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. 2014 May 9;9(5):e96938.
doi: 10.1371/journal.pone.0096938. eCollection 2014.

Interactions between BMP-7 and USAG-1 (uterine sensitization-associated gene-1) regulate supernumerary organ formations

Honoka Kiso  1 , Katsu Takahashi  1 , Kazuyuki Saito  1 , Yumiko Togo  1 , Hiroko Tsukamoto  1 , Boyen Huang  2 , Manabu Sugai  3 , Akira Shimizu  4 , Yasuhiko Tabata  5 , Aris N Economides  6 , Harold C Slavkin  7 , Kazuhisa Bessho  1
Affiliations

Interactions between BMP-7 and USAG-1 (uterine sensitization-associated gene-1) regulate supernumerary organ formations

Honoka Kiso et al. PLoS One. .

Abstract

Bone morphogenetic proteins (BMPs) are highly conserved signaling molecules that are part of the transforming growth factor (TGF)-beta superfamily, and function in the patterning and morphogenesis of many organs including development of the dentition. The functions of the BMPs are controlled by certain classes of molecules that are recognized as BMP antagonists that inhibit BMP binding to their cognate receptors. In this study we tested the hypothesis that USAG-1 (uterine sensitization-associated gene-1) suppresses deciduous incisors by inhibition of BMP-7 function. We learned that USAG-1 and BMP-7 were expressed within odontogenic epithelium as well as mesenchyme during the late bud and early cap stages of tooth development. USAG-1 is a BMP antagonist, and also modulates Wnt signaling. USAG-1 abrogation rescued apoptotic elimination of odontogenic mesenchymal cells. BMP signaling in the rudimentary maxillary incisor, assessed by expressions of Msx1 and Dlx2 and the phosphorylation of Smad protein, was significantly enhanced. Using explant culture and subsequent subrenal capsule transplantation of E15 USAG-1 mutant maxillary incisor tooth primordia supplemented with BMP-7 demonstrated in USAG-1+/- as well as USAG-1-/- rescue and supernumerary tooth development. Based upon these results, we conclude that USAG-1 functions as an antagonist of BMP-7 in this model system. These results further suggest that the phenotypes of USAG-1 and BMP-7 mutant mice reported provide opportunities for regenerative medicine and dentistry.

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Conflict of interest statement

Competing Interests: Aris N. Economides is employed by Regeneron Pharmaceuticals, Inc. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Figures

Figure 1
Figure 1. BMP-7 co-localization with USAG-1 in the mesenchymal and epithelial cells of maxillary rudimentary incisor.
(A–F) Whole-mount X-Gal expression in tooth germs of E13 –15 maxillary. (A’–F’) Parasagittal sections (anterior to the left) of the tooth germs from panels A–F show X-Gal expression in the rudimentary incisor epithelium. USAG-1 (A–C, A’–C’) and BMP-7 (D–F, D’–F’) were expressed in the tooth organ of rudimentary maxillary incisor (red arrow) in addition to the tooth organ of characteristic incisor (black arrow). At E13, USAG-1 and BMP-7 transcripts were prominent in the labial epithelium in addition to the dental epithelium (A, D, A’ and D’). At E14, USAG-1 and BMP-7 started to be expressed in the mesenchymal cells of the maxillary rudimentary incisor to the surface of the epithelium (B, E, B’ and E’). At E15, the expression of both USAG-1 and BMP-7 increased in the mesenchymal cells of the maxillary rudimentary incisor (C, F, C’ and F’). BMP-7 co-localized with USAG-1 in the area of the tooth germ of maxillary rudimentary incisor in addition to the tooth organ of regular maxillary incisor. White dotted line indicates the interface between epithelium and mesenchyme.
Figure 2
Figure 2. USAG-1 antagonises BMP-7 in maxillary supernumerary incisors formation.
Sagittal sections of E15 (A–D) embryos and frontal sections of mice on the day of birth (E–H). (A’–H’) Higher magnification of the boxed regions in (A–H). USAG-1+/+/BMP-7+/+, (A, A’, E, E’); USAG-1−/−/BMP-7+/+, (B, B’, F, F’); USAG-1+/+/BMP-7−/−, (C, C’, G, G’) and USAG-1−/−/BMP-7−/− (D, D’, H, H’). The area of rudimentary incisor was measured in transverse sections of USAG-1+/+/BMP-7+/+ (white bars), USAG-1−/−/BMP-7+/+ (right grey bars), USAG-1+/+/BMP-7−/− (dark grey bars) and USAG-1−/−/BMP-7−/− (black bars) mice (n = 5) in E15 (I) and P0 (J). At E15, the area of the maxillary deciduous incisor was identified in wild type as well as all mutant mice in the labial border of the epithelial invagination. The size of rudimentary incisor is similar except USAG-1+/+/BMP-7−/− at E15 (A, A’, B, B’, C, C’, D, D’ and I).Rudimentary tooth primordia in USAG-1−/−/BMP-7−/− and USAG-1+/+/BMP-7+/+ regressed and its size became smaller at birth, whereas the teeth in USAG-1−/−/BMP-7+/+ continued to develop and enamel organ was formed (E, E’, F, F’, H, H’ and J).
Figure 3
Figure 3. USAG-1 abrogation rescues apoptotic elimination of odontogenic mesenchymal cells.
Sagittal sections of E15 embryo maxillary rudimentary incisor in transferase-mediated dUTP nick end-labelling method (TUNEL) staining; Cell nuclei were counterstained with Dapi (A–D), and TUNEL-positive cells in mesenchymal cells of maxillary rudimentary incisor (A’–D’). USAG-1+/+/BMP-7+/+, (A, A’); USAG-1−/−/BMP-7+/+, (B, B’); USAG-1+/+/BMP-7−/−, (C, C’) and USAG-1−/−/BMP-7−/− (D, D’). White line indicates the interface between epithelium and mesenchyme. The number of TUNEL-positive cells per section was counted in transverse section of USAG-1+/+/BMP-7+/+ (white bars), USAG-1−/−/BMP-7+/+ (right grey bars), USAG-1+/+/BMP-7−/− (dark grey bars), and USAG-1−/−/BMP-7−/− (black bars) mice (n = 3; E). USAG-1 abrogation rescued the apoptotic elimination of odontogenic mesenchymal cells in the tooth primordia of rudimentary maxillary incisor at E15, whereas these size are comparable (A, A’, B and B’). The apoptotic odontogenic mesenchymal cells in USAG-1−/−/BMP-7−/− are similar to USAG-1+/+/BMP-7+/+ in contrast to those in USAG-1−/−/BMP-7+/+ (A, A’, B, B’, D and D’).
Figure 4
Figure 4. Intensive expression of Msx1 and Dlx2 in the rudimentary incisors of USAG-1 and BMP-7 mutants.
Occlusal view of the tooth organ of rudimentary maxillary incisor primordium at E13 on whole mount in situ hybridization (A–H). Msx1 (A–D) and Dlx2 (E–H) transcription factors were expressed in the tooth organ of rudimentary maxillary incisor (black arrow) in addition to the tooth organ of characteristic incisor (white arrow). At E13, Msx1 and Dlx2 expression in the rudimentary maxillary incisors of USAG-1−/−/BMP-7−/− mice was comparable with that of USAG-1+/+/BMP-7+/+, whereas that of USAG-1−/−/BMP-7+/+ appeared more intense as compared with that of controls (A–H).
Figure 5
Figure 5. Enhanced BMP signal transduction in maxillary incisors of USAG-1 and BMP-7 mutants.
Immunolocalisation of phosphorylated Smad (1/5/8 (A–D) and Smad 2/3 (E–H) at E15. USAG-1+/+/BMP-7+/+, (A, E); USAG-1−/−/BMP-7+/+, (B, F); USAG-1+/+/BMP-7−/−, (C, G) and USAG-1−/−/BMP-7−/− (D, H). The number of pSmad 1/5/8– and pSmad 2/3- positive nuclei per section was counted in transverse sections of USAG-1+/+/BMP-7+/+ (white bars), USAG-1−/−/BMP-7+/+ (right grey bars), USAG-1+/+/BMP-7−/− (dark grey bars), and USAG-1−/−/BMP-7−/− (black bars) mice (n = 5; I). Compared with USAG-1−/−/BMP-7+/+ embryos, USAG-1−/−/BMP-7−/− embryos iibited increased phosphorylated Smad 1/5/8- positive cells in odontogenic mesenchymal cells within the rudimentary maxillary incisor primordia at E15 (A–D). Employed immunostaining using anti-phospho-Smad 2/3 showed no difference among mutant mice (E–H). Enhanced BMP signalling in supernumerary teeth of the USAG-1-deficient mice could be inhibited by BMP-7 abrogation.
Figure 6
Figure 6. BMP-7 has potential to partially induce the formation of maxillary supernumerary incisors formation in vitro.
Enhanced BMP-7 rescue the formation of maxillary incisor supernumerary tooth in E15 USAG-1 mutant mice in organ culture and subrenal capsule assay. The incisor explants supplemented with BMP-7 in USAG-1+/− (E and H) and USAG-1−/− (F and I) have supernumerary tooth in similar incidence after 20 days of culture, whereas these cultured explants in USAG-1+/+ (D and G) maintained the normal tooth number. (A–C) Explant appearance. (D–F) Coronal and (G–I) sagittal sections of explant. (J–L) Sagittal sections of control explant. (M) Table showing the relationship between number of teeth of explants and USAG-1 phenotypes.
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This work was supported by Grant-in-Aid for Scientific Research(C): 22592213 and 25463081 and A-STEP (Adaptable & Seamless Technology Transfer Program through Target-driven R&D) FS stage: AS231Z01061G and AS242Z02645Q. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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