- Email: [email protected]
βTrCP during mouse tooth development
Archives of Oral Biology (2005) 50, 147—151
www.intl.elsevierhealth.com/journals/arob
Expression of the Hedgehog antagonists Rab23 and Slimb/bTrCP during mouse tooth development Isabelle Mileticha,*, Martyn T. Cobourneb, Marium Abdeena, Paul T. Sharpea a
Department of Craniofacial Development, Dental Institute, King’s College London, Floor 28, Guy’s Hospital, London SE1 9RT, UK b Department of Craniofacial Development and Orthodontics, Dental Institute, King’s College London, Floor 28, Guy’s Hospital, London SE1 9RT, UK Accepted 15 September 2004
KEYWORDS Shh; Tooth development; Rab23; Slimb/bTrCP
Summary The sonic hedgehog signalling peptide has been demonstrated to play an important role in the growth and patterning of several organs including the tooth. Inappropriate activation of Shh signalling in the embryo causes various patterning defects and complex regulation of this pathway is important during normal development. A growing list of diverse antagonists have been identified that restrict Shh signalling in the embryo, however, only Ptc1, Gas1 and Hip1 have been studied during tooth development. We have examined the expression pattern of the putative antagonists Rab23 and Slimb/bTrCP during early murine odontogenesis and find that these molecules are expressed in the developing tooth. Interestingly, Rab23 demonstrates contrasting expression domains in the incisor and molar dentition during the cap stage, being restricted to the mesenchymal compartment of molar teeth and the epithelium of the enamel knot in incisor teeth. These findings provide the first evidence of distinct regulatory pathways for Shh in teeth of different classes. # 2004 Elsevier Ltd. All rights reserved.
Introduction The Hedgehog (Hh) family of secreted peptides comprises of three members in mouse; Sonic, Indian and Desert, of which Sonic hedgehog (Shh) has been shown to play a crucial role during tooth development. Teeth form as a result of reciprocal interactions between the ectoderm lining the primitive oral * Corresponding author. Tel.: +44 207 1881795; fax: +44 207 1881674. E-mail address: [email protected] (I. Miletich).
cavity and the underlying neural crest-derived mesenchyme1,2 and throughout tooth initiation, morphogenesis and differentiation, Shh expression is restricted to epithelial cells of the developing tooth germ.3 Shh is highly localised to the epithelial thickenings that mark the sites of future tooth formation and as these epithelial ingrowths bud into the underlying mesenchyme, expression continues at the tip of the buds. Early Shh expression at the presumptive tooth sites suggests a role during tooth initiation and in vitro evidence has indicated that Shh mitogenic activity may be responsible for loca-
0003–9969/$ — see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.archoralbio.2004.09.006
148
lised epithelial proliferation at the dental sites. Indeed, loss of Shh prevents tooth bud formation4,5 and conversely, application of ectopic Shh protein6 or induction of ectopic Shh transcription by electroporation (unpublished observations) induces ectopic budding of the oral epithelium into the underlying mesenchyme. Therefore, tight control of Shh expression or activity may play a key role in the positioning of future teeth. The discovery of two independent mechanisms of Shh restriction, acting at different levels in the signalling cascade at the epithelial thickening stage, further reinforces the idea that a strict control of Shh signalling is required during early tooth development. The first mechanism spatially restricts Shh transcription to discrete areas of dental epithelium where teeth will develop. Wnt7b, expressed in a reciprocal pattern to Shh represses Shh expression, establishing boundaries between dental and non-dental epithelium.5 Identification of the second mechanism took advantage of a toothless area or diastema situated between molars and incisors in the mouse mandible. Shh signalling peptides are able to move over considerable distance from producing cells7 and at the epithelial thickening stage, Shh protein diffuses from the molar and incisor tooth buds into the diastema mesenchyme. To prevent tooth formation in this non-odontogenic area, Shh protein is rendered inactive in the diastema mesenchyme by a mechanism that requires an intact diastema epithelium.8 Inactivation of Shh may be relayed in the diastema mesenchyme by Gas1, an antagonist of Shh signalling,9 expression of which in the diastema mesenchyme depends upon the presence of diastema epithelium.8 Transduction of the Shh signal10 (Fig. 1) involves two transmembrane proteins, Patched1 (Ptc1) and Smoothened (Smo), which are present at the surface of cells capable of responding to Shh. Ptc1 is the receptor for Shh, while Smo is a transmembrane protein that signals downstream through a mechanism that is poorly understood. In vertebrates, Shh signalling is mediated by transcription factors of the Gli family, of which there are three in mouse; Gli1, Gli2 and Gli3. In the resting state, Ptc1 inhibits the activity of Smo, but binding of Shh ligand to Ptc1 releases this inhibition, allowing signal transduction and transcription of the target genes Ptc1, Gli1 and Hip1 (Hedgehog-interacting protein). As previously emphasised, this signalling pathway is restricted during normal tooth development and thus far only three antagonists have been studied in murine dental tissues, Ptc1,6 Gas18 and Hip1.11 To gain insight into how Shh signalling is regulated during tooth development, we studied the expression pattern of two additional inhibitors, Rab23 and Slimb/bTrCP.
I. Miletich et al.
Figure 1 Model of Shh signal transduction. (A) At the plasma membrane, in the absence of Shh ligand, Ptc1 inhibits the positive signalling activity of Smo. Following proteasomal targeting of Ci/Gli by Slimb/bTrCP, Ci/Gli is cleaved into a smaller fragment that enters into the nucleus and represses Shh target genes. (B) Upon secretion, Shh binds to Ptc1 and relieves the inhibitory effect of Ptc1 on Smo. This allows downstream Smo signalling to Gli transcription factors through a poorly understood mechanism and transcription of the Shh targets Ptc1, Gli1 and Hip1. Hip1 and Gas1 are proteins localised at the plasma membrane that are also capable of binding Shh and attenuating signalling. Rab23 may inhibit Shh by regulating endocytosis or vesicle transport of Ptc1. Note that Shh signalling auto-regulates itself by increasing production of the Ptc1 receptor, which accelerates the clearing of Shh ligand at the plasma membrane and leads to subsequent inhibition of Shh signalling by Ptc1 in absence of Shh. Events described in the cytoplasm deduced from data obtained in Drosophila, but yet to be confirmed in vertebrates are indicated with dashed lines.
Rab23, encoded by the mouse open brain (opb) gene, belongs to the Rab family of GTPases involved in vesicle transport. Rab23 has been shown to antagonise Shh signalling in the mouse spinal cord by acting intracellularly, downstream of Shh.12 Rab23 localises to the plasma membrane and the endocytic pathway13 and may negatively regulate Shh signalling by acting on the endocytosis or vesicle trafficking of complexes from the cell surface. Slimb/bTrCP is a Fbox/WD40-repeat-containing protein identified in Drosophila.14—16 Slimb/bTrCP binds to specific proteins and facilitates their ubiquitination, resulting in the targeting of these proteins for degradation or proteolytic processing by the ubiquitin/proteasome pathway. In Drosophila, Slimb/ bTrCP targets the Gli homolog Cubitus interruptus (Ci) to the proteasome, where a truncated form of Ci is generated, which represses transcription of the Hh target genes. To evaluate the roles of Rab23 and Slimb/bTrCP during tooth development, we ana-
Expression of the Hedgehog antagonists
lysed their expression in the mouse embryo, from tooth initiation to crown morphogenesis.
149
antibody (Santa Cruz) detected using a biotinylated monoclonal anti-rabbit IgG (Sigma) and visualised with DAB (Vector Laboratories). Slides were counterstained with haematoxylin.
Materials and methods In situ hybridisation
Results
Wild type mouse embryos were collected from CD-I mice. The embryonic age was determined by the day when the vaginal plug was detected and designated as embryonic day (E) 0.5 (E0.5). Radioactive section in situ hybridisation using 35S-UTP radiolabelled riboprobes has previously been described.17 Shh and Ptc1 antisense riboprobes were made as in Cobourne et al.8 Rab23 and bTrCP antisense riboprobes were generated from mouse cDNA clones that were gifts respectively from J. Eggenschwiller12 and J. Jiang.18 For in situ hybridisation on newborn teeth, false colour images were generated with Adobe Photoshop 7.0 software in order to help visualising layers of differentiated ameloblasts and odontoblasts. Slides were counterstained with haematoxylin (Fluka).
Expression of Rab23 and Slimb/bTrCP in developing molar teeth
Immunohistochemistry Immunochemistry was carried out according to Gritli-Linde et al.7 with a primary Shh rabbit polyclonal
To investigate the possible roles of Rab23 and Slimb/bTrCP in the regulation of Shh signalling during tooth development, we analysed their expression from initiation at E11.5, to an advanced stage of crown morphogenesis at postnatal day 0 (P0) in the mouse mandibular first molar. Rab23 and Slimb/bTrCP expression was compared with the expression patterns of Shh and Ptc1. Ptc1 is upregulated in response to Shh signalling, but acts as an inhibitor of Shh in the absence of Shh ligand. At E11.5, when localised thickenings of the oral epithelium mark the future sites of tooth development, Rab23 and Slimb/bTrCP showed uniform expression throughout the dental epithelium and the underlying mesenchyme of the mandibular primordia (data not shown). Generally, Rab23 and Slimb/bTrCP were ubiquitously expressed at low levels in all tissues of the developing mandible
Figure 2 Expression of Shh, Ptc1, Rab23, Slimb/bTrCP and Shh protein during embryonic and postnatal development of teeth. In situ hybridisation on coronal sections of E13.5 (A—D), E14.5 (E—H) mandibular first molar tooth germs and P0 (I— L) maxillary molar tooth germs. Mandibular incisor tooth germs at E14.5 (M—R); haematoxylin and eosin (M and P); Rab23 expression in the enamel knot (N and Q); Shh protein in the enamel knot (O) and Rab23 expression superimposed upon a light field view of the incisor (R). Tooth epithelium is outlined in red. Note gene expression superimposed in red for sections (I—L).
150
at all developmental stages studied. At E13.5, when proliferation of the epithelial thickenings has reached the late bud stage, associated with localised condensation of the underlying mesenchyme to form the tooth germs, Shh was expressed in epithelial cells at the tip of the molar tooth bud (Fig. 2A). Rab23 was upregulated in both the tooth buds and associated ectomesenchyme (Fig. 2C), whilst Slimb/bTrCP appeared to be upregulated in the dental mesenchyme only (Fig. 2D). Ptc1 was intensely expressed in the condensing ectomesenchyme, with weaker expression in the epithelial cells of the molar bud (Fig. 2B). At E14.5, developing molars had reached the cap stage of development and the position of the enamel knot, an epithelial signalling centre involved in the formation of tooth crown cusps, was clearly demarcated by the localised expression of Shh (Fig. 2E). Expression of Rab23 was increased in the dental papilla and follicle at this stage (Fig. 2G), whilst Slimb/bTrCP was strongly expressed in the whole tooth bud (Fig. 2H). Expression of Ptc1 was widespread throughout the epithelium and the mesenchyme of cap stage molar germs, but excluded from the enamel knot and inner enamel epithelium (Fig. 2F). At birth, crown morphogenesis is almost complete and ameloblasts and odontoblasts (responsible for formation of the dental hard tissues enamel and dentine, respectively) have differentiated. Shh was strongly expressed in the amelobasts (Fig. 2I), whilst Ptc1 exhibited a complementary pattern in the dental pulp, odontoblasts and dental sac surrounding the teeth (Fig. 2J). Similar to Ptc1, Slimb/bTrCP exhibited upregulation in the dental pulp (Fig. 2L). Rab23 predominantly localised to the ameloblasts, with stronger expression at the tip and bottom of the molar cusps (Fig. 2K). Interestingly, the tips of the molar cusps are areas devoid of enamel in mice.
I. Miletich et al.
Discussion Our in situ hybridisation analysis reveals that in common with other components of the Shh signalling pathway, Rab23 and Slimb/bTrCP are upregulated in the developing tooth germ. During the early stages of development, these two genes were ubiquitously expressed in the mandible, which may reflect the fact that products of these genes are required for various regulatory cell processes. Indeed, Slimb/bTrCP participates in other signalling pathways such as Wnt14—16 and NF-kB18 signalling and also plays a role in regulating the cell cycle.19,20 At the initiation stage of tooth development, Rab23 and Slimb/bTrCP exhibited weak but uniform expression. At the bud and cap stages, they were upregulated in the mesenchyme and the epithelium of the tooth germs, similar to Ptc1. At an advanced stage of crown morphogenesis, Slimb/bTrCP showed a spatial expression identical to Ptc1, whilst Rab23 upregulated in the ameloblast population, where Shh is strongly expressed. Transcription of Rab23 in the ameloblasts was stronger at the tip of the cusps, which is consistent with an absence of enamel deposition in these regions of the mouse tooth. More striking was the strong upregulation of Rab23 in the incisor enamel knot, an upregulation that could not be identified in the molar enamel knot. This is the first time that a difference in enamel knot gene expression has been reported between incisor and molar teeth and invites speculation that differences in the fine-tuning of Shh activity may exist between teeth of different classes. Indeed, it is interesting to note that differences in phenotype have been reported between the incisor and molar teeth of conditional Shh knockout mice.21 The additional complexity of the molar dentition may, therefore, require higher levels of Shh signalling activity.
Rab23 is strongly upregulated in the enamel knot of mandibular incisors
Acknowledgements
The analysis of consecutive sections of the mandibular incisor regions (Fig. 2M—R) revealed that, in contrast to the other genes examined, Rab23 expression exhibited marked differences between molar and incisor tooth germs at the cap stage. Rab23 was strongly upregulated in the incisor enamel knot (Fig. 2N, Q and R), which was not observed in molar tooth germs (Fig. 2G). Rab23 expression therefore coincided with Shh secretion in the incisor tooth germs at the cap stage (Fig. 2N and O). Expression of Rab23 at other stages of tooth development was the same in developing incisors and molars.
This work was supported by the Medical Research Council.
References 1. Miletich I, Sharpe PT. Neural crest contribution to mammalian tooth formation. Birth Defects Res Part C Embryol Today 2004;72:200—12. 2. Peters H, Balling RT. Where and how to make them. Trends Genet 1999;15:59—65. 3. Bitgood MJ, McMahon AP. Hedgehog and Bmp genes are coexpressed at many diverse sites of cell—cell interaction in the mouse embryo. Dev Biol 1995;172:126—38.
Expression of the Hedgehog antagonists
4. Cobourne MT, Hardcastle Z, Sharpe PT. Sonic hedgehog regulates epithelial proliferation and cell survival in the developing tooth germ. J Dent Res 2001;80:1974—9. 5. Sarkar L, Cobourne M, Naylor S, Smalley M, Dale T, Sharpe PT. Wnt/Shh interactions regulate ectodermal boundary formation during mammalian tooth development. Proc Natl Acad Sci USA 2000;97:4520—4. 6. Hardcastle Z, Mo R, Hui CC, Sharpe PT. The Shh signalling pathway in tooth development: defects in Gli2 and Gli3 mutants. Development 1998;125:2803—11. 7. Gritli-Linde A, Lewis P, McMahon AP, Linde A. The whereabouts of a morphogen: direct evidence for short- and graded long-range activity of hedgehog signalling peptides. Dev Biol 2001;236:364—86. 8. Cobourne MT, Miletich I, Sharpe PT. Restriction of sonic hedgehog signalling during early tooth development. Development 2004;131:2875—85. 9. Lee CS, Buttitta L, Fan CM. Evidence that the WNT-inducible growth arrest-specific gene 1 encodes an antagonist of sonic hedgehog signaling in the somite. Proc Natl Acad Sci USA 2001;98:11347—52. 10. Nybakken K, Perrimon N. Hedgehog signal transduction: recent findings. Curr Opin Genet Dev 2002;12:503—11. 11. Cobourne MT, Sharpe PT. Expression and regulation of hedgehog-interacting protein during early tooth development. Connect Tissue Res 2002;43:143—7. 12. Eggenschwiler JT, Espinoza E, Anderson KV. Rab23 is an essential negative regulator of the mouse sonic hedgehog signalling pathway. Nature 2001;412:194—8. 13. Evans TM, Ferguson C, Wainwright BJ, Parton RG, Wicking C. Rab23, a negative regulator of hedgehog signalling, localises
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to the plasma membrane and the endocytic pathway. Traffic 2003;4:869—84. Jiang J, Struhl G. Regulation of the hedgehog and wingless signalling pathways by the F-box/WD40-repeat protein slimb. Nature 1998;391:493—6. Miletich I, Limbourg-Bouchon B. Drosophila null slimb clones transiently deregulate hedgehog-independent transcription of wingless in all limb discs, and induces decapentaplegic transcription linked to imaginal disc regeneration. Mech Dev 2000;93:15—26. Theodosiou NA, Zhang S, Wang WY, Xu T. Slimb coordinates wg and dpp expression in the dorsal-ventral and anterior-posterior axes during limb development. Development 1998;125: 3411—6. Wilkinson DG. In situ hybridisation: a practical approach. Oxford, UK: IRL Press; 1992. Spencer E, Jiang J, Chen ZJ. Signal-induced ubiquitination of IkappaBalpha by the F-box protein Slimb/beta-TrCP. Genes Dev 1999;13:284—94. Heriche JK, Ang D, Bier E, O’Farrell PH. Involvement of an SCFSlmb complex in timely elimination of E2F upon initiation of DNA replication in Drosophila. BMC Genet 2003;4:9. Margottin-Goguet F, Hsu JY, Loktev A, Hsieh HM, Reimann JD, Jackson PK. Prophase destruction of Emil by the SCF(betaTrCP/Slimb) ubiquitin ligase activates the anaphase promoting complex to allow progression beyond prometaphase. Dev Cell 2003;4:813—26. Gritli-Linde A, Bei M, Maas R, Zhang XM, Linde A, McMahon AP. Shh signalling within the dental epithelium is necessary for cell proliferation, growth and polarization. Development 2002;129:5323—37.
www.intl.elsevierhealth.com/journals/arob
Expression of the Hedgehog antagonists Rab23 and Slimb/bTrCP during mouse tooth development Isabelle Mileticha,*, Martyn T. Cobourneb, Marium Abdeena, Paul T. Sharpea a
Department of Craniofacial Development, Dental Institute, King’s College London, Floor 28, Guy’s Hospital, London SE1 9RT, UK b Department of Craniofacial Development and Orthodontics, Dental Institute, King’s College London, Floor 28, Guy’s Hospital, London SE1 9RT, UK Accepted 15 September 2004
KEYWORDS Shh; Tooth development; Rab23; Slimb/bTrCP
Summary The sonic hedgehog signalling peptide has been demonstrated to play an important role in the growth and patterning of several organs including the tooth. Inappropriate activation of Shh signalling in the embryo causes various patterning defects and complex regulation of this pathway is important during normal development. A growing list of diverse antagonists have been identified that restrict Shh signalling in the embryo, however, only Ptc1, Gas1 and Hip1 have been studied during tooth development. We have examined the expression pattern of the putative antagonists Rab23 and Slimb/bTrCP during early murine odontogenesis and find that these molecules are expressed in the developing tooth. Interestingly, Rab23 demonstrates contrasting expression domains in the incisor and molar dentition during the cap stage, being restricted to the mesenchymal compartment of molar teeth and the epithelium of the enamel knot in incisor teeth. These findings provide the first evidence of distinct regulatory pathways for Shh in teeth of different classes. # 2004 Elsevier Ltd. All rights reserved.
Introduction The Hedgehog (Hh) family of secreted peptides comprises of three members in mouse; Sonic, Indian and Desert, of which Sonic hedgehog (Shh) has been shown to play a crucial role during tooth development. Teeth form as a result of reciprocal interactions between the ectoderm lining the primitive oral * Corresponding author. Tel.: +44 207 1881795; fax: +44 207 1881674. E-mail address: [email protected] (I. Miletich).
cavity and the underlying neural crest-derived mesenchyme1,2 and throughout tooth initiation, morphogenesis and differentiation, Shh expression is restricted to epithelial cells of the developing tooth germ.3 Shh is highly localised to the epithelial thickenings that mark the sites of future tooth formation and as these epithelial ingrowths bud into the underlying mesenchyme, expression continues at the tip of the buds. Early Shh expression at the presumptive tooth sites suggests a role during tooth initiation and in vitro evidence has indicated that Shh mitogenic activity may be responsible for loca-
0003–9969/$ — see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.archoralbio.2004.09.006
148
lised epithelial proliferation at the dental sites. Indeed, loss of Shh prevents tooth bud formation4,5 and conversely, application of ectopic Shh protein6 or induction of ectopic Shh transcription by electroporation (unpublished observations) induces ectopic budding of the oral epithelium into the underlying mesenchyme. Therefore, tight control of Shh expression or activity may play a key role in the positioning of future teeth. The discovery of two independent mechanisms of Shh restriction, acting at different levels in the signalling cascade at the epithelial thickening stage, further reinforces the idea that a strict control of Shh signalling is required during early tooth development. The first mechanism spatially restricts Shh transcription to discrete areas of dental epithelium where teeth will develop. Wnt7b, expressed in a reciprocal pattern to Shh represses Shh expression, establishing boundaries between dental and non-dental epithelium.5 Identification of the second mechanism took advantage of a toothless area or diastema situated between molars and incisors in the mouse mandible. Shh signalling peptides are able to move over considerable distance from producing cells7 and at the epithelial thickening stage, Shh protein diffuses from the molar and incisor tooth buds into the diastema mesenchyme. To prevent tooth formation in this non-odontogenic area, Shh protein is rendered inactive in the diastema mesenchyme by a mechanism that requires an intact diastema epithelium.8 Inactivation of Shh may be relayed in the diastema mesenchyme by Gas1, an antagonist of Shh signalling,9 expression of which in the diastema mesenchyme depends upon the presence of diastema epithelium.8 Transduction of the Shh signal10 (Fig. 1) involves two transmembrane proteins, Patched1 (Ptc1) and Smoothened (Smo), which are present at the surface of cells capable of responding to Shh. Ptc1 is the receptor for Shh, while Smo is a transmembrane protein that signals downstream through a mechanism that is poorly understood. In vertebrates, Shh signalling is mediated by transcription factors of the Gli family, of which there are three in mouse; Gli1, Gli2 and Gli3. In the resting state, Ptc1 inhibits the activity of Smo, but binding of Shh ligand to Ptc1 releases this inhibition, allowing signal transduction and transcription of the target genes Ptc1, Gli1 and Hip1 (Hedgehog-interacting protein). As previously emphasised, this signalling pathway is restricted during normal tooth development and thus far only three antagonists have been studied in murine dental tissues, Ptc1,6 Gas18 and Hip1.11 To gain insight into how Shh signalling is regulated during tooth development, we studied the expression pattern of two additional inhibitors, Rab23 and Slimb/bTrCP.
I. Miletich et al.
Figure 1 Model of Shh signal transduction. (A) At the plasma membrane, in the absence of Shh ligand, Ptc1 inhibits the positive signalling activity of Smo. Following proteasomal targeting of Ci/Gli by Slimb/bTrCP, Ci/Gli is cleaved into a smaller fragment that enters into the nucleus and represses Shh target genes. (B) Upon secretion, Shh binds to Ptc1 and relieves the inhibitory effect of Ptc1 on Smo. This allows downstream Smo signalling to Gli transcription factors through a poorly understood mechanism and transcription of the Shh targets Ptc1, Gli1 and Hip1. Hip1 and Gas1 are proteins localised at the plasma membrane that are also capable of binding Shh and attenuating signalling. Rab23 may inhibit Shh by regulating endocytosis or vesicle transport of Ptc1. Note that Shh signalling auto-regulates itself by increasing production of the Ptc1 receptor, which accelerates the clearing of Shh ligand at the plasma membrane and leads to subsequent inhibition of Shh signalling by Ptc1 in absence of Shh. Events described in the cytoplasm deduced from data obtained in Drosophila, but yet to be confirmed in vertebrates are indicated with dashed lines.
Rab23, encoded by the mouse open brain (opb) gene, belongs to the Rab family of GTPases involved in vesicle transport. Rab23 has been shown to antagonise Shh signalling in the mouse spinal cord by acting intracellularly, downstream of Shh.12 Rab23 localises to the plasma membrane and the endocytic pathway13 and may negatively regulate Shh signalling by acting on the endocytosis or vesicle trafficking of complexes from the cell surface. Slimb/bTrCP is a Fbox/WD40-repeat-containing protein identified in Drosophila.14—16 Slimb/bTrCP binds to specific proteins and facilitates their ubiquitination, resulting in the targeting of these proteins for degradation or proteolytic processing by the ubiquitin/proteasome pathway. In Drosophila, Slimb/ bTrCP targets the Gli homolog Cubitus interruptus (Ci) to the proteasome, where a truncated form of Ci is generated, which represses transcription of the Hh target genes. To evaluate the roles of Rab23 and Slimb/bTrCP during tooth development, we ana-
Expression of the Hedgehog antagonists
lysed their expression in the mouse embryo, from tooth initiation to crown morphogenesis.
149
antibody (Santa Cruz) detected using a biotinylated monoclonal anti-rabbit IgG (Sigma) and visualised with DAB (Vector Laboratories). Slides were counterstained with haematoxylin.
Materials and methods In situ hybridisation
Results
Wild type mouse embryos were collected from CD-I mice. The embryonic age was determined by the day when the vaginal plug was detected and designated as embryonic day (E) 0.5 (E0.5). Radioactive section in situ hybridisation using 35S-UTP radiolabelled riboprobes has previously been described.17 Shh and Ptc1 antisense riboprobes were made as in Cobourne et al.8 Rab23 and bTrCP antisense riboprobes were generated from mouse cDNA clones that were gifts respectively from J. Eggenschwiller12 and J. Jiang.18 For in situ hybridisation on newborn teeth, false colour images were generated with Adobe Photoshop 7.0 software in order to help visualising layers of differentiated ameloblasts and odontoblasts. Slides were counterstained with haematoxylin (Fluka).
Expression of Rab23 and Slimb/bTrCP in developing molar teeth
Immunohistochemistry Immunochemistry was carried out according to Gritli-Linde et al.7 with a primary Shh rabbit polyclonal
To investigate the possible roles of Rab23 and Slimb/bTrCP in the regulation of Shh signalling during tooth development, we analysed their expression from initiation at E11.5, to an advanced stage of crown morphogenesis at postnatal day 0 (P0) in the mouse mandibular first molar. Rab23 and Slimb/bTrCP expression was compared with the expression patterns of Shh and Ptc1. Ptc1 is upregulated in response to Shh signalling, but acts as an inhibitor of Shh in the absence of Shh ligand. At E11.5, when localised thickenings of the oral epithelium mark the future sites of tooth development, Rab23 and Slimb/bTrCP showed uniform expression throughout the dental epithelium and the underlying mesenchyme of the mandibular primordia (data not shown). Generally, Rab23 and Slimb/bTrCP were ubiquitously expressed at low levels in all tissues of the developing mandible
Figure 2 Expression of Shh, Ptc1, Rab23, Slimb/bTrCP and Shh protein during embryonic and postnatal development of teeth. In situ hybridisation on coronal sections of E13.5 (A—D), E14.5 (E—H) mandibular first molar tooth germs and P0 (I— L) maxillary molar tooth germs. Mandibular incisor tooth germs at E14.5 (M—R); haematoxylin and eosin (M and P); Rab23 expression in the enamel knot (N and Q); Shh protein in the enamel knot (O) and Rab23 expression superimposed upon a light field view of the incisor (R). Tooth epithelium is outlined in red. Note gene expression superimposed in red for sections (I—L).
150
at all developmental stages studied. At E13.5, when proliferation of the epithelial thickenings has reached the late bud stage, associated with localised condensation of the underlying mesenchyme to form the tooth germs, Shh was expressed in epithelial cells at the tip of the molar tooth bud (Fig. 2A). Rab23 was upregulated in both the tooth buds and associated ectomesenchyme (Fig. 2C), whilst Slimb/bTrCP appeared to be upregulated in the dental mesenchyme only (Fig. 2D). Ptc1 was intensely expressed in the condensing ectomesenchyme, with weaker expression in the epithelial cells of the molar bud (Fig. 2B). At E14.5, developing molars had reached the cap stage of development and the position of the enamel knot, an epithelial signalling centre involved in the formation of tooth crown cusps, was clearly demarcated by the localised expression of Shh (Fig. 2E). Expression of Rab23 was increased in the dental papilla and follicle at this stage (Fig. 2G), whilst Slimb/bTrCP was strongly expressed in the whole tooth bud (Fig. 2H). Expression of Ptc1 was widespread throughout the epithelium and the mesenchyme of cap stage molar germs, but excluded from the enamel knot and inner enamel epithelium (Fig. 2F). At birth, crown morphogenesis is almost complete and ameloblasts and odontoblasts (responsible for formation of the dental hard tissues enamel and dentine, respectively) have differentiated. Shh was strongly expressed in the amelobasts (Fig. 2I), whilst Ptc1 exhibited a complementary pattern in the dental pulp, odontoblasts and dental sac surrounding the teeth (Fig. 2J). Similar to Ptc1, Slimb/bTrCP exhibited upregulation in the dental pulp (Fig. 2L). Rab23 predominantly localised to the ameloblasts, with stronger expression at the tip and bottom of the molar cusps (Fig. 2K). Interestingly, the tips of the molar cusps are areas devoid of enamel in mice.
I. Miletich et al.
Discussion Our in situ hybridisation analysis reveals that in common with other components of the Shh signalling pathway, Rab23 and Slimb/bTrCP are upregulated in the developing tooth germ. During the early stages of development, these two genes were ubiquitously expressed in the mandible, which may reflect the fact that products of these genes are required for various regulatory cell processes. Indeed, Slimb/bTrCP participates in other signalling pathways such as Wnt14—16 and NF-kB18 signalling and also plays a role in regulating the cell cycle.19,20 At the initiation stage of tooth development, Rab23 and Slimb/bTrCP exhibited weak but uniform expression. At the bud and cap stages, they were upregulated in the mesenchyme and the epithelium of the tooth germs, similar to Ptc1. At an advanced stage of crown morphogenesis, Slimb/bTrCP showed a spatial expression identical to Ptc1, whilst Rab23 upregulated in the ameloblast population, where Shh is strongly expressed. Transcription of Rab23 in the ameloblasts was stronger at the tip of the cusps, which is consistent with an absence of enamel deposition in these regions of the mouse tooth. More striking was the strong upregulation of Rab23 in the incisor enamel knot, an upregulation that could not be identified in the molar enamel knot. This is the first time that a difference in enamel knot gene expression has been reported between incisor and molar teeth and invites speculation that differences in the fine-tuning of Shh activity may exist between teeth of different classes. Indeed, it is interesting to note that differences in phenotype have been reported between the incisor and molar teeth of conditional Shh knockout mice.21 The additional complexity of the molar dentition may, therefore, require higher levels of Shh signalling activity.
Rab23 is strongly upregulated in the enamel knot of mandibular incisors
Acknowledgements
The analysis of consecutive sections of the mandibular incisor regions (Fig. 2M—R) revealed that, in contrast to the other genes examined, Rab23 expression exhibited marked differences between molar and incisor tooth germs at the cap stage. Rab23 was strongly upregulated in the incisor enamel knot (Fig. 2N, Q and R), which was not observed in molar tooth germs (Fig. 2G). Rab23 expression therefore coincided with Shh secretion in the incisor tooth germs at the cap stage (Fig. 2N and O). Expression of Rab23 at other stages of tooth development was the same in developing incisors and molars.
This work was supported by the Medical Research Council.
References 1. Miletich I, Sharpe PT. Neural crest contribution to mammalian tooth formation. Birth Defects Res Part C Embryol Today 2004;72:200—12. 2. Peters H, Balling RT. Where and how to make them. Trends Genet 1999;15:59—65. 3. Bitgood MJ, McMahon AP. Hedgehog and Bmp genes are coexpressed at many diverse sites of cell—cell interaction in the mouse embryo. Dev Biol 1995;172:126—38.
Expression of the Hedgehog antagonists
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