Novel Golgi Protein,GoPro49,Is a Specific Dental Follicle Marker

Abstract

GoPro49 is a recently identified, novel Golgi protein that is expressed in embryonic mesenchymal tissues, including dental follicle. In the present study, we have tested the hypothesis that the gene is a specific marker for the dental follicle, and examined its expression during the development of mouse incisors and molars. In situ hybridization showed that GoPro49 is expressed in dental follicles from bud to post-eruption stages. The expression is intense throughout the dental follicle during crown development, and persists in the root follicle during root development. In the forming periodontal ligament, GoPro49 expression is down-regulated upon differentiation of the follicle cells to cementoblasts and osteoblasts marked by Bsp1. In cultured dental follicle cells, the GoPro49 protein co-localizes with β-COP, suggesting that GoPro49 may function in the secretory pathway. We conclude that GoPro49 is a novel, specific marker for the dental follicle and can be used to identify this tissue. Abbreviations: Bsp1, bone sialoprotein 1; GoPro49, Golgi protein 49 kDa; E16, embryonic day 16; HERS, Hertwig’s epithelial root sheath; PDL, periodontal ligament; dpn, day post-natal.

INTRODUCTION

The dental follicle is a mesenchymal tissue that gives rise to the periodontal ligament (PDL), a specialized connective tissue between cementum and alveolar bone. The PDL is composed of a heterogeneous population of mesenchymal cells, including fibroblasts, cementoblasts, and osteoblasts (Nanci and Bosshardt, 2006; Ten Cate, 1997). In addition, undifferentiated mesenchymal stem or progenitor cells have been identified in the PDL (Seo et al., 2004). There is also an epithelial cell population in the PDL, the epithelial cell rests of Malassez (ERM), which derive from dental epithelium, and form a fenestrated network around the root (Rincon et al., 2006).

The dental follicle is an important contributor to root development and essential to tooth eruption (Wise et al., 2002). While the molecular mechanisms involved in the regulation of tooth crown formation are quite well-known (Jernvall and Thesleff, 2000), this is not the case for root formation. It has been demonstrated, by tissue recombination experiments, that interactions between the dental follicle and Hertwig’s epithelial root sheath regulate the root formation (Thomas, 1995). However, the molecular mechanisms involved remain elusive.

The expression of numerous proteins has been reported in the dental follicle and periodontal ligament, but these are either limited to a certain developmental period or are also present in other tissues of the developing teeth, most often in the dental papilla. The ‘Gene expression in tooth’ database (http://bite-it.helsinki.fi/) shows the expression patterns of 92 different genes in the dental follicle during tooth morphogenesis, and none is expressed solely in the follicle or present throughout follicle development. ActivinβA has previously been reported as a possible dental follicle marker for early stages of tooth development (Wang et al., 2004b), but it is also expressed intensely in the dental papilla mesenchyme. Bone sialoprotein 1 (Bsp1) is specifically expressed in the terminally differentiated cells forming mineralized tissue, i.e., osteoblasts, odontoblasts, and cementoblasts (Yamashiro et al., 2003), and therefore it serves as a marker for the differentiated components of the PDL bordering the dental follicle.

Golgi protein 49kDa (GoPro49) was identified in our Golgi proteomic study (Takatalo et al., 2006). We have recently reported the expression of GoPro49 in a selection of embryonic mesenchymal tissues, including the cartilage of vertebrae and limbs (Takatalo et al., 2008). In cartilage, the gene was found mostly in columnar chondrocytes, which might indicate a function before terminal differentiation. Interestingly, expression was also noted in the dental follicles of embryonic mouse molar tooth germs. In the present study, we have tested the hypothesis that the gene is a specific marker for the dental follicle, and examined its expression in early and late incisor development and during root development of the molar.

MATERIALS & METHODS

Tissue Processing and in situ Hybridization

For radioactive in situ hybridization analysis, wild-type NMRI mouse jaws (E13-12 dpn) were fixed in 4% paraformaldehyde overnight and embedded in paraffin; serial sections were cut (7 μm). Radioactive in situ hybridization procedures were carried out as previously described (Åberg et al., 2004). All mice were handled in accordance with the institutional animal care policy of the University of Helsinki. All animal protocols were approved by the University of Helsinki Review Board for Animal Experiments.

Probes for GoPro49 (Takatalo et al., 2008), Bsp1 (Young et al., 1994), and ActivinβA (Wang et al., 2004a) have been described previously. Plasmids were linearized, and for radioactive in situ hybridization, cRNA probes were synthesized with ³⁵S-labeled nucleotides (Wilkinson and Green, 1991) with the appropriate RNA polymerase (Promega, Madison, WI, USA).

Hematoxylin and eosin staining was performed in the non- hybridized histological consecutive sections.

Images of the sections were taken with an Olympus digital camera and processed with Adobe Photoshop CS version 8.0.

Cell Culture and Indirect Immunofluorescence

Dental follicle cells were isolated from E18.5 embryonic NMRI mice as described previously (Thesleff, 1986) and fixed with MeOH at − 20°C. The endogenous GoPro49 protein was visualized with a rabbit polyclonal antibody raised against GST-fusion protein of human GoPro49 (Takatalo et al., 2008). Specificity of the antibody was tested with immunostaining in transfected HeLa cells, where affinity-purified antibody detected the fusion protein, but no endogenous protein. The purified antibody was diluted to 2% FBS-PBS for staining, followed by Cy3-coupled secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA). For double-staining, β-COP (clone M3A5, Sigma, St. Louis, MO, USA) or GM130 (Transduction Laboratories, Lexington, KY, USA) antibodies were used, followed by Alexa488-coupled secondary antibody (Invitrogen Molecular Probes, Eugene, OR, USA). Images were collected with the use of a Leica TCS SP laser scanning confocal microscope with 63x oil immersion objective.

RESULTS

In this study, we show that GoPro49 has a highly specific expression pattern between E13 and 12 dpn. GoPro49 expression was observed in the mouse first molar and incisor and was restricted mainly to the dental follicle (Figs. 1, 2 .). Expression was observed primarily as a complementary layer between Bsp1 in the bone and tooth root, with little or no expression in the dental papilla. The strongest GoPro49 expression was seen surrounding the 4-dpn incisors, where expression was observed on both labial and lingual sides, comparable with the crown and root follicle, respectively (Fig. 1G ). The expression patterns of incisors and molars corresponded well. Furthermore, GoPro49 co-localized with β-COP in the dental follicle cells.

Expression during Embryogenesis

GoPro49 expression has been reported in embryonic mouse molars (Takatalo et al., 2008). We examined the expression during the development of incisors, showing strong and even expression restricted to the dental follicle surrounding the dental epithelium and dental papilla mesenchyme (Fig. 1). At E13 (Fig. 1A ), expression was restricted to the peripheral cells of the condensed dental mesenchyme, which form the dental follicle. The mesenchyme directly underlining the bud epithelium that forms the dental papilla was completely negative. In E14 and E16 incisors (Figs. 1C, 1E ), GoPro49 expression was restricted to the dental follicle.

ActivinβA was co-expressed with GoPro49 in the incisor dental follicle from E13 to E16, but appeared more intense in the peripheral cells of the follicle bordering the area of bone formation. In addition, strong ActivinβA expression was seen in the mesenchyme forming the dental papilla, in particular at E13 and E14 (Figs. 1B, 1D, 1F ). ActivinβA expression was seen lining bone at E16 (Fig. 1F ). Bsp1, in contrast, was co-expressed with GoPro49 only in the peripheral follicle representing the osteoblast cell lineage, but not in the follicle cells close to the tooth (Appendix Figs. 1A–1C).

Expression in Post-natal Tissues

In 4-dpn incisors, GoPro49 expression was limited to the dental follicle on both labial and lingual sides (Figs. 1G–1I , 2B ). In 4-dpn first molars, strong GoPro49 expression was observed in the dental follicle and some in the stellate reticulum cells (Fig. 2B ). A large cluster of GoPro49-expressing cells was present in the dental follicle mesenchyme at the site of initial root formation (Fig. 2E ), while the expression in the crown follicle had decreased. There was also faint, but specific, expression of GoPro49 in osteoblasts throughout the alveolar bone (Fig. 2B ).

Bsp1 was expressed in cementoblasts and osteoblasts, with no expression in the dental follicle cells (Appendix Figs. 1D, 1E). The cluster of follicular mesenchyme beneath the molar root tip showed no Bsp1 expression (Appendix Fig. 1E). In incisors, Bsp1 expression was intense in odontoblasts and in bone cells. In addition, Bsp1 expression was observed on the labial side of the incisor in a layer of dental follicle cells near the ameloblasts, possibly representing cementoblast precursors. Again, Bsp1 and GoPro49 patterns were largely complementary in the dental follicle. At 4 dpn, ActivinβA expression was observed in the dental follicles of molars and incisors, and in the mesenchyme bordering bone (Figs. 2C, 2F ). The ActivinβA expression levels were similar in both crown and root follicle.

At 7 dpn, GoPro49 expression continued in the molar dental follicle and lined the surface of the forming root. The expression in the stellate reticulum was reduced to the area near the dental lamina (Appendix Fig. 2C). A higher magnification showed continued expression of GoPro49 in the dental follicle bordering the crown and root domain (Appendix Fig. 2D). Comparable with the situation in the molar, there was a higher expression of GoPro49 on the lingual (root analogue) side of the incisor, compared with the labial (crown analogue) side (Appendix Fig. 2C).

Strong Bsp1 expression was observed in the pre-odontoblasts of the forming root and in the cementoblasts and osteoblasts lining the cementum and alveolar bone, respectively (Appendix Figs. 2E, 2F). Again, Bsp1 was complementary to GoPro49 expression. ActivinβA expression at 7 dpn (Appendix Figs. 2G, 2H) was observed in the dental follicle and in the mesenchyme near bone, similar to previous stages, and additional expression was found in the ameloblasts.

At 12 dpn, the molar root had elongated, and the PDL was developing. The expression of GoPro49 seemed reduced compared with that in earlier stages. However, it was still prominent in the dental follicle, both in the crown area and at the root tip (Figs. 2H, 2K ). There was also some expression in the PDL along the root. ActivinβA (Figs. 2I, 2L ) was observed in the crown pulp, and it was absent from the PDL around the roots. Relatively strong expression was seen in the ameloblasts (Fig. 2L ). Bsp1 expression was present in the cementoblasts and osteoblasts, and in the pre-odontoblasts of the root (Appendix Fig. 1F, 1G).

Subcellular Localization

We have previously shown that the GoPro49 protein co-localizes with β-COP in a chondrosarcoma cell line (Takatalo et al., 2008). We analyzed the intracellular localization of GoPro49 protein in dental follicle cells isolated from E18.5-old embryos and compared GoPro49 location with that of β-COP and GM130 (Golgi matrix 130 kDa) using indirect immunofluorescence. A clear co-localization with β-COP (Fig. 3C), but not with GM130 (Fig. 3F ), was detected as also previously in the chondrosarcoma cell line (Takatalo et al., 2008). The staining intensity of GoPro49 was highly dependent on the cell passage and decreased rapidly, with highest intensity in primary culture. This decrease suggests a loss of dental follicle identity and dedifferentiation during extended culture.

DISCUSSION

We show that the Golgi protein GoPro49 is expressed specifically in the dental follicle mesenchyme from the bud stage until 12 dpn in molars and incisors, thus encompassing the main developmental stages of the dental follicle. GoPro49 expression was largely restricted to the dental follicle cells and was not detected in the dental papilla. Interestingly, while GoPro49 was expressed intensely in most of the condensed mesenchyme surrounding the bud-stage tooth germ, it was absent in the presumptive dental papilla mesenchyme. The condensed dental mesenchyme is a morphologically homogenous cell population at this stage, showing the usefulness of GoPro49 as a marker for early stages of tooth development distinguishing between dental follicle and papilla cells.

We used Bsp1 as a marker for the hard tissues bordering the dental follicle during later stages of morphogenesis. Bsp1 is known to be restricted to the cementoblasts lining the root surface and osteoblasts lining the bone surface (Yamashiro et al., 2003). We showed that Bsp1 expression was complementary to GoPro49 in the dental follicle, indicating that the follicle cells lost GoPro49 expression during their terminal differentiation. We also compared GoPro49 expression with that of ActivinβA, which is expressed in the dental follicle during early tooth development (Wang et al., 2004b). Although ActivinβA was co-expressed with GoPro49 in the dental follicle during embryonic and post-natal development, ActivinβA was also expressed in the dental papilla, in osteogenic mesenchyme, and in ameloblasts. ActivinβA was also down-regulated from the root follicle earlier than GoPro49. Hence, GoPro49 was a more specific marker for the dental follicle during both pre- and post-natal stages of development in molars and incisors.

Our immunofluorescent studies show that the GoPro49 protein has a clear co-localization with the β-COP protein involved in the retrograde transport between ER and the Golgi complex. The COPI staining in these cells showed a highly disperse pattern and a high number of tubular structures. The COPI co-localization suggests that the GoPro49 protein may be involved in membrane traffic, with a possible role in the retention of proteins in the ERGIC/ER. The down-regulation of GoPro49 upon follicle cell differentiation into cementoblasts and osteoblasts, and in more mature post-natal tissues, suggests a function for the GoPro49 gene in cell populations before terminal differentiation. This is in line with our previous observation that GoPro49 expression in cartilage was strongest in columnar, proliferating chondrocytes that are still differentiating (Takatalo et al., 2008). Combined with a possible role in membrane traffic, the expression in the dental follicle suggests a role in the secretion or retention of specific PDL matrix molecules.

Both the crown and root follicle have been shown to be important for tooth eruption in dogs (Marks and Cahill, 1987; Marks, 1995). Interestingly, the follicle has different functions in the root and crown areas. While the crown follicle regulates bone resorption, allowing for the eruption of the tooth through the bone, the root follicle stimulates bone formation at the tip of the root, which is necessary for the eruptive movement of the tooth toward the oral cavity (Marks and Cahill, 1987). The intense expression of GoPro49 in the root follicle, but not in the crown follicle, could indicate a specific function for GoPro49 in the root follicle function that could be related to tooth eruption, perhaps to the regulation of bone apposition at the base of the root.

In conclusion, GoPro49 is a novel, specific marker for the dental follicle and early periodontal ligament. To date, it is the only reported dental follicle gene which is not expressed in other mesenchymal cell populations in the developing teeth. The COPI co-localization, together with the expression in secretory cell types, would suggest the protein to have a role in membrane trafficking and perhaps secretion of specific matrix molecules. The function of GoPro49 in the follicle remains unknown at present, but its intense expression throughout the follicle during early morphogenesis and the continuing preferential expression in the root follicle at the post-natal stages may provide clues to the in vivo function of GoPro49.

Figure 1.

GoPro49 expression during incisor morphogenesis. At the E13 late bud stage, GoPro49 is expressed in the peripheral cells of the dental mesenchyme, but not in the forming dental papilla (A). At E14 and E16, the expression continues in the dental follicle (C,E). ActivinβA is expressed in the dental follicle and is also strong in the dental papilla at E13 and E14 (B,D). At E16, ActivinβA is down-regulated in dental papilla and is expressed around the bone (F). In the 4-dpn incisor and molars, strong GoPro49 expression is present in the dental follicle, and weak expression in the bone (G–I). Higher magnifications of the boxed areas in H and I show the restricted expression in incisor dental follicle in the labial and lingual sides, as well as in the molar follicle. Bsp1 is expressed in osteoblasts and odontoblasts (J–L) and in the cell layer close to ameloblasts, possibly representing cementoblast precursor cells (arrow, L), and is complementary to the GoPro49 expression pattern in the dental follicle (G–l). Black indicates intense expression. E, tooth epithelium; Ab, ameloblast; DP, dental papilla; DF, dental follicle; I, incisor; Lab, labial; Lin, lingual; M, molar; Ob, odontoblast; Bone. Scale bars: A–F, 200 μm; G and J, 500 μm; H, I, K, and L, 100 μm.

Figure 2.

GoPro49 expression in the first molar during root development. Hematoxylin/eosin staining of frontal sections of the 4-dpn first molars and incisors (initiation of root development) (A), with higher magnification of the root follicle in the boxed area (D), and during advanced root development in the 12-dpn molar (G), with a detailed view of the dental follicle bordering the crown-root junction (J). At 4 dpn, strong expression of GoPro49 is seen in the dental follicle. Expression in the stellate reticulum of the first molar (B). A large cluster of GoPro49-expressing cells is present in the dental follicle mesenchyme at the site of initial root formation (E). ActivinβA is expressed in the dental follicle and osteogenic mesenchyme bordering the bone (C). ActivinβA co-expressed with GoPro49 in the forming root follicle (F). At 12 dpn, the expression of GoPro49 seems reduced compared with that in earlier stages, but is still present in the dental follicle and most prominent in the root follicle (H,K). ActivinβA is expressed in some scattered cells in the dental follicle, in crown pulp, and in ameloblasts (I,L). The junction between crown and root domain is marked with a yellow dashed line in G, cementoblasts with a white arrowhead, and odontoblasts with a white arrow (K). Ab, ameloblast; cr, crown; r, root; D, dentin; DP, dental papilla; DF, dental follicle; E, enamel; I, incisor; PDL, periodontal ligament; M, first molar; Lab, labial; Lin, lingual; Ob, odontoblast; Sr, stellate reticulum. Scale bars: A–C and G–I, 500 μm; D–F and J–L, 100 μm.

Figure 3.

Localization of GoPro49, β-COP, and GM130 in cultured dental follicle cells by indirect immunofluorescence. The dental follicle cells were fixed after 5 days in primary culture (A–C, β-COP) or after two passages (D–F, GM130). The staining intensity for GoPro49 was highly dependent on the passage (A,D) and decreased rapidly during culture. GoPro49 shows clear co-localization with β-COP, a vesicle coat protein, while little or no overlap is seen with GM130, the marker for the stacked part of the Golgi complex. Scale bar: 25 μm.

Footnotes

Notes

+

authors contributing equally to this work

A supplemental appendix to this article is published electronically only at .

Acknowledgements

We thank Riikka Santalahti, Raija Savolainen, and Merja Mäkinen for excellent technical assistance. This work was supported by the Magnus Ehrnrooth Foundation, the Finnish Society of Science and Letters, the Viikki Graduate School in Molecular Biosciences, and The Academy of Finland.

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