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Class III β-tubulin expression during hard tissue formation in developing mouse teeth
pediatric dental journal xxx (xxxx) xxx
Available online at www.sciencedirect.com
Pediatric Dental Journal journal homepage: www.elsevier.com/locate/pdj
Research Paper
Class III b-tubulin expression during hard tissue formation in developing mouse teeth Shohei Oshima*, Yasutaka Yawaka Department of Dentistry for Children and Disabled Person, Graduate School of Dental Medicine, Hokkaido University, Sapporo 060-8586, Japan
article info
abstract
Article history:
Microtubules of the eukaryotic cytoskeleton are involved in critical cellular processes,
Received 5 September 2019
including mitosis, cell motility, and intracellular transport. They are assembled from
Received in revised form
tubulin heterodimers that contain different a-tubulin (TUBA) and b-tubulin (TUBB) iso-
25 October 2019
types. Among these isotypes, Class III b-tubulin (TUBB3) is highly expressed in the brain
Accepted 15 December 2019
and testes, and aberrant expression has been reported in a multitude of human cancer
Available online xxx
types. In this study, we investigated TUBB3 expression in the developing mouse teeth using immunohistochemistry. Similar to previous research, immunohistochemistry with TUBB3-
Keywords:
specific antibodies showed immunoreactivity in the nerve fiber around the tooth germ.
Tubb3 protein
However, TUBB3 immunoreactivity was minimal or not seen at all in the tooth germ at
Mouse
early cytological and histological differentiation stages of developing teeth. However, the
Tooth calcification
immunoreactivity was intense in odontoblasts during dentin formation and in most am-
Microtubules
eloblasts at the secretory stage. Intense immunoreactivity was observed only in some ameloblasts at the maturation stage. Similar to the immunoreactivity in ameloblasts and odontoblasts, the immunoreactivity was intense in cementoblasts during cementum formation. The spaciotemporal profiles of TUBB3 expression suggested that TUBB3 may be correlated to some cellular process at the mineralization stage of tooth development. © 2020 Japanese Society of Pediatric Dentistry. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
Microtubules, which are the key elements of the eukaryotic cytoskeleton, are involved in several critical cellular processes, including mitosis, cell motility, and intracellular transport. They are assembled from tubulin heterodimers containing different a-tubulin (TUBA) and b-tubulin (TUBB) isotypes. Of these isotypes, several are encoded by separate genes [1]. Tubulin isotypes reportedly form individual
microtubules with specialized functions in cells and tissues [2], but this viewpoint has not yet been confirmed because different isotypes can freely intermingle into mosaic microtubules [3]. The impact of specific tubulin isotypes on microtubule properties and functions remains poorly researched and reported. However, specialized microtubules, such as axonemes [4], neuronal microtubules [5], and platelet marginal band microtubules [6,7], are known to be selectively enriched in specific b-tubulin isotypes. This process might be responsible, in part, for microtubule functions.
* Corresponding author. E-mail address: [email protected] (S. Oshima). https://doi.org/10.1016/j.pdj.2019.12.002 0917-2394/© 2020 Japanese Society of Pediatric Dentistry. Published by Elsevier Ltd. All rights reserved. Please cite this article as: Oshima S, Yawaka Y, Class III b-tubulin expression during hard tissue formation in developing mouse teeth, Pediatric Dental Journal, https://doi.org/10.1016/j.pdj.2019.12.002
2
pediatric dental journal xxx (xxxx) xxx
Class III b-tubulin (TUBB3) is highly expressed in the brain and testes. In the brain, it exhibits a distinct neuronal specificity [8], and TUBB3 mutations have been implicated in various neurological disorders where TUBB3 is required for proper axon guidance [9]. In the testes, TUBB3 has been localized in Sertoli cells, but its role still remains largely unclear [10]. Moreover, TUBB3 expression is aberrant in several human cancers, and high levels of it have been associated with poor clinical outcomes and poor efficacy when using microtubule-targeting anticancer drugs [11]. In a preliminary study, we found that TUBB3 was expressed in developing teeth, but we could find no reports supporting this association. In the current study, we therefore aimed to investigate TUBB3 expression further in the developing mouse tooth, focusing on its spaciotemporal expression.
2.
Material and methods
All experimental procedures were approved by the Committee on the Ethics of Animal Experiments of Hokkaido University. We used C57BL/6 N mice at embryonic d (E) 13 and 18 and postnatal d (P) 3, 7, 10, 14, 21, and 28. The day following overnight mating was counted as E0 and the date of birth was counted as P1. Mice were purchased from Sankyo Labo Service, Tokyo, Japan. Immunohistochemical procedures were carried out as follows. Mice were killed by deep anesthesia with intraperitoneal pentobarbital (100 mg/kg of body weight) at E13, E15, and E18. Their heads were then freshly dissected and fixed by overnight immersion in 4% paraformaldehyde in 0.1 M phosphate buffer at 4 C. The heads were then processed for paraffin embedding. Postnatal mice underwent transcardial perfusion with the same fixative under deep pentobarbital anesthesia and were fixed by overnight immersion. The heads were dissected and decalcified at 4 C in 10% ethylenediaminetetraacetic acid (pH 7.4) for 2e4 wks. The heads were then processed for paraffin embedding. Paraffin sections were cut through tooth germs in the mesiodistal plane on a sliding microtome and mounted on silane-coated glass slides. Sections were deparaffinized and incubated for 20 min at room temperature (22 C) with 10% normal goat serum. Sections were incubated overnight at room temperature with TUBB3 antibodies (ab18207, Abcam, Cambridge, UK) at a concentration of 0.05e0.1 mg/mL and then incubated for 30 min with biotinylated secondary antibodies to rabbit IgG (host species, goat) (Histofine Simple Stain MAX-PO(R), Nichirei, Tokyo, Japan). Immunoreaction was visualized with 3,30 -diaminobenzidine (SK4100, Vector Laboratories, Burlingame, CA, USA). Negative controls were prepared from other sections by omitting the primary antibody, and no positive immunostaining was detected. Adjacent sections were counterstained with haematoxylin and eosin, as necessary. Sections were photographed using a light microscope (ECLIPSE 80i, Nikon, Tokyo, Japan).
3.
Results
3.1.
Bud stage of mouse molar
First, we examined the molar tooth germ at the bud stage, which was represented by epithelial incursion into the ectomesenchyme (Fig. 1A, D). Intense immunostaining was observed in the nerve fiber around the tooth germ and the maxillary nerve, indicating that the antibody could target TUBB3 specifically in this region. However, no TUBB3 immunostaining was observed in the dental epithelium or condensed mesenchyme at the bud stage.
3.2.
Cap stage of mouse molar
The dental organ is constituted by the enamel organ, the dental papilla, and the dental follicle. Immunostaining for TUBB3 was observed slightly in the enamel knot of the enamel organ, and it was dotted in the nerve fibers around the dental follicle; however, no staining was seen in the dental mesenchyme (Fig. 1B, E).
3.3.
Bell stage of mouse molar
The enamel organ forms the inner and outer enamel epithelia, the stellate intermedium, and the stellate reticulum. At the bell stage, immunostaining for TUBB3 was observed moderately in the inner enamel epithelium and the dental papilla adjunct at the cusp portion. No or slight staining was seen in the outer enamel epithelium, the stellate intermedium, the stellate reticulum, and the other dental papilla. Intense immunostaining for TUBB3 was observed in the nerve fiber around the dental follicle (Fig. 1C, F).
3.4.
Hard tissue formation stage
The inner enamel epithelium differentiates to ameloblasts, whereas the dental papilla cells adjcent to the inner enamel epithelium differentiate to odontoblasts. At P3, the initial phase of immature enamel and dentin secretion, intense TUBB3 immunostaining spread over all ameloblasts and odontoblasts. In the ameloblasts, TUBB3 was observed in the whole cytoplasm of the cell body, especially close to the basal lamina. In the odontoblasts, it was prominent in the supranuclear compartment near to the predentin layer. No or slight staining was seen in the outer enamel epithelium, the stellate intermedium, the stellate reticulum, and the dental papilla (Fig. 2). At P7, the immature enamel and dentin formation proceeded, with odontoblasts exhibiting morphological change into bowling pin shapes, in which the perikarya were located from the bottom to the top of the supranuclear compartment. In the odontoblasts, TUBB3 was not only prominent in the supranuclear compartment near the predentin layer but also in the predentin layer. The ameloblasts showed intense immunostaining for TUBB3 and sporadic TUBB3-positive puncta were observed in the pulp, probably representing the nerve fiber (Fig. 3A, C). At P14, before tooth
Please cite this article as: Oshima S, Yawaka Y, Class III b-tubulin expression during hard tissue formation in developing mouse teeth, Pediatric Dental Journal, https://doi.org/10.1016/j.pdj.2019.12.002
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Fig. 1 e Immunohistochemistry for TUBB3 in the developing molar tooth germ from the bud stage to the bell stage. AeC are counterstained with haematoxylin and eosin (HE). A and D (bud stage), B and E (cap stage) and C and F (bell stage) are adjacent sections respectively. Arrowheads indicate peripheral nerve fibers. TB, tooth bud; CM, condensed mesenchymal cell; V2, maxillary nerve; EK, enamel knot; DP, dental papilla; IEE, inner enamel epithelium. Scale bars, 200 mm.
Fig. 2 e Immunohistochemistry for TUBB3 in the developing molar tooth germ at the initiation of hard tissue formation. A and B are counterstained with HE. A and C are adjacent sections. B and D are magnified of A and C, respectively. V2, maxillary nerve; Od, odontoblasts; Am, ameloblast. Scale bars, A and C, 500 mm, B and D, 100 mm.
Please cite this article as: Oshima S, Yawaka Y, Class III b-tubulin expression during hard tissue formation in developing mouse teeth, Pediatric Dental Journal, https://doi.org/10.1016/j.pdj.2019.12.002
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Fig. 3 e Immunohistochemistry for TUBB3 in the developing molar tooth germ at the hard tissue formation. A and B are counterstained with HE. A and C (during dentinogenesis) and B and D (completion of dentinogenesis) are adjacent sections respectively. B and D, Dot or beads like immunoreactivity of TUBB3 in the pulp indicates nerve fibers. P, dental pulp; Od, odontoblasts; Am, ameloblast; En, enamel; De, dentin. Scale bars, 100 mm.
eruption but when crown dentinogenesis has finished, the strength of TUBB3 immunostaining decreased substantially in the supranuclear compartment of odontoblasts in the tooth crown. By contrast, the number of TUBB3 immunopositive nerve fibers increased around the odontoblasts (Fig. 3B, D). TUBB3 was also observed in the tooth crown and the area of tooth root formation. At P10, radicular dentin formation was initiated, and intense immunostaining for TUBB3 was observed in the odontoblasts of the radicular dentin and Hertwig’s epithelial root sheath (Fig. 4A, C). During tooth root development, intense immunostaining for TUBB3 was observed in the differentiated odontoblasts of the rim of the root sheath near Hertwig’s epithelial root sheath. However, the strength of TUBB3 immunostaining was substantially less in odontoblasts not in the root sheath, where root formation was advanced (Fig. 4B, D).
During tooth root development, intense immunostaining for TUBB3 was observed in the cementoblasts on the root surface. The cementum consists of acellular and cellular parts: the acellular part covers the cervical half of the root and provides attachment to the tooth; and the cellular part covers an apical half, plays an adaptive role in response to tooth wear and movement, and is associated with periodontal tissue repair. Because it was difficult to identify cementoblasts by shape alone, we considered any relatively large cuboidal cells lining the cervical or apical root surface as the acellular or cellular cementum, respectively. During tooth root development, intense immunostaining for TUBB3 was observed in the cementoblasts on the root surface (Fig. 5A and B). After tooth eruption, the number of TUBB3 immunopositive cementoblasts was substantially decreased in the acellular cementum of the cervical half of the root (Fig. 5C and D). By
Please cite this article as: Oshima S, Yawaka Y, Class III b-tubulin expression during hard tissue formation in developing mouse teeth, Pediatric Dental Journal, https://doi.org/10.1016/j.pdj.2019.12.002
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Fig. 4 e Immunohistochemistry for TUBB3 in the developing root dentin. A and B are counterstained with HE. A and C (Hertwig’s epithelial root sheath, HERS) and B and D (radicular dentinogenesis) are adjacent sections respectively. Arrowheads indicate HERS. P, dental pulp; Od, odontoblasts; Am, ameloblast; Ce, cementoblasts; En, enamel; De, dentin. Scale bars, 100 mm.
contrast, cementoblasts continued to show TUBB3 expression in the cellular cementum (Fig. 5C, E), while cementocytes entrapped in the matrix of the cellular cementum showed no or only slightly positive immunostaining. Through all stages, osteoblasts lining the surface of the jawbone matrix and entrapped in the bone matrix were not immunopositive for TUBB3 in the bone around the tooth.
3.5.
The ameloblasts of mouse incisors
We investigated TUBB3 expression in the ameloblasts of the mouse incisors because the spaciotemporal changes were clear. Similar to the molar teeth, ameloblasts stained positive for TUBB3 at the secretary stage (Fig. 6C). TUBB3 was also observed in the whole cytoplasm of the cell body during the transition and secretary stages (Fig. 6D), while the TUBB3 expression pattern varied during the maturation stage (Fig. 6EeL). One TUBB3 immunostaining pattern was observed in the whole cytoplasm of the cell body and was similar to that
observed in the secretary and transition stage ameloblasts. However, some ameloblasts exhibited TUBB3 immunostaining in the distal portion adjacent to the enamel (Fig. 6G), some did so in both the distal and medial portions (Fig. 6I, K), and some did so in the perikarya. As with this variation in expression patterns, the intensity of TUBB3 immunostaining also varied from intense to slight. Immunostaining for TUBB3 was observed in ameloblasts at the protective stage (Fig. 6M and N); however, not all ameloblasts showed TUBB3 expression, with some showing no or slight positivity.
4.
Discussion
In the present study, we found that the neurospecific betatubulin subtype, TUBB3, was expressed in the developing teeth of mice, especially in the cells involved with hard tissue formation. As previously reported, TUBB3 was expressed in the nerve fibers of teeth, but we also observed high expression
Please cite this article as: Oshima S, Yawaka Y, Class III b-tubulin expression during hard tissue formation in developing mouse teeth, Pediatric Dental Journal, https://doi.org/10.1016/j.pdj.2019.12.002
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Fig. 5 e Immunohistochemistry for TUBB3 in the developing root dentin. Sections are counterstained with HE. B is magnified of A (P14, before eruption, during radicular dentinogenesis). D and E are magnified of C (4 wks, after eruption, completion of radicular dentinogenesis). Arrowheads indicate HERS. P, dental pulp; Od, odontoblasts; Am, ameloblast; Ce, cementoblasts; En, enamel; De, dentin; Al, alveolar bone; Pl, periodontal ligament. Scale bars, 100 mm.
levels in ameloblasts, odontoblasts, and cementoblasts during hard tissue formation. The coronal odontoblasts expressed TUBB3 abundantly during primary dentin formation, but scarcely thereafter. In the root dentin, similar to the crown, odontoblasts expressed abundant TUBB3 near Hertwig’s epithelial root sheath, where the root was forming, but scarcely expressed it after primary dentin formation had completed. Enamel formation is a two-step process. Although enamel develops its full thickness during the secretory stage, only 30% of enamel is mineralized at this stage. Subsequently, enamel mineralization is increased to beyond 96% by the removal of water and organic materials and the addition of minerals during the maturation stage [12]. In the secretory stage, all ameloblasts expressed abundant TUBB3 in the present study, but this varied in the maturation stage. Although cyclical variations occur between ruffle-ended and smooth-ended ameloblasts in the maturation stage, the expression patterns of TUBB3 were not periodic and seemed unrelated to these cyclical variations.
The cementoblasts showed similar expression patterns to those of odontoblasts. During tooth root formation, the cementoblasts of the acellular part covering the cervical half of the root expressed abundant TUBB3, but the number of cementoblasts in this region decreased when root formation completed. Cementoblasts in the cellular part, which covers the apical half and plays an adaptive role in response to tooth wear and movement, continued to express TUBB3 abundantly. No reports have described either the association between tooth development and TUBB3 or the association of tooth abnormality with TUBB3 mutation. The functions of TUBB3 are not fully understood. In neurons, TUBB3 mutations may cause congenital fibrosis of the extraocular muscles type 3. It may also be involved in neurogenesis and in axon guidance and maintenance [9]. Furthermore, TUBB3 is expressed in Sertoli cells and is regulated by androgen receptors [13], but its function in these cells is still unclear. TUBB3 expression has been reported in other normal extraneuronal tissues, with low levels in rodent lungs [14] and in the columnar epithelium of the human colon [15]. High
Please cite this article as: Oshima S, Yawaka Y, Class III b-tubulin expression during hard tissue formation in developing mouse teeth, Pediatric Dental Journal, https://doi.org/10.1016/j.pdj.2019.12.002
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Fig. 6 e Immunohistochemistry for TUBB3 in the lower inscisor. A and B are combined photos from two consecutive photos. A is counterstained with HE. A and B are adjacent sections. C (secretory stage), D (transitional stage), E, F, G, H, I, J, K, L (maturation stage), M and N (protection stage) are magnified ameloblasts images of c’, d’, e’, f ’, g’, h’, i’, j’, k’, l’, m’ and n’ in B respectively. P, dental pulp; Od, odontoblasts; Am, ameloblast; Ce, cementoblasts; En, Enamel; De, dentin Scale bars in A and B, 1000 mm. Scale bars in CeN, 50 mm. levels of TUBB3 in normal extraneuronal tissues, such as teeth and Sertoli cells, have not yet been reported. As stated, increased TUBB3 expression is associated with poor clinical outcomes in various cancers, such as non-small cell lung cancer [16,17], ovarian cancer [18], urothelial carcinoma of the bladder [19], and prostate cancer [20]. TUBB3 overexpression has also been linked to a reduced response to taxane-based microtubule-targeting anticancer drugs [21,22]. Using a cell-free assay, Lu and Luduena have shown that removing TUBB3 by immunoprecipitation with specific antibodies can enhance paclitaxel-induced microtubule polymerization [23]. The physiologic role of TUBB3 seems to be associated with microtubule dynamic instability [24], with microtubules of the cell cytoskeleton playing an important role in cancer cell migration [25]. Kanojia et al. for example, reported that TUBB3 was associated with brain metastases in patients with breast cancer [26]. In the developing tooth, TUBB3 is distinctly expressed in cells responsible for hard tissue formation, but only when hard tissue is forming. TUBB3 appears to be associated with ameloblast, odontoblast, and cementoblast function during this stage, even though osteoblasts in the alveolar bone
showed no TUBB3 expression. This suggests that TUBB3 may be associated with hard tissue formation in the toot, but not in the bone. Both ameloblasts and odontoblasts change their shape during differentiation and hard tissue formation, with the microtubules that assemble with aand beIIIeheterodimers being more dynamic [24]. Thus, TUBB3 may be required to change ameloblast and odontoblast cell shapes when hard tissue is forming. Unfortunately, our study was not able to confirm the roles of TUBB3 in developing teeth due to technical limitations. Although we did demonstrate a previously unknown function of TUBB3, further research is required to clarify the role of TUBB3 in normal tooth development.
5.
Conclusions
In developing teeth, TUBB3 is specifically expressed in ameloblasts, odontoblasts, and cementoblasts during hard tissue formation. Their spatiotemporal profiles suggest that TUBB3 may be correlated to some cellular process at the mineralization stage of tooth development.
Please cite this article as: Oshima S, Yawaka Y, Class III b-tubulin expression during hard tissue formation in developing mouse teeth, Pediatric Dental Journal, https://doi.org/10.1016/j.pdj.2019.12.002
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Declaration of Competing Interest The authors declare no conflict of interest.
Acknowledgments This work was supported by JSPS KAKENHI Grant Number 25463162. The authors would like to thank Enago (www.enago. jp) for the English language review.
references
[1] Gadadhar S, Bodakuntla S, Natarajan K, Janke C. The tubulin code at a glance. J Cell Sci 2017;130:1347e53. [2] Lewis SA, Lee MG, Cowan NJ. Five mouse tubulin isotypes and their regulated expression during development. J Cell Biol 1985;101:852e61. [3] Lewis SA, Gu W, Cowan NJ. Free intermingling of mammalian beta-tubulin isotypes among functionally distinct microtubules. Cell 1987;49:539e48. [4] Raff EC, Hoyle HD, Popodi EM, Turner FR. Axoneme betatubulin sequence determines attachment of outer dynein arms. Curr Biol 2008;18:911e4. [5] Joshi HC, Cleveland DW. Differential utilization of betatubulin isotypes in differentiating neurites. J Cell Biol 1989;109:663e73. [6] Lecine P, Italiano Jr JE, Kim SW, Villeval JL, Shivdasani RA. Hematopoietic-specific beta 1 tubulin participates in a pathway of platelet biogenesis dependent on the transcription factor NF-E2. Blood 2000;96:1366e73. [7] Schwer HD, Lecine P, Tiwari S, Italiano Jr JE, Hartwig JH, Shivdasani RA. A lineage-restricted and divergent betatubulin isoform is essential for the biogenesis, structure and function of blood platelets. Curr Biol 2001;11:579e86. € rk SJ. Class III beta-tubulin in [8] Katsetos CD, Herman MM, Mo human development and cancer. Cell Motil Cytoskelet 2003;55:77e96. [9] Tischfield MA, Baris HN, Wu C, Rudolph G, Van Maldergem L, He W, et al. Human TUBB3 mutations perturb microtubule dynamics, kinesin interactions, and axon guidance. Cell 2010;140:74e87. [10] Lewis SA, Cowan NJ. Complex regulation and functional versatility of mammalian alpha- and beta-tubulin isotypes during the differentiation of testis and muscle cells. J Cell Biol 1988;106:2023e33. [11] Mariani M, Karki R, Spennato M, Pandya D, He S, Andreoli M, et al. Class III b-tubulin in normal and cancer tissues. Gene 2015;563:109e14. [12] Nanci A. Ten Cate’s oral histology : development, structure, and function. St louis, Missouri: Mosby; 2008.
[13] De Gendt K, Denolet E, Willems A, Daniels VW, Clinckemalie L, Denayer S, et al. Expression of Tubb3, a betatubulin isotype, is regulated by androgens in mouse and rat Sertoli cells. Biol Reprod 2011;85:934e45. ~ a RF. Multiple forms of tubulin: different gene [14] Luduen products and covalent modifications. Int Rev Cytol 1998;178:207e75. ~ a RF. [15] Roach MC, Boucher VL, Walss C, Ravdin PM, Luduen Preparation of a monoclonal antibody specific for the class I isotype of beta-tubulin: the beta isotypes of tubulin differ in their cellular distributions within human tissues. Cell Motil Cytoskelet 1998;39:273e85. [16] Koh Y, Jang B, Han SW, Kim TM, Oh DY, Lee SH, et al. Expression of class III beta-tubulin correlates with unfavorable survival outcome in patients with resected nonsmall cell lung cancer. J Thorac Oncol 2010;5:320e5. [17] Ohashi T, Yoshimasu T, Oura S, Kokawa Y, Kawago M, Hirai Y, et al. Class III beta-tubulin expression in non-small cell lung cancer: a predictive factor for paclitaxel response. Anticancer Res 2015;35:2669e74. C, Davidson B, [18] Hetland TE, Hellesylt E, Flørenes VA, Trope Kærn J. Class III b-tubulin expression in advanced-stage serous ovarian carcinoma effusions is associated with poor survival and primary chemoresistance. Hum Pathol 2011;42:1019e26. [19] Choi JW, Kim Y, Lee JH, Kim YS. Expression of b-tubulin isotypes in urothelial carcinoma of the bladder. World J Urol 2014;32:347e52. [20] Tsourlakis MC, Weigand P, Grupp K, Kluth M, Steurer S, Schlomm T, et al. bIII-tubulin overexpression is an independent predictor of prostate cancer progression tightly linked to ERG fusion status and PTEN deletion. Am J Pathol 2014;184:609e17. [21] Zhang HL, Ruan L, Zheng LM, Whyte D, Tzeng CM, Zhou XW. Association between class III b-tubulin expression and response to paclitaxel/vinorebine-based chemotherapy for non-small cell lung cancer: a meta-analysis. Lung Cancer 2012;77:9e15. [22] Zhou J, Li J, Wang Z, Yin C, Zhang W. Metadherin is a novel prognostic marker for bladder cancer progression and overall patient survival. Asia Pac J Clin Oncol 2012;8:e42e8. [23] Lu Q, Luduena RF. Removal of beta III isotype enhances taxol induced microtubule assembly. Cell Struct Funct 1993;18:173e82. ~ a RF, Wilson L. [24] Panda D, Miller HP, Banerjee A, Luduen Microtubule dynamics in vitro are regulated by the tubulin isotype composition. Proc Natl Acad Sci U S A 1994;91:11358e62. [25] Fife CM, McCarroll JA, Kavallaris M. Movers and shakers: cell cytoskeleton in cancer metastasis. Br J Pharmacol 2014;171:5507e23. [26] Kanojia D, Morshed RA, Zhang L, Miska JM, Qiao J, Kim JW, et al. bIII-tubulin regulates breast cancer metastases to the brain. Mol Cancer Ther 2015;14:1152e61.
Please cite this article as: Oshima S, Yawaka Y, Class III b-tubulin expression during hard tissue formation in developing mouse teeth, Pediatric Dental Journal, https://doi.org/10.1016/j.pdj.2019.12.002
Available online at www.sciencedirect.com
Pediatric Dental Journal journal homepage: www.elsevier.com/locate/pdj
Research Paper
Class III b-tubulin expression during hard tissue formation in developing mouse teeth Shohei Oshima*, Yasutaka Yawaka Department of Dentistry for Children and Disabled Person, Graduate School of Dental Medicine, Hokkaido University, Sapporo 060-8586, Japan
article info
abstract
Article history:
Microtubules of the eukaryotic cytoskeleton are involved in critical cellular processes,
Received 5 September 2019
including mitosis, cell motility, and intracellular transport. They are assembled from
Received in revised form
tubulin heterodimers that contain different a-tubulin (TUBA) and b-tubulin (TUBB) iso-
25 October 2019
types. Among these isotypes, Class III b-tubulin (TUBB3) is highly expressed in the brain
Accepted 15 December 2019
and testes, and aberrant expression has been reported in a multitude of human cancer
Available online xxx
types. In this study, we investigated TUBB3 expression in the developing mouse teeth using immunohistochemistry. Similar to previous research, immunohistochemistry with TUBB3-
Keywords:
specific antibodies showed immunoreactivity in the nerve fiber around the tooth germ.
Tubb3 protein
However, TUBB3 immunoreactivity was minimal or not seen at all in the tooth germ at
Mouse
early cytological and histological differentiation stages of developing teeth. However, the
Tooth calcification
immunoreactivity was intense in odontoblasts during dentin formation and in most am-
Microtubules
eloblasts at the secretory stage. Intense immunoreactivity was observed only in some ameloblasts at the maturation stage. Similar to the immunoreactivity in ameloblasts and odontoblasts, the immunoreactivity was intense in cementoblasts during cementum formation. The spaciotemporal profiles of TUBB3 expression suggested that TUBB3 may be correlated to some cellular process at the mineralization stage of tooth development. © 2020 Japanese Society of Pediatric Dentistry. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
Microtubules, which are the key elements of the eukaryotic cytoskeleton, are involved in several critical cellular processes, including mitosis, cell motility, and intracellular transport. They are assembled from tubulin heterodimers containing different a-tubulin (TUBA) and b-tubulin (TUBB) isotypes. Of these isotypes, several are encoded by separate genes [1]. Tubulin isotypes reportedly form individual
microtubules with specialized functions in cells and tissues [2], but this viewpoint has not yet been confirmed because different isotypes can freely intermingle into mosaic microtubules [3]. The impact of specific tubulin isotypes on microtubule properties and functions remains poorly researched and reported. However, specialized microtubules, such as axonemes [4], neuronal microtubules [5], and platelet marginal band microtubules [6,7], are known to be selectively enriched in specific b-tubulin isotypes. This process might be responsible, in part, for microtubule functions.
* Corresponding author. E-mail address: [email protected] (S. Oshima). https://doi.org/10.1016/j.pdj.2019.12.002 0917-2394/© 2020 Japanese Society of Pediatric Dentistry. Published by Elsevier Ltd. All rights reserved. Please cite this article as: Oshima S, Yawaka Y, Class III b-tubulin expression during hard tissue formation in developing mouse teeth, Pediatric Dental Journal, https://doi.org/10.1016/j.pdj.2019.12.002
2
pediatric dental journal xxx (xxxx) xxx
Class III b-tubulin (TUBB3) is highly expressed in the brain and testes. In the brain, it exhibits a distinct neuronal specificity [8], and TUBB3 mutations have been implicated in various neurological disorders where TUBB3 is required for proper axon guidance [9]. In the testes, TUBB3 has been localized in Sertoli cells, but its role still remains largely unclear [10]. Moreover, TUBB3 expression is aberrant in several human cancers, and high levels of it have been associated with poor clinical outcomes and poor efficacy when using microtubule-targeting anticancer drugs [11]. In a preliminary study, we found that TUBB3 was expressed in developing teeth, but we could find no reports supporting this association. In the current study, we therefore aimed to investigate TUBB3 expression further in the developing mouse tooth, focusing on its spaciotemporal expression.
2.
Material and methods
All experimental procedures were approved by the Committee on the Ethics of Animal Experiments of Hokkaido University. We used C57BL/6 N mice at embryonic d (E) 13 and 18 and postnatal d (P) 3, 7, 10, 14, 21, and 28. The day following overnight mating was counted as E0 and the date of birth was counted as P1. Mice were purchased from Sankyo Labo Service, Tokyo, Japan. Immunohistochemical procedures were carried out as follows. Mice were killed by deep anesthesia with intraperitoneal pentobarbital (100 mg/kg of body weight) at E13, E15, and E18. Their heads were then freshly dissected and fixed by overnight immersion in 4% paraformaldehyde in 0.1 M phosphate buffer at 4 C. The heads were then processed for paraffin embedding. Postnatal mice underwent transcardial perfusion with the same fixative under deep pentobarbital anesthesia and were fixed by overnight immersion. The heads were dissected and decalcified at 4 C in 10% ethylenediaminetetraacetic acid (pH 7.4) for 2e4 wks. The heads were then processed for paraffin embedding. Paraffin sections were cut through tooth germs in the mesiodistal plane on a sliding microtome and mounted on silane-coated glass slides. Sections were deparaffinized and incubated for 20 min at room temperature (22 C) with 10% normal goat serum. Sections were incubated overnight at room temperature with TUBB3 antibodies (ab18207, Abcam, Cambridge, UK) at a concentration of 0.05e0.1 mg/mL and then incubated for 30 min with biotinylated secondary antibodies to rabbit IgG (host species, goat) (Histofine Simple Stain MAX-PO(R), Nichirei, Tokyo, Japan). Immunoreaction was visualized with 3,30 -diaminobenzidine (SK4100, Vector Laboratories, Burlingame, CA, USA). Negative controls were prepared from other sections by omitting the primary antibody, and no positive immunostaining was detected. Adjacent sections were counterstained with haematoxylin and eosin, as necessary. Sections were photographed using a light microscope (ECLIPSE 80i, Nikon, Tokyo, Japan).
3.
Results
3.1.
Bud stage of mouse molar
First, we examined the molar tooth germ at the bud stage, which was represented by epithelial incursion into the ectomesenchyme (Fig. 1A, D). Intense immunostaining was observed in the nerve fiber around the tooth germ and the maxillary nerve, indicating that the antibody could target TUBB3 specifically in this region. However, no TUBB3 immunostaining was observed in the dental epithelium or condensed mesenchyme at the bud stage.
3.2.
Cap stage of mouse molar
The dental organ is constituted by the enamel organ, the dental papilla, and the dental follicle. Immunostaining for TUBB3 was observed slightly in the enamel knot of the enamel organ, and it was dotted in the nerve fibers around the dental follicle; however, no staining was seen in the dental mesenchyme (Fig. 1B, E).
3.3.
Bell stage of mouse molar
The enamel organ forms the inner and outer enamel epithelia, the stellate intermedium, and the stellate reticulum. At the bell stage, immunostaining for TUBB3 was observed moderately in the inner enamel epithelium and the dental papilla adjunct at the cusp portion. No or slight staining was seen in the outer enamel epithelium, the stellate intermedium, the stellate reticulum, and the other dental papilla. Intense immunostaining for TUBB3 was observed in the nerve fiber around the dental follicle (Fig. 1C, F).
3.4.
Hard tissue formation stage
The inner enamel epithelium differentiates to ameloblasts, whereas the dental papilla cells adjcent to the inner enamel epithelium differentiate to odontoblasts. At P3, the initial phase of immature enamel and dentin secretion, intense TUBB3 immunostaining spread over all ameloblasts and odontoblasts. In the ameloblasts, TUBB3 was observed in the whole cytoplasm of the cell body, especially close to the basal lamina. In the odontoblasts, it was prominent in the supranuclear compartment near to the predentin layer. No or slight staining was seen in the outer enamel epithelium, the stellate intermedium, the stellate reticulum, and the dental papilla (Fig. 2). At P7, the immature enamel and dentin formation proceeded, with odontoblasts exhibiting morphological change into bowling pin shapes, in which the perikarya were located from the bottom to the top of the supranuclear compartment. In the odontoblasts, TUBB3 was not only prominent in the supranuclear compartment near the predentin layer but also in the predentin layer. The ameloblasts showed intense immunostaining for TUBB3 and sporadic TUBB3-positive puncta were observed in the pulp, probably representing the nerve fiber (Fig. 3A, C). At P14, before tooth
Please cite this article as: Oshima S, Yawaka Y, Class III b-tubulin expression during hard tissue formation in developing mouse teeth, Pediatric Dental Journal, https://doi.org/10.1016/j.pdj.2019.12.002
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Fig. 1 e Immunohistochemistry for TUBB3 in the developing molar tooth germ from the bud stage to the bell stage. AeC are counterstained with haematoxylin and eosin (HE). A and D (bud stage), B and E (cap stage) and C and F (bell stage) are adjacent sections respectively. Arrowheads indicate peripheral nerve fibers. TB, tooth bud; CM, condensed mesenchymal cell; V2, maxillary nerve; EK, enamel knot; DP, dental papilla; IEE, inner enamel epithelium. Scale bars, 200 mm.
Fig. 2 e Immunohistochemistry for TUBB3 in the developing molar tooth germ at the initiation of hard tissue formation. A and B are counterstained with HE. A and C are adjacent sections. B and D are magnified of A and C, respectively. V2, maxillary nerve; Od, odontoblasts; Am, ameloblast. Scale bars, A and C, 500 mm, B and D, 100 mm.
Please cite this article as: Oshima S, Yawaka Y, Class III b-tubulin expression during hard tissue formation in developing mouse teeth, Pediatric Dental Journal, https://doi.org/10.1016/j.pdj.2019.12.002
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Fig. 3 e Immunohistochemistry for TUBB3 in the developing molar tooth germ at the hard tissue formation. A and B are counterstained with HE. A and C (during dentinogenesis) and B and D (completion of dentinogenesis) are adjacent sections respectively. B and D, Dot or beads like immunoreactivity of TUBB3 in the pulp indicates nerve fibers. P, dental pulp; Od, odontoblasts; Am, ameloblast; En, enamel; De, dentin. Scale bars, 100 mm.
eruption but when crown dentinogenesis has finished, the strength of TUBB3 immunostaining decreased substantially in the supranuclear compartment of odontoblasts in the tooth crown. By contrast, the number of TUBB3 immunopositive nerve fibers increased around the odontoblasts (Fig. 3B, D). TUBB3 was also observed in the tooth crown and the area of tooth root formation. At P10, radicular dentin formation was initiated, and intense immunostaining for TUBB3 was observed in the odontoblasts of the radicular dentin and Hertwig’s epithelial root sheath (Fig. 4A, C). During tooth root development, intense immunostaining for TUBB3 was observed in the differentiated odontoblasts of the rim of the root sheath near Hertwig’s epithelial root sheath. However, the strength of TUBB3 immunostaining was substantially less in odontoblasts not in the root sheath, where root formation was advanced (Fig. 4B, D).
During tooth root development, intense immunostaining for TUBB3 was observed in the cementoblasts on the root surface. The cementum consists of acellular and cellular parts: the acellular part covers the cervical half of the root and provides attachment to the tooth; and the cellular part covers an apical half, plays an adaptive role in response to tooth wear and movement, and is associated with periodontal tissue repair. Because it was difficult to identify cementoblasts by shape alone, we considered any relatively large cuboidal cells lining the cervical or apical root surface as the acellular or cellular cementum, respectively. During tooth root development, intense immunostaining for TUBB3 was observed in the cementoblasts on the root surface (Fig. 5A and B). After tooth eruption, the number of TUBB3 immunopositive cementoblasts was substantially decreased in the acellular cementum of the cervical half of the root (Fig. 5C and D). By
Please cite this article as: Oshima S, Yawaka Y, Class III b-tubulin expression during hard tissue formation in developing mouse teeth, Pediatric Dental Journal, https://doi.org/10.1016/j.pdj.2019.12.002
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Fig. 4 e Immunohistochemistry for TUBB3 in the developing root dentin. A and B are counterstained with HE. A and C (Hertwig’s epithelial root sheath, HERS) and B and D (radicular dentinogenesis) are adjacent sections respectively. Arrowheads indicate HERS. P, dental pulp; Od, odontoblasts; Am, ameloblast; Ce, cementoblasts; En, enamel; De, dentin. Scale bars, 100 mm.
contrast, cementoblasts continued to show TUBB3 expression in the cellular cementum (Fig. 5C, E), while cementocytes entrapped in the matrix of the cellular cementum showed no or only slightly positive immunostaining. Through all stages, osteoblasts lining the surface of the jawbone matrix and entrapped in the bone matrix were not immunopositive for TUBB3 in the bone around the tooth.
3.5.
The ameloblasts of mouse incisors
We investigated TUBB3 expression in the ameloblasts of the mouse incisors because the spaciotemporal changes were clear. Similar to the molar teeth, ameloblasts stained positive for TUBB3 at the secretary stage (Fig. 6C). TUBB3 was also observed in the whole cytoplasm of the cell body during the transition and secretary stages (Fig. 6D), while the TUBB3 expression pattern varied during the maturation stage (Fig. 6EeL). One TUBB3 immunostaining pattern was observed in the whole cytoplasm of the cell body and was similar to that
observed in the secretary and transition stage ameloblasts. However, some ameloblasts exhibited TUBB3 immunostaining in the distal portion adjacent to the enamel (Fig. 6G), some did so in both the distal and medial portions (Fig. 6I, K), and some did so in the perikarya. As with this variation in expression patterns, the intensity of TUBB3 immunostaining also varied from intense to slight. Immunostaining for TUBB3 was observed in ameloblasts at the protective stage (Fig. 6M and N); however, not all ameloblasts showed TUBB3 expression, with some showing no or slight positivity.
4.
Discussion
In the present study, we found that the neurospecific betatubulin subtype, TUBB3, was expressed in the developing teeth of mice, especially in the cells involved with hard tissue formation. As previously reported, TUBB3 was expressed in the nerve fibers of teeth, but we also observed high expression
Please cite this article as: Oshima S, Yawaka Y, Class III b-tubulin expression during hard tissue formation in developing mouse teeth, Pediatric Dental Journal, https://doi.org/10.1016/j.pdj.2019.12.002
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Fig. 5 e Immunohistochemistry for TUBB3 in the developing root dentin. Sections are counterstained with HE. B is magnified of A (P14, before eruption, during radicular dentinogenesis). D and E are magnified of C (4 wks, after eruption, completion of radicular dentinogenesis). Arrowheads indicate HERS. P, dental pulp; Od, odontoblasts; Am, ameloblast; Ce, cementoblasts; En, enamel; De, dentin; Al, alveolar bone; Pl, periodontal ligament. Scale bars, 100 mm.
levels in ameloblasts, odontoblasts, and cementoblasts during hard tissue formation. The coronal odontoblasts expressed TUBB3 abundantly during primary dentin formation, but scarcely thereafter. In the root dentin, similar to the crown, odontoblasts expressed abundant TUBB3 near Hertwig’s epithelial root sheath, where the root was forming, but scarcely expressed it after primary dentin formation had completed. Enamel formation is a two-step process. Although enamel develops its full thickness during the secretory stage, only 30% of enamel is mineralized at this stage. Subsequently, enamel mineralization is increased to beyond 96% by the removal of water and organic materials and the addition of minerals during the maturation stage [12]. In the secretory stage, all ameloblasts expressed abundant TUBB3 in the present study, but this varied in the maturation stage. Although cyclical variations occur between ruffle-ended and smooth-ended ameloblasts in the maturation stage, the expression patterns of TUBB3 were not periodic and seemed unrelated to these cyclical variations.
The cementoblasts showed similar expression patterns to those of odontoblasts. During tooth root formation, the cementoblasts of the acellular part covering the cervical half of the root expressed abundant TUBB3, but the number of cementoblasts in this region decreased when root formation completed. Cementoblasts in the cellular part, which covers the apical half and plays an adaptive role in response to tooth wear and movement, continued to express TUBB3 abundantly. No reports have described either the association between tooth development and TUBB3 or the association of tooth abnormality with TUBB3 mutation. The functions of TUBB3 are not fully understood. In neurons, TUBB3 mutations may cause congenital fibrosis of the extraocular muscles type 3. It may also be involved in neurogenesis and in axon guidance and maintenance [9]. Furthermore, TUBB3 is expressed in Sertoli cells and is regulated by androgen receptors [13], but its function in these cells is still unclear. TUBB3 expression has been reported in other normal extraneuronal tissues, with low levels in rodent lungs [14] and in the columnar epithelium of the human colon [15]. High
Please cite this article as: Oshima S, Yawaka Y, Class III b-tubulin expression during hard tissue formation in developing mouse teeth, Pediatric Dental Journal, https://doi.org/10.1016/j.pdj.2019.12.002
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Fig. 6 e Immunohistochemistry for TUBB3 in the lower inscisor. A and B are combined photos from two consecutive photos. A is counterstained with HE. A and B are adjacent sections. C (secretory stage), D (transitional stage), E, F, G, H, I, J, K, L (maturation stage), M and N (protection stage) are magnified ameloblasts images of c’, d’, e’, f ’, g’, h’, i’, j’, k’, l’, m’ and n’ in B respectively. P, dental pulp; Od, odontoblasts; Am, ameloblast; Ce, cementoblasts; En, Enamel; De, dentin Scale bars in A and B, 1000 mm. Scale bars in CeN, 50 mm. levels of TUBB3 in normal extraneuronal tissues, such as teeth and Sertoli cells, have not yet been reported. As stated, increased TUBB3 expression is associated with poor clinical outcomes in various cancers, such as non-small cell lung cancer [16,17], ovarian cancer [18], urothelial carcinoma of the bladder [19], and prostate cancer [20]. TUBB3 overexpression has also been linked to a reduced response to taxane-based microtubule-targeting anticancer drugs [21,22]. Using a cell-free assay, Lu and Luduena have shown that removing TUBB3 by immunoprecipitation with specific antibodies can enhance paclitaxel-induced microtubule polymerization [23]. The physiologic role of TUBB3 seems to be associated with microtubule dynamic instability [24], with microtubules of the cell cytoskeleton playing an important role in cancer cell migration [25]. Kanojia et al. for example, reported that TUBB3 was associated with brain metastases in patients with breast cancer [26]. In the developing tooth, TUBB3 is distinctly expressed in cells responsible for hard tissue formation, but only when hard tissue is forming. TUBB3 appears to be associated with ameloblast, odontoblast, and cementoblast function during this stage, even though osteoblasts in the alveolar bone
showed no TUBB3 expression. This suggests that TUBB3 may be associated with hard tissue formation in the toot, but not in the bone. Both ameloblasts and odontoblasts change their shape during differentiation and hard tissue formation, with the microtubules that assemble with aand beIIIeheterodimers being more dynamic [24]. Thus, TUBB3 may be required to change ameloblast and odontoblast cell shapes when hard tissue is forming. Unfortunately, our study was not able to confirm the roles of TUBB3 in developing teeth due to technical limitations. Although we did demonstrate a previously unknown function of TUBB3, further research is required to clarify the role of TUBB3 in normal tooth development.
5.
Conclusions
In developing teeth, TUBB3 is specifically expressed in ameloblasts, odontoblasts, and cementoblasts during hard tissue formation. Their spatiotemporal profiles suggest that TUBB3 may be correlated to some cellular process at the mineralization stage of tooth development.
Please cite this article as: Oshima S, Yawaka Y, Class III b-tubulin expression during hard tissue formation in developing mouse teeth, Pediatric Dental Journal, https://doi.org/10.1016/j.pdj.2019.12.002
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Declaration of Competing Interest The authors declare no conflict of interest.
Acknowledgments This work was supported by JSPS KAKENHI Grant Number 25463162. The authors would like to thank Enago (www.enago. jp) for the English language review.
references
[1] Gadadhar S, Bodakuntla S, Natarajan K, Janke C. The tubulin code at a glance. J Cell Sci 2017;130:1347e53. [2] Lewis SA, Lee MG, Cowan NJ. Five mouse tubulin isotypes and their regulated expression during development. J Cell Biol 1985;101:852e61. [3] Lewis SA, Gu W, Cowan NJ. Free intermingling of mammalian beta-tubulin isotypes among functionally distinct microtubules. Cell 1987;49:539e48. [4] Raff EC, Hoyle HD, Popodi EM, Turner FR. Axoneme betatubulin sequence determines attachment of outer dynein arms. Curr Biol 2008;18:911e4. [5] Joshi HC, Cleveland DW. Differential utilization of betatubulin isotypes in differentiating neurites. J Cell Biol 1989;109:663e73. [6] Lecine P, Italiano Jr JE, Kim SW, Villeval JL, Shivdasani RA. Hematopoietic-specific beta 1 tubulin participates in a pathway of platelet biogenesis dependent on the transcription factor NF-E2. Blood 2000;96:1366e73. [7] Schwer HD, Lecine P, Tiwari S, Italiano Jr JE, Hartwig JH, Shivdasani RA. A lineage-restricted and divergent betatubulin isoform is essential for the biogenesis, structure and function of blood platelets. Curr Biol 2001;11:579e86. € rk SJ. Class III beta-tubulin in [8] Katsetos CD, Herman MM, Mo human development and cancer. Cell Motil Cytoskelet 2003;55:77e96. [9] Tischfield MA, Baris HN, Wu C, Rudolph G, Van Maldergem L, He W, et al. Human TUBB3 mutations perturb microtubule dynamics, kinesin interactions, and axon guidance. Cell 2010;140:74e87. [10] Lewis SA, Cowan NJ. Complex regulation and functional versatility of mammalian alpha- and beta-tubulin isotypes during the differentiation of testis and muscle cells. J Cell Biol 1988;106:2023e33. [11] Mariani M, Karki R, Spennato M, Pandya D, He S, Andreoli M, et al. Class III b-tubulin in normal and cancer tissues. Gene 2015;563:109e14. [12] Nanci A. Ten Cate’s oral histology : development, structure, and function. St louis, Missouri: Mosby; 2008.
[13] De Gendt K, Denolet E, Willems A, Daniels VW, Clinckemalie L, Denayer S, et al. Expression of Tubb3, a betatubulin isotype, is regulated by androgens in mouse and rat Sertoli cells. Biol Reprod 2011;85:934e45. ~ a RF. Multiple forms of tubulin: different gene [14] Luduen products and covalent modifications. Int Rev Cytol 1998;178:207e75. ~ a RF. [15] Roach MC, Boucher VL, Walss C, Ravdin PM, Luduen Preparation of a monoclonal antibody specific for the class I isotype of beta-tubulin: the beta isotypes of tubulin differ in their cellular distributions within human tissues. Cell Motil Cytoskelet 1998;39:273e85. [16] Koh Y, Jang B, Han SW, Kim TM, Oh DY, Lee SH, et al. Expression of class III beta-tubulin correlates with unfavorable survival outcome in patients with resected nonsmall cell lung cancer. J Thorac Oncol 2010;5:320e5. [17] Ohashi T, Yoshimasu T, Oura S, Kokawa Y, Kawago M, Hirai Y, et al. Class III beta-tubulin expression in non-small cell lung cancer: a predictive factor for paclitaxel response. Anticancer Res 2015;35:2669e74. C, Davidson B, [18] Hetland TE, Hellesylt E, Flørenes VA, Trope Kærn J. Class III b-tubulin expression in advanced-stage serous ovarian carcinoma effusions is associated with poor survival and primary chemoresistance. Hum Pathol 2011;42:1019e26. [19] Choi JW, Kim Y, Lee JH, Kim YS. Expression of b-tubulin isotypes in urothelial carcinoma of the bladder. World J Urol 2014;32:347e52. [20] Tsourlakis MC, Weigand P, Grupp K, Kluth M, Steurer S, Schlomm T, et al. bIII-tubulin overexpression is an independent predictor of prostate cancer progression tightly linked to ERG fusion status and PTEN deletion. Am J Pathol 2014;184:609e17. [21] Zhang HL, Ruan L, Zheng LM, Whyte D, Tzeng CM, Zhou XW. Association between class III b-tubulin expression and response to paclitaxel/vinorebine-based chemotherapy for non-small cell lung cancer: a meta-analysis. Lung Cancer 2012;77:9e15. [22] Zhou J, Li J, Wang Z, Yin C, Zhang W. Metadherin is a novel prognostic marker for bladder cancer progression and overall patient survival. Asia Pac J Clin Oncol 2012;8:e42e8. [23] Lu Q, Luduena RF. Removal of beta III isotype enhances taxol induced microtubule assembly. Cell Struct Funct 1993;18:173e82. ~ a RF, Wilson L. [24] Panda D, Miller HP, Banerjee A, Luduen Microtubule dynamics in vitro are regulated by the tubulin isotype composition. Proc Natl Acad Sci U S A 1994;91:11358e62. [25] Fife CM, McCarroll JA, Kavallaris M. Movers and shakers: cell cytoskeleton in cancer metastasis. Br J Pharmacol 2014;171:5507e23. [26] Kanojia D, Morshed RA, Zhang L, Miska JM, Qiao J, Kim JW, et al. bIII-tubulin regulates breast cancer metastases to the brain. Mol Cancer Ther 2015;14:1152e61.
Please cite this article as: Oshima S, Yawaka Y, Class III b-tubulin expression during hard tissue formation in developing mouse teeth, Pediatric Dental Journal, https://doi.org/10.1016/j.pdj.2019.12.002