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Dynamic expression patterns of the new protocadherin families CNRs and Pcdh-γ during mouse odontogenesis: comparison with reelin expression
Mechanisms of Development 106 (2001) 181±184
www.elsevier.com/locate/modo
Gene expression pattern
Dynamic expression patterns of the new protocadherin families CNRs and Pcdh-g during mouse odontogenesis: comparison with reelin expression Robert Heymann a,b, Sacha Kallenbach c, Serge Alonso c, Patrick Carroll c, Thimios A. Mitsiadis b,* a
Department of Cell and Molecular Biology, Karolinska Institutet, 171 77 Stockholm, Sweden b Faculte d'Odontologie, Universite de la MeÂditerraneÂe, 13385 Marseille Cedex 05, France c INSERM U. 382, Developmental Biology Institute of Marseille (IBDM), Campus de Luminy, Marseille Cedex 09, France Received 5 March 2001; received in revised form 2 April 2001; accepted 15 May 2001
Abstract Protocadherins are transmembrane glycoproteins belonging to the cadherin superfamily of molecules, which are involved in many biological processes such as cell adhesion, cytoskeletal organization and morphogenesis. Protocadherins generally exhibit only moderate adhesive activity and are highly expressed in the nervous system. Here, we report on the expression pattern of two novel families of protocadherins (CNRs and Pcdh-g) during rodent teeth development. Furthermore, we compare their expression with that of reelin, which is the potential ligand of CNRs. Throughout odontogenesis, CNRs, Pcdh-g and reelin show dynamic spatiotemporal expression patterns, which relate to both morphogenesis and cell differentiation events. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cadherins; Protocadherins; Reelin; CNRs; Mouse; Odontogenesis; Odontoblast; Tooth; Incisor; Molar; Development; Adhesion; Cell
Cadherin-like proteins constitute a rapidly expanding family of transmembrane molecules, which have been shown to play roles in processes as diverse as cell adhesion, control of cell division and migration, differentiation and axon path-®nding (reviewed by Yagi and Takeichi, 2000). Recently, three novel families of protocadherins with unusual gene organizations have been described: protocadherin-a, -b and -g (Wu and Maniatis, 1999). It has been suggested that protocadherin-a proteins can, via heterophilic interactions, act as receptors for the extracellular matrix protein, reelin (Senzaki et al., 1999), which is involved in the positioning of neuronal cells (reviewed by Gilmore and Herrup, 2000). Members of the protocadherin-a (called CNRs in mouse) and -g (Pcdh-g) families are found to be expressed in the developing and adult nervous system (Obata et al., 1995; Kohmura et al., 1998; Carroll et al., 2001). The detailed analysis of their expression in tissues other than neuronal ones will contribute towards a better understanding of the developmental functions of the protocadherin-a and -g families. Teeth develop as a result of sequential and reciprocal interactions between the oral ectoderm and neural crestderived mesenchyme. Mouse odontogenesis starts at the embryonic day 11.5 (E11.5) as local thickenings of the stomodeal epithelium. The thickened epithelium invagi* Corresponding author. Tel.: 133-791-784670; fax: 133-491-804343. E-mail address: [email protected] (T.A. Mitsiadis).
nates into the underlying mesenchyme and progressively acquires the bud (E13.5), cap (E14.5±E15.5) and bell (E16.5±18.5) con®gurations. The epithelium gives rise to the enamel organ and the mesenchyme forms the dental papilla. At the late bell stage, cells of the dental papilla start to differentiate into odontoblasts, responsible for dentin matrix synthesis. It has been shown that reelin is expressed in odontoblasts (Ikeda and Terashima, 1997; Buchaille et al., 2000), but no data are available for earlier stages. Here, we report the expression patterns of the CNRs and Pcdh-g and compare them with those of reelin and its intracellular signalling molecule, Dab1, during the different stages of mouse molar and incisor development. 1. Results and discussion CNRs, Pcdh-g , reelin and Dab1 expression during mouse odontogenesis were examined by in situ hybridization using digoxigenin-labelled probes. At E12, reelin and CNRs expression was restricted in the dental epithelium, whereas Pcdh-g mRNA was detected in the underlying mesenchyme (Fig. 1). Dab1 transcripts were found in both epithelial and mesenchymal components. At E13.5, reelin, Pcdh-g and Dab1 transcripts were observed in both dental epithelium and mesenchyme (Fig. 1). CNRs mRNA was detected only in epithelial cells in the
0925-4773/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(01)00433-6
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R. Heymann et al. / Mechanisms of Development 106 (2001) 181±184
Fig. 1. Expression patterns of the CNRs, Pcdh-g , reelin and Dab1 genes during the initiation (E12), bud (E13.5) and cap (E15.5) stages of molar development. The red lines represent the borders between the dental epithelium and underlying dental mesenchyme, while the green lines represent the borders between the dental epithelium and the mesenchyme of the dental follicle. Abbreviations: b, epithelial dental bud; bm, bone mesenchyme; cm, condensed dental mesenchyme; de, dental epithelium; eo, enamel organ; m, mesenchyme; md, mandibular process; mx, maxillary process; n, nerve; oe, oral epithelium; p, dental papilla mesenchyme.
middle of the dental bud. CNRs mRNA was also found in mesenchymal cells forming the alveolar bone. At E15.5, the reelin, CNRs and Pcdh-g genes were expressed in the enamel organ, while only Pcdh-g transcripts were found in the dental papilla (Fig. 1). The Dab1 hybridization signal was very weak in both epithelium and mesenchyme (Fig. 1). At E18.5, cytodifferentiation starts at the tip of the cusps. The reelin and Pcdh-g genes were detected in preameloblasts and differentiating odontoblasts (Fig. 2). By contrast, CNRs transcripts were detected in cells of the outer dental epithelium and stratum intermedium. The signal for Dab1 was very faint in dental tissues (Fig. 2). In incisors, reelin and Pcdh-g transcripts were detected in differentiating odontoblasts, preameloblasts and cells of the outer dental epithelium (Figs. 2 and 3). CNRs transcripts were found in stratum intermedium and stellate reticulum. The hybridization signal for the Dab1 gene was absent. The expression of reelin and absence of Dab1 have already been shown in the developing retina (Rice and Curran, 2000; Carroll et al., 2001). These results show that the changing patterns of CNRs, Pcdh-g , reelin and Dab1 expression during the different stages of mouse odontogenesis are related to morphogenetic and cell differentiation phenomena (Fig. 4).
2. Experimental procedures Swiss mice were used at embryonic stages (E12±E18.5).
In situ hybridization on cryosections, using digoxigeninlabelled antisense riboprobes for CNRs, Dab1 and reelin
Fig. 2. Expression patterns of the CNRs, Pcdh-g , reelin and Dab1 genes during the bell stage (E18.5) of both molar and incisor development. Longitudinal sections. Abbreviations: b, bone; bm, bone mesenchyme; df, incisor furrow; Hs, Hertwig's epithelial root sheath; ide, inner dental epithelium; o, odontoblasts; ode, outer dental epithelium; oe, oral epithelium; p, dental papilla mesenchyme; pa, preameloblasts; si, stratum intermedium; sr, stellate reticulum.
R. Heymann et al. / Mechanisms of Development 106 (2001) 181±184
183
Fig. 3. Expression patterns of the CNRs, Pcdh-g and reelin genes in incisors. Frontal sections through the upper and lower E17 incisors. Abbreviations: d, dentin; ide, inner dental epithelium; m, mesenchyme; o, odontoblasts; ode, outer dental epithelium; oe, oral epithelium; p, dental papilla mesenchyme; pa, preameloblasts; si, stratum intermedium; sr, stellate reticulum.
Fig. 4. Schematic representation of the reelin, CNRs, Pcdh-g and Dab1 gene expression in the ®rst mouse molar, during the successive stages of odontogenesis. Expression in the epithelium is indicated by the red colour, while the green colour shows expression in the mesenchyme. Abbreviations: b, epithelial dental bud; bm, bone mesenchyme; cl, cervical loop region; cm, condensed dental mesenchyme; de, dental epithelium; ide, inner dental epithelium; m, mesenchyme; o, odontoblasts; ode, outer dental epithelium; oe, oral epithelium; p, dental papilla mesenchyme; pa, preameloblasts; si, stratum intermedium; sr, stellate reticulum.
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R. Heymann et al. / Mechanisms of Development 106 (2001) 181±184
(as described in Carroll et al., 2001) was carried out using the method previously described (Mitsiadis et al., 1998). The CNRs probe is derived from the `constant' region. A 450 bp fragment from the `constant' region of Pcdh-g (accession number, U88550) was generated by PCR using oligonucleotides, sense 5 0 -CGATACGGACCCCAGTTTAC and antisense 5 0 -GCTTATCCATCTCCTGCCGC, and cloned into the pGEM-easy vector (Promega) for riboprobe synthesis. Acknowledgements This work was supported by grants of the Association pour la Recherche sur le Cancer (ARC), the Association FrancËaise contre les Myopathies (AFM), INSERM, the Swedish Medical Research Council (project 05200) and the Swedish Cancer Foundation (project 3957). R.H. was supported by fellowships from the Swedish Medical Research Council (project K2000-24F-13358-01), Swedish Dental Society, Erik and Edith FernstroÈms stiftelse, and Karolinska Institutet. References Buchaille, R., Couble, M.L., Magloire, H., Bleicher, F., 2000. A subtractive PCR-based cDNA library from human odontoblast cells: identi®cation
of novel genes expressed in tooth forming cells. Matrix Biol. 19, 421± 430. Carroll, P., Gayet, O., Feuillet, C., Kallenbach, S., de Bovis, B., Dudley, K., Alonso, S., 2001. Juxtaposition of CNR protocadherins and reelin expression in the developing spinal cord. Mol. Cell. Neurosci. in press. Gilmore, E.C., Herrup, K., 2000. Cortical development: receiving reelin. Curr. Biol. 10, 162±166. Ikeda, Y., Terashima, T., 1997. Expression of reelin, the gene responsible for the reeler mutation, in embryonic development and adulthood in the mouse. Dev. Dyn. 210, 157±172. Kohmura, N., Senzaki, K., Hamada, S., Kai, N., Yasuda, R., Watanabe, M., Ishii, H., Yasuda, M., Mishina, M., Yagi, T., 1998. Diversity revealed by a novel family of cadherins expressed in neurons at a synaptic complex. Neuron 20, 1137±1151. Mitsiadis, T.A., Hirsinger, E., Lendahl, U., Goridis, C., 1998. Delta-Notch signaling in odontogenesis: correlation with cytodifferentiation and evidence for feed-back regulation. Dev. Biol. 204, 420±431. Obata, S., Sago, H., Mori, N., Rochelle, J.M., Seldin, M.F., Davidson, M., St. John, T., Taketani, S., Suzuki, S.T., 1995. Protocadherin Pcdh2 shows properties similar to, but distinct from, those of classical cadherins. J. Cell Sci. 108, 3765±3773. Rice, D.S., Curran, T., 2000. Disabled-1 is expressed in type AII amacrine cells in the mouse retina. J. Comp. Neurol. 424, 327±338. Senzaki, K., Ogawa, M., Yagi, T., 1999. Proteins of the CNR family are multiple receptors for Reelin. Cell 99, 635±647. Wu, Q., Maniatis, T., 1999. A striking organization of a large family of human neural cadherin-like cell adhesion genes. Cell 97, 779±790. Yagi, T., Takeichi, M., 2000. Cadherin superfamily genes: functions, genomic organization, and neurologic diversity. Genes Dev. 14, 1169±1180.
www.elsevier.com/locate/modo
Gene expression pattern
Dynamic expression patterns of the new protocadherin families CNRs and Pcdh-g during mouse odontogenesis: comparison with reelin expression Robert Heymann a,b, Sacha Kallenbach c, Serge Alonso c, Patrick Carroll c, Thimios A. Mitsiadis b,* a
Department of Cell and Molecular Biology, Karolinska Institutet, 171 77 Stockholm, Sweden b Faculte d'Odontologie, Universite de la MeÂditerraneÂe, 13385 Marseille Cedex 05, France c INSERM U. 382, Developmental Biology Institute of Marseille (IBDM), Campus de Luminy, Marseille Cedex 09, France Received 5 March 2001; received in revised form 2 April 2001; accepted 15 May 2001
Abstract Protocadherins are transmembrane glycoproteins belonging to the cadherin superfamily of molecules, which are involved in many biological processes such as cell adhesion, cytoskeletal organization and morphogenesis. Protocadherins generally exhibit only moderate adhesive activity and are highly expressed in the nervous system. Here, we report on the expression pattern of two novel families of protocadherins (CNRs and Pcdh-g) during rodent teeth development. Furthermore, we compare their expression with that of reelin, which is the potential ligand of CNRs. Throughout odontogenesis, CNRs, Pcdh-g and reelin show dynamic spatiotemporal expression patterns, which relate to both morphogenesis and cell differentiation events. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cadherins; Protocadherins; Reelin; CNRs; Mouse; Odontogenesis; Odontoblast; Tooth; Incisor; Molar; Development; Adhesion; Cell
Cadherin-like proteins constitute a rapidly expanding family of transmembrane molecules, which have been shown to play roles in processes as diverse as cell adhesion, control of cell division and migration, differentiation and axon path-®nding (reviewed by Yagi and Takeichi, 2000). Recently, three novel families of protocadherins with unusual gene organizations have been described: protocadherin-a, -b and -g (Wu and Maniatis, 1999). It has been suggested that protocadherin-a proteins can, via heterophilic interactions, act as receptors for the extracellular matrix protein, reelin (Senzaki et al., 1999), which is involved in the positioning of neuronal cells (reviewed by Gilmore and Herrup, 2000). Members of the protocadherin-a (called CNRs in mouse) and -g (Pcdh-g) families are found to be expressed in the developing and adult nervous system (Obata et al., 1995; Kohmura et al., 1998; Carroll et al., 2001). The detailed analysis of their expression in tissues other than neuronal ones will contribute towards a better understanding of the developmental functions of the protocadherin-a and -g families. Teeth develop as a result of sequential and reciprocal interactions between the oral ectoderm and neural crestderived mesenchyme. Mouse odontogenesis starts at the embryonic day 11.5 (E11.5) as local thickenings of the stomodeal epithelium. The thickened epithelium invagi* Corresponding author. Tel.: 133-791-784670; fax: 133-491-804343. E-mail address: [email protected] (T.A. Mitsiadis).
nates into the underlying mesenchyme and progressively acquires the bud (E13.5), cap (E14.5±E15.5) and bell (E16.5±18.5) con®gurations. The epithelium gives rise to the enamel organ and the mesenchyme forms the dental papilla. At the late bell stage, cells of the dental papilla start to differentiate into odontoblasts, responsible for dentin matrix synthesis. It has been shown that reelin is expressed in odontoblasts (Ikeda and Terashima, 1997; Buchaille et al., 2000), but no data are available for earlier stages. Here, we report the expression patterns of the CNRs and Pcdh-g and compare them with those of reelin and its intracellular signalling molecule, Dab1, during the different stages of mouse molar and incisor development. 1. Results and discussion CNRs, Pcdh-g , reelin and Dab1 expression during mouse odontogenesis were examined by in situ hybridization using digoxigenin-labelled probes. At E12, reelin and CNRs expression was restricted in the dental epithelium, whereas Pcdh-g mRNA was detected in the underlying mesenchyme (Fig. 1). Dab1 transcripts were found in both epithelial and mesenchymal components. At E13.5, reelin, Pcdh-g and Dab1 transcripts were observed in both dental epithelium and mesenchyme (Fig. 1). CNRs mRNA was detected only in epithelial cells in the
0925-4773/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(01)00433-6
182
R. Heymann et al. / Mechanisms of Development 106 (2001) 181±184
Fig. 1. Expression patterns of the CNRs, Pcdh-g , reelin and Dab1 genes during the initiation (E12), bud (E13.5) and cap (E15.5) stages of molar development. The red lines represent the borders between the dental epithelium and underlying dental mesenchyme, while the green lines represent the borders between the dental epithelium and the mesenchyme of the dental follicle. Abbreviations: b, epithelial dental bud; bm, bone mesenchyme; cm, condensed dental mesenchyme; de, dental epithelium; eo, enamel organ; m, mesenchyme; md, mandibular process; mx, maxillary process; n, nerve; oe, oral epithelium; p, dental papilla mesenchyme.
middle of the dental bud. CNRs mRNA was also found in mesenchymal cells forming the alveolar bone. At E15.5, the reelin, CNRs and Pcdh-g genes were expressed in the enamel organ, while only Pcdh-g transcripts were found in the dental papilla (Fig. 1). The Dab1 hybridization signal was very weak in both epithelium and mesenchyme (Fig. 1). At E18.5, cytodifferentiation starts at the tip of the cusps. The reelin and Pcdh-g genes were detected in preameloblasts and differentiating odontoblasts (Fig. 2). By contrast, CNRs transcripts were detected in cells of the outer dental epithelium and stratum intermedium. The signal for Dab1 was very faint in dental tissues (Fig. 2). In incisors, reelin and Pcdh-g transcripts were detected in differentiating odontoblasts, preameloblasts and cells of the outer dental epithelium (Figs. 2 and 3). CNRs transcripts were found in stratum intermedium and stellate reticulum. The hybridization signal for the Dab1 gene was absent. The expression of reelin and absence of Dab1 have already been shown in the developing retina (Rice and Curran, 2000; Carroll et al., 2001). These results show that the changing patterns of CNRs, Pcdh-g , reelin and Dab1 expression during the different stages of mouse odontogenesis are related to morphogenetic and cell differentiation phenomena (Fig. 4).
2. Experimental procedures Swiss mice were used at embryonic stages (E12±E18.5).
In situ hybridization on cryosections, using digoxigeninlabelled antisense riboprobes for CNRs, Dab1 and reelin
Fig. 2. Expression patterns of the CNRs, Pcdh-g , reelin and Dab1 genes during the bell stage (E18.5) of both molar and incisor development. Longitudinal sections. Abbreviations: b, bone; bm, bone mesenchyme; df, incisor furrow; Hs, Hertwig's epithelial root sheath; ide, inner dental epithelium; o, odontoblasts; ode, outer dental epithelium; oe, oral epithelium; p, dental papilla mesenchyme; pa, preameloblasts; si, stratum intermedium; sr, stellate reticulum.
R. Heymann et al. / Mechanisms of Development 106 (2001) 181±184
183
Fig. 3. Expression patterns of the CNRs, Pcdh-g and reelin genes in incisors. Frontal sections through the upper and lower E17 incisors. Abbreviations: d, dentin; ide, inner dental epithelium; m, mesenchyme; o, odontoblasts; ode, outer dental epithelium; oe, oral epithelium; p, dental papilla mesenchyme; pa, preameloblasts; si, stratum intermedium; sr, stellate reticulum.
Fig. 4. Schematic representation of the reelin, CNRs, Pcdh-g and Dab1 gene expression in the ®rst mouse molar, during the successive stages of odontogenesis. Expression in the epithelium is indicated by the red colour, while the green colour shows expression in the mesenchyme. Abbreviations: b, epithelial dental bud; bm, bone mesenchyme; cl, cervical loop region; cm, condensed dental mesenchyme; de, dental epithelium; ide, inner dental epithelium; m, mesenchyme; o, odontoblasts; ode, outer dental epithelium; oe, oral epithelium; p, dental papilla mesenchyme; pa, preameloblasts; si, stratum intermedium; sr, stellate reticulum.
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R. Heymann et al. / Mechanisms of Development 106 (2001) 181±184
(as described in Carroll et al., 2001) was carried out using the method previously described (Mitsiadis et al., 1998). The CNRs probe is derived from the `constant' region. A 450 bp fragment from the `constant' region of Pcdh-g (accession number, U88550) was generated by PCR using oligonucleotides, sense 5 0 -CGATACGGACCCCAGTTTAC and antisense 5 0 -GCTTATCCATCTCCTGCCGC, and cloned into the pGEM-easy vector (Promega) for riboprobe synthesis. Acknowledgements This work was supported by grants of the Association pour la Recherche sur le Cancer (ARC), the Association FrancËaise contre les Myopathies (AFM), INSERM, the Swedish Medical Research Council (project 05200) and the Swedish Cancer Foundation (project 3957). R.H. was supported by fellowships from the Swedish Medical Research Council (project K2000-24F-13358-01), Swedish Dental Society, Erik and Edith FernstroÈms stiftelse, and Karolinska Institutet. References Buchaille, R., Couble, M.L., Magloire, H., Bleicher, F., 2000. A subtractive PCR-based cDNA library from human odontoblast cells: identi®cation
of novel genes expressed in tooth forming cells. Matrix Biol. 19, 421± 430. Carroll, P., Gayet, O., Feuillet, C., Kallenbach, S., de Bovis, B., Dudley, K., Alonso, S., 2001. Juxtaposition of CNR protocadherins and reelin expression in the developing spinal cord. Mol. Cell. Neurosci. in press. Gilmore, E.C., Herrup, K., 2000. Cortical development: receiving reelin. Curr. Biol. 10, 162±166. Ikeda, Y., Terashima, T., 1997. Expression of reelin, the gene responsible for the reeler mutation, in embryonic development and adulthood in the mouse. Dev. Dyn. 210, 157±172. Kohmura, N., Senzaki, K., Hamada, S., Kai, N., Yasuda, R., Watanabe, M., Ishii, H., Yasuda, M., Mishina, M., Yagi, T., 1998. Diversity revealed by a novel family of cadherins expressed in neurons at a synaptic complex. Neuron 20, 1137±1151. Mitsiadis, T.A., Hirsinger, E., Lendahl, U., Goridis, C., 1998. Delta-Notch signaling in odontogenesis: correlation with cytodifferentiation and evidence for feed-back regulation. Dev. Biol. 204, 420±431. Obata, S., Sago, H., Mori, N., Rochelle, J.M., Seldin, M.F., Davidson, M., St. John, T., Taketani, S., Suzuki, S.T., 1995. Protocadherin Pcdh2 shows properties similar to, but distinct from, those of classical cadherins. J. Cell Sci. 108, 3765±3773. Rice, D.S., Curran, T., 2000. Disabled-1 is expressed in type AII amacrine cells in the mouse retina. J. Comp. Neurol. 424, 327±338. Senzaki, K., Ogawa, M., Yagi, T., 1999. Proteins of the CNR family are multiple receptors for Reelin. Cell 99, 635±647. Wu, Q., Maniatis, T., 1999. A striking organization of a large family of human neural cadherin-like cell adhesion genes. Cell 97, 779±790. Yagi, T., Takeichi, M., 2000. Cadherin superfamily genes: functions, genomic organization, and neurologic diversity. Genes Dev. 14, 1169±1180.