intermedium layer (si) is indicated by bars (A,D). In contrast, mRNAs for Msx2 (D), Ptc1 (E) and Dlx3 (F) are enriched apically (red arrow). Scale bars: 100 µm.

 


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  		Fig. 4. Analysis of Shh expression and Shh responsiveness at the cap stage in Smo mutants. Schematic drawings showing tissue organization of molars at the cap stage and Shh expression (stippled) and responsiveness (yellow) within the dental epithelium and mesenchyme (A,B). The green arrows show movement of Shh protein. Alterations of Shh responsiveness in the dental epithelium and its preservation in the mesenchyme in Smo mutants are summarized in B. In situ hybridizations on frontal sections of molars for Shh (C,D); Ptc1 (E,F); Gli1 (G,H); Ptc2 (I,J) and Hip1 (K,L). DM, dental mesenchyme; DS, dental sac; EK, enamel knot; IDE, inner dental epithelium; ODE, outer dental epithelium; SR, stellate reticulum. Scale bars: 100 µm.

 


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  		Fig. 6. Premature cell-cycle exit and differentiation of Smo mutant preameloblasts. Molars (A-H) and incisors (I-L) from control (A,C,E,G,I,K) and mutant (B,D,F,H,J,L) pups. Parasagittal sections (C-F). Frontal sections (A,B,G-L). Immunohistochemistry showing phosphorylated histone H3 (PH-H3; A,B) and calbindin D28K (G,H) distribution. The insets in A, and B show high magnifications of the boxed areas. Arrows in A and B point to preameloblasts adjacent to post-mitotic odontoblasts prior to predentin secretion. The arrowhead in A indicates differentiated ameloblasts in a more mature cusp. In situ hybridization for cyclin D1 (C,D) and amelin (E,F,I-L). In control molars, cyclin D1 expression is lost in post-mitotic ameloblasts facing the first layer of predentin matrix in the most advanced cusp (red arrow in C), whereas preameloblasts and the overlying stratum intermedium (black arrowhead in C) facing post-mitotic odontoblasts (black arrow in C) continue to express cyclin D1. cyclin D1 expression is prematurely lost in Smo mutant preameloblasts and stratum intermedium (black arrowhead in D) adjacent to post-mitotic odontoblasts (arrow in D) prior to predentin secretion. Asterisks in C and D show proliferating cells at the cervical loops and red arrowheads show preodontoblasts. In D the section is through the less-developed cusps of the first molar region. In control molars (E), at the level of the less mature cusps, amelin is expressed in polarizing odontoblasts and is barely detectable in preameloblasts adjacent to them. In the Smo mutant molars (F), amelin is expressed prematurely at high amounts in preameloblasts facing polarizing odontoblasts in several cusps. Note that the cusps in the molar in F are at the same developmental stage as the left cusp of the molar in E. Arrow in H indicates preameloblasts adjacent to preodontoblasts. The insets in G and H show high magnifications of the boxed areas. Sections through the middle segment of the incisors showing absence of amelin expression in control preameloblasts (I) and premature expression by mutant preameloblasts (J). Sections through the anterior segment of incisors (K,L) showing amelin expression by ameloblasts. Scale bars: 200 µm (A-L).

 


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  		Fig. 7. Normal differentiation of odontoblasts and altered differentiation of ameloblasts in Smo mutant teeth. Parasagittal (A,B,K,L,O,P) and frontal (C-J,M,N,Q-T) sections at 1 dpp. Sections from control (ctrl; A,C,E,G,I,K,M,O,Q,S) and mutant (mut, B,D,F,H,J,L,N,P,R,T) teeth. von Kossa staining of mineralized dentin matrix (A,B). In situ hybridization for Bmp2 (C-F), Dlx7 (G-J), DSP (K-P) and Bmp5 (Q-T). Bmp2 and DSP are expressed normally in odontoblasts in Smo mutant teeth (C-F,K-P). Dlx7 expression is severely decreased in mutant ameloblasts (G-J). DSP is expressed in preameloblasts facing predentin matrix (arrows in K and M) and is downregulated in secretory ameloblasts (a in O) in control teeth. In mutants, DSP expression is severely decreased in preameloblasts facing predentin matrix (arrows in L and N). The arrowhead in L indicates the start of decline of DSP expression in preameloblasts. Bmp5 is expressed in secretory ameloblasts (Q) and in differentiating ameloblasts (S) in control teeth. At the anterior segment of mutant incisors, Bmp5 expression is severely decreased in ameloblasts (R) but is normal in the less-mature ameloblasts in molars (T). Signals outside the tooth are sometimes due to refractile structures such as erythrocytes, cellular fragments or tissue folding. Scale bar: 200 µm.

 


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  		Fig. 8. Schematic drawing showing Shh expression (stippled green) and responsiveness (yellow) as well as the cytological changes occuring in the enamel organ and odontoblast layer during the gradual differentiation process in a normal (upper panel) and in a Smo mutant (lower panel) tooth. Proliferating cells are represented by smiling nuclei and post-mitotic cells are represented by non-smiling or plain nuclei. In a normal tooth, at stage 4, preameloblasts facing the first layer of predentin matrix (PD, pink) withdraw from the cell cycle and become postmitotic (PmA). This occurs about 24 hours after the adjacent odontoblasts become postmitotic (stage 2), polarize (stage 3) and secrete PD (stage 4). Polarizing ameloblasts (PoA) continue to grow, they attain their full size at stage 6 and become secretory ameloblasts (SA) displaying Tomes' processes and secrete enamel matrix (E, dark red). Cells of the stratum intermedium overlying PoA and SA increase in size and become cuboidal (cSI). Shortly before enamel secretion, gaps appear in the outer dental epithelium (ODE) and blood vessels and fibroblasts from the dental sac (DS) start to invade the stellate reticulum (SR). Shh is produced in large amounts in proliferating preameloblasts (PA), PmA, PoA, squamous stratum intermedium (sSI) and cSI. Shh expression and Shh synthesis decline thereafter in presecretory ameloblasts (not represented) and SA (stage 6). In the normal tooth, both the enamel organ and dental mesenchyme are responsive to Shh signaling. In the Smo mutant tooth, Shh responsiveness, cell proliferation, growth and differentiation are preserved in the dental mesenchyme, including the dental papilla (not represented), dental sac and the odontoblast layer. However, Shh responsiveness is absent in the enamel organ at all stages (stages 1-6). Mutant preameloblasts become post-mitotic prematurely (stage 3) before predentin secretion by the adjacent odontoblasts. Mutant ameloblasts fail to grow in size, are unpolarized and unable to produce enamel matrix. The mutant stratum intermedium cells remain small and squamous. The outer dental epithelium layer remains continuous, and the stellate reticulum remains avascular. In addition, a premature gradual decrease of Shh production occurs in the mutant ameloblasts, stratum intermedium and stellate reticulum. A, ameloblast; BM, basement membrane (light green); D, dentin (dark pink); O, odontoblast; PmO, post-mitotic odontoblast; PO, proliferating preodontoblast; PoO, polarizing odontoblast.

 

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