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Expression pattern of Sox2 during mouse tooth development
Gene Expression Patterns 12 (2012) 273–281
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Expression pattern of Sox2 during mouse tooth development Li Zhang 1, Guohua Yuan 1, Huan Liu, Heng Lin, Chunyan Wan, Zhi Chen ⇑ State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory for Oral Biomedicine of Ministry of Education (KLOBM), School and Hospital of Stomatology, Wuhan University, Wuhan 430079, China
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Article history: Received 26 December 2011 Received in revised form 4 July 2012 Accepted 14 July 2012 Available online 24 July 2012 Keywords: Sox2 Epithelium stem cell Apical bud Mouse odontogenesis
a b s t r a c t The transcription factor Sox2 plays important roles in maintaining the pluripotency of embryonic stem cells and adult progenitors. However, whether Sox2 is involved in odontogenesis has not been reported. In this study, we examined the expression pattern of Sox2 during mouse incisor and molar development using real-time PCR, in situ hybridization and immunohistochemistry. Sox2 mRNA was expressed in the dental epithelium and mesenchyme while Sox2 protein was mainly detected in the epithelium from embryonic day (E) 11.5 to postnatal (PN) day 20. In the case of incisor, Sox2 mRNA and protein were expressed in most of dental epithelial cells from E11.5 to E14.5, and they were both highly expressed in the labial cervical loop area from E16.5 to PN20. During molar development, we observed an asymmetrical distribution of Sox2 protein in the epithelium from E13.5 to E16.5, with stronger signals in the lingual side. From E18.5 to PN2, Sox2 was expressed within the cervical loop area, and the stellate intermediate layer. From PN6 to PN14, Sox2 expression was confined mainly to the apical end of hertwig’s epithelium root sheath (HERS) cells. Sox2 was also detected within the perivascular region of the dental pulp at PN14 and PN20. Our results suggested that: (1) Sox2 was involved in mouse odontogenesis, and (2) it might participate in maintaining the pluripotency of the epithelial stem cells of labial cervical loop in mouse incisor development and the epithelium progenitors during molar development, (3) Sox2 might be regulated at post-transcription level during mouse odontogenesis. Ó 2012 Elsevier B.V. All rights reserved.
Odontogenesis is regulated by the complex interactions between an ectoderm, the dental epithelium and the cranial neural crest-derived mesenchyme (Slavkin et al., 1984; Thesleff et al., 1995; Vainio and Thesleff, 1992). Tooth morphogenesis goes through initiation stage, bud stage, cap stage and bell stage, and this process exhibits many morphological and molecular similarities with other developing epithelial appendages, such as hair follicles, salivary glands, lungs, mammary glands and kidneys (Chuong and Noveen, 1999; Iseki et al., 1996; Pispa and Thesleff, 2003; Thesleff et al., 1995). Sox2 belongs to the family of SRY (sex-determining region on the Y chromosome)-related HMG (high-mobility-group) transcription factors which are important for cell fate and differentiation in multiple developmental and physiological processes (Kamachi et al., 2000; Lefebvre et al., 2007). During embryogenesis, Sox2 mRNA is initially expressed in morula cells at embryonic day (E) 2.5, then specifically within the inner cell mass (ICM) at E3.5 (Avilion et al., 2003). Embryos deficient for Sox2 lack a pluripotent ICM and fail to survive shortly after implantation (Avilion et al., 2003; Masui et al., 2007). Sox2 is also widely expressed in adult tis-
⇑ Corresponding author. Tel.: +86 27 87686198; fax: +86 27 87873849. 1
E-mail address: [email protected] (Z. Chen). Both authors contributed equally to this paper.
1567-133X/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gep.2012.07.001
sues, such as the progenitors in brain (Brazel et al., 2005; Ellis et al., 2004), tongue epithelium (Okubo et al., 2009), neural retina (Taranova et al., 2006), dermal papilla of the hair follicle (Driskell et al., 2009), glandular stomach, cervix and tests (Arnold et al., 2011). Recently, it has been reported that forced expression of Sox2, together with Oct-3/4, c-myc and klf4, can endow adult mouse fibroblasts with pluripotency, giving rise to induced pluripotent stem cells (Takahashi and Yamanaka, 2006). However, whether Sox2 is involved in tooth development is unknown. In this study, we for the first time examined detailed Sox2 expression profile during mouse incisor and molar development by real-time PCR, in situ hybridization and immunohistochemistry.
1. Results 1.1. Sox2 mRNA expressed in mouse tooth germs To examine a possible involvement of Sox2 during tooth development, Sox2 mRNA expression was first confirmed in the tissues harvested from the whole incisor or molar germs (Fig. 1A, cartoon depiction of the mouse molar (M1) and incisor (dashed line) at different stages from embryonic day (E) 11.5 to postnatal day (PN) 20 by real-time PCR. The result showed that Sox2 was expressed at all these stages investigated (Fig. 1B and C). The expression level of
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Fig. 1. Real-time PCR analysis of Sox2 mRNA expression in mouse incisors and molars. (A) Cartoon depiction of the mouse molar (M1) and incisor (dashed line) Sox2 transcript was detected from the tissues taken from the whole mandibular mouse incisors from E11.5 to PN20. (B) Sox2 transcript was expressed in the tissues taken from first mandibular molars (M1) from E11.5 to PN20. E, embryonic day and PN, postnatal day. All error bars represent mean ± SD.
Sox2 mRNA during incisor development was gradually down-regulated with age (E11.5 to PN20), and maintained in a certain moderate level at postnatal stages (PN2 to PN20) (Fig. 1B). During molar development, the expression level of Sox2 mRNA was upregulated from E11.5 to E13.5, and then down-regulated from E13.5 to E18.5, and after postnatal life (PN2 to PN20), Sox2 mRNA expression was reduced to a much lower level compared with embryonic stages (Fig. 1C).
1.2. Expression pattern of Sox2 mRNA during mouse incisor development To further characterize in situ expression of Sox2 transcript, we examined the expression of Sox2 mRNA during mouse incisor development using in situ hybridization from E11.5 to PN20. At the lamina stage (E11.5), the late bud stage (E13.5) and the cap stage (E14.5), Sox2 mRNA was broadly detected both in the dental epithelium and mesenchyme (Fig. 2A–E). And the signal in the epithelium was slightly stronger than that in the dental mesenchyme. Sox2 mRNA was highly expressed within the labial cervical loop area of dental epithelium at E14.5 (Fig. 2D and E). From the early bell stage (E16.5) to PN20, the signal of Sox2 mRNA was much stronger in the dental epithelium than that in the dental mesenchyme. The labial side of the dental epithelium, especially the labial cervical loop area (laCL), including the basal epithelium (BE), the stellate reticulum (SR) and the outer enamel epithelium (OEE) showed strong Sox2 mRNA signal (Fig. 2G, H, J, K, M, N, Q and R). The transit-amplifying (TA) cells of the inner enamel epithelium adjacent to the apical bud and the stellate intermediate (SI) also exhibited expression of Sox2 mRNA (Fig. 2G, H, J, K, M, N, P, Q and R). In addition, we found Sox2 mRNA in the epithelium cells of lingual cervical loop (liCL) (Fig. 2G, I, J, L, M and O), as well as in the pre-ameloblasts and ameloblasts (Fig. 2P and U, red arrows). We also observed Sox2 mRNA expression in the odontoblasts (Fig. 2T and U, black arrows) and the cells in the perivascular region (Fig. 2S, red asterisks). The eye, brain and dermal papillae of hair follicle were used as positive control (Fig. S1A–C). Strong signals of Sox2 mRNA were observed in these tissues. Sense probe was used as negative control (Fig. 2F) and no positive signal was observed.
1.3. Expression pattern of Sox2 mRNA during mouse molar development In situ hybridization was performed to further investigate the in situ expression of Sox2 mRNA during the mouse molar development from E11.5 to PN20. At the lamina stage (E11.5), Sox2 mRNA was expressed both in the epithelium (Fig. 3A, black arrow) including the dental lamina (Fig. 3A, red arrow), and in the underlying mesenchyme. At the bud stage (E13.5) and the cap stage (E14.5), the expression of Sox2 in the dental epithelium of the enamel organ was stronger than that in the underlying mesenchyme. At the early bell stage (E16.5), the inner enamel epithelium showed strong signal of Sox2 mRNA (Fig. 3D and E). At the late bell stage (E18.5) and PN2, Sox2 was primarily localized in the inner enamel epithelium, especially in the cervical loop area, the stellate intermediate (SI) layer and the stellate reticulum that adjacent to the SI layer (Figs. 3F–H; 4A–C). From PN6 to PN14, the cells of the hertwig’s epithelium root sheath (HERS) showed positive expression of Sox2 (Fig. 4D, F and G, blue arrows). Sox2 mRNA was also detected in the cells around blood vessels marked by CD31 within the dental pulp at PN14 and PN20 (Fig. 4G–J, red asterisks). In addition, we also found Sox2 mRNA signal in the odontoblasts and ameloblasts (Fig. 4B and E, black and red asterisks, respectively). Sense probe was used as negative control (Fig. 3I) and no positive signal was observed.
1.4. Localization of Sox2 protein during mouse incisor development Based on the expression pattern of Sox2 mRNA, immunohistochemistry was performed to further elucidate the translation expression of Sox2 during mouse incisor development. Interestingly, Sox2 protein was mainly detected in the dental epithelium. At the lamina stage (E11.5), the late bud stage (E13.5) and the cap stage (E14.5), Sox2 protein was highly observed in most of the cells within the dental epithelium (Fig. 5A–C, red arrows), while the protein signal was only observed in a limited number of mesenchymal cells adjacent to the epithelium (Fig. 5A–E, red arrowheads). At E14.5, Sox2 protein was strongly expressed within the labial cervical loop area of dental epithelium (Fig. 5D and E,
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Fig. 2. Expression of Sox2 mRNA during mouse incisor development from E11.5 to PN20. The samples are sagittal sections. At E11.5, E13.5 and E14.5, Sox2 mRNA was broadly detected both in the dental epithelium and mesenchyme (A–E). The expression of Sox2 in the epithelium was slightly stronger than that in the mesenchyme. At E14.5, Sox2 signal was strongly expressed within the labial cervical loop area of dental epithelium (D and E). (F) Sense probe was used as negative control and no positive signal was observed. (G–U) From E16.5 to PN20, Sox2 mRNA signal was much stronger in the dental epithelium than in the dental mesenchyme. The labial side of the dental epithelium, especially the labial cervical loop area (laCL), including the basal epithelium (BE), the stellate reticulum (SR) and the outer enamel epithelium (OEE) (G, H, J, K, M, N, P–R) showed strong Sox2 mRNA expression. The transit-amplifying (TA) cells and the stellate intermediate (SI) also expressed Sox2 mRNA (G, H, J, K, M, N, P–R). In addition, Sox2 mRNA was found in the epithelium cells of lingual cervical loop (liCL) (G, I, J, L, M, O), as well as in the pre-ameloblasts and ameloblasts (P and U, red arrows). Sox2 mRNA expression was also expressed in the odontoblasts (T and U, black arrows) and the cells in the perivascular region (S, red asterisks). (C, E, H, I, K, L, N, O) Higher-magnification images of black and red boxes in panels B, D, G, J, M, respectively. Black and red dashed line outlined the dental epithelium and blood vessel, respectively. de, Dental epithelium; dm, dental mesenchyme; cm, condensed mesenchyme; vl, vestibular lamina; liCL, lingual cervical loop; laCL, labial cervical loop; BE, basal epithelium; OEE, outer dental epithelium; SR, stellate reticulum; SI, stratum intermedium and TA, transit-amplifying cells. Od, odontoblast; Am, ameloblast; pre-Am, pre-ameloblast; dp, dental pulp and BV, blood vessel. Scale bars: A, C, E, H, I, K, L, N–U: 50 lm; B and D: 100 lm; F, G, J, M: 200 lm.
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Fig. 3. Expression of Sox2 mRNA during mouse molar development from E11.5 to E18.5. The samples are frontal sections from E11.5 to E16.5, and sagittal sections at E18.5. At E11.5, Sox2 mRNA was expressed both in the epithelium (A, black arrow) including the dental lamina (A, red arrow) and in the underlying mesenchyme (dm). At E13.5 and E14.5, the expression of Sox2 in the dental epithelium was stronger than that in the underlying mesenchyme (B and C). At E16.5, the inner enamel epithelium showed strong signal of Sox2 (D–F). At E18.5, Sox2 was primarily localized in the stellate intermediate (SI) layer, the stellate reticulum adjacent to SI layer, and the inner enamel epithelium, especially in the cervical loop area (G and H). (I) Sense probe was used as negative control (F) and no positive signal was observed. Dashed line outlined the dental epithelium. (E, G and H) Higher-magnification images in panel D and F, respectively. oe, oral epithelium; de, dental epithelium; dm, dental mesenchyme; cm, condensed mesenchyme; dp, dental papilla; pek, primary enamel knot; T, tongue; CL, cervical loop; IEE, inner enamel epithelium; SR: stellate reticulum and SI: stratum intermedium. Scale bars: A, B, C, E, G, H: 50 lm; D and I: 100 lm; F: 200 lm.
blue arrow).The vestibular lamina also showed strong signal of Sox2 (Fig. 5D and F, red arrow). At the early bell stage (E16.5), the expression of Sox2 was restricted to the labial side of the dental epithelium, especially the labial cervical loop area, including the basal epithelium (BE), the stellate reticulum (SR) and the outer enamel epithelium (OEE) (Fig. 5G and I). The transit-amplifying (TA) cells of the inner enamel epithelium adjacent to the apical bud and the stellate intermediate (SI) also exhibited expression of Sox2 (Fig. 5G–I, red arrows and blue arrow, respectively). From the middle bell stage (E18.5) to PN20, the expression of Sox2 was primarily and obviously maintained in the cells within the labial cervical loop (Fig. 5J–U), the TA cells adjacent to the labial cervical loop (Fig. 5J–U, red arrows) and the SI layer (Fig. 5G, H, J, K, M, N, P, Q, S and T, blue arrows). Some mesenchymal cells adjacent to the labial cervical loop were also found to express weak Sox2 protein signal (Fig. 5G, I, J, L, M, O, P, R, S and U, red arrowheads).
1.5. Localization of Sox2 protein during mouse molar development At the lamina stage (E11.5), Sox2 was highly expressed in the epithelium (Fig. 6A, black arrow) including the dental lamina (Fig. 6A, red arrow). At the bud stage (E13.5), Sox2 protein strongly expressed in the lingual side of the dental epithelium, only weak signal of Sox2 was found in the buccal side of the dental epithelium (Fig. 6B). From the cap stage (E14.5), Sox2 started to be expressed in the stellate reticulum (Fig. 6C). Similar to E13.5, asymmetric distribution of Sox2 signal was found, with stronger staining in the lingual side than the buccal side of the tooth germ (Fig. 6C). No staining was found in the cells of the primary enamel knot. We also
found some small number of mesenchymal cells adjacent to the epithelium expressed Sox2 protein at E11.5, E13.5 and E14.5 (Fig. 6A–E, red arrowheads). At the early bell stage (E16.5), preferential expression of Sox2 was also observed in the lingual side of the outer enamel epithelium, the cervical loop, and the inner enamel epithelium between secondary enamel knots (Fig. 6D–F). At the late bell stage (E18.5), Sox2 was primarily localized in the inner enamel epithelium, especially the cervical loop area, the stellate intermediate (SI) layer and the stellate reticulum adjacent to SI layer (Fig. 6G–I). At PN2, Sox2 was localized in the stellate intermediate layer (Fig. 7A–C, black arrows), and the stellate reticulum adjacent to the SI layer (Fig. 7A–C, red arrows). Besides, a few cells within the cervical loop were stained by Sox2 (Fig. 7C, blue arrow). At PN6, the cells of the hertwig’s epithelium root sheath (HERS) showed nuclei expression of Sox2 (Fig. 7D–F, blue arrows). At PN14, Sox2 expression was maintained in the cells within the apical end of HERS (Fig. 7F and G, blue arrows). Sox2 signal was detected in the cells at the perivascular region of the dental pulp at PN14 and PN20 (Fig. 7G–L, red asterisks).We could sometimes found Sox2 positive cells within the perivascular region as early as at E18.5 (data not shown). The blood vessels were marked by CD31 within the dental pulp (Fig. 7I and L). We could never found the expression of Sox2 protein in odontoblasts or ameloblasts.
2. Discussion In our present study, we clearly showed the spatial and temporal expression pattern of Sox2 mRNA and protein during mouse incisor and molar development. Sox2 mRNA was expressed at both
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Fig. 4. Expression of Sox2 mRNA during mouse molar development from PN2 to PN20. At PN2, Sox2 mRNA was primarily localized in the stellate intermediate (SI) layer, the stellate reticulum adjacent to SI layer and the inner enamel epithelium, especially in the cervical loop area (A–C, black, red and blue arrows, respectively). From PN6 to PN14, the cells of the hertwig’s epithelium root sheath (HERS) showed positive expression of Sox2 (D, F and G, blue arrows). Sox2 mRNA was also detected in the cells around blood vessels (G–J, red asterisks) marked by CD31 (H and J) within the dental pulp at PN14 and PN20. In addition, Sox2 mRNA signal was observed in the odontoblasts and ameloblasts (B, D and E, black and red asterisks, respectively). Dashed line outlined the structure of HERS. (B, C, E and F) Higher-magnification images of boxes in panel A and D, respectively. SR, stellate reticulum; SI, stratum intermedium; Am, ameloblasts; Od, odontoblasts; HERS, Hertwig’s epithelium root sheath; PN, postnatal day. Scale bars: A and D: 200 lm; B, C, F–J: 50 lm.
dental epithelium and mesenchyme, while Sox2 protein was mainly expressed in the dental epithelial cells. Both Sox2 mRNA and protein were highly expressed in the labial cervical loop area of the mouse incisor and the dental epithelium during molar development. Sox2 has been reported to play important roles in maintaining the pluripotency of embryonic stem cells and adult progenitors (Arnold et al., 2011; Avilion et al., 2003; Masui et al., 2007). Mouse incisors are continuously growing teeth throughout life. The labial cervical loop area is believed to contain stem cells that are responsible for the continuous growth (Harada et al., 1999; Harada and Ohshima, 2004; Ohshima et al., 2005). Some stem cell related markers, such as Oct-3/4, Bmi-1, and Yap have been reported to be localized in the apical bud of developing mouse incisor (Li et al., 2011). And it is also well known that Sox2 associates with Oct-3/4 to maintain self-renewal of ES cells (Masui et al., 2007; Okumura-Nakanishi et al., 2005; Rodda et al., 2005). Combined with our results, the persistent expression of Sox2 within the apical bud of mouse incisor (Figs. 2H, K, N, Q, R and 5G–U) suggested that Sox2 might be responsible for maintaining the stem cell niche in this specific epithelium structure, either by itself or by interactive cooperation with other stem cell molecules, such as Oct-3/4, Yap and Nanog (Gee et al., 2011; Masui et al., 2007; Rodda et al., 2005). Compared with the continuously growing incisor, mouse molar has limited growth ability. Our results suggested Sox2 was also involved in molar development. The asymmetric expression of Sox2 protein in molar germs from E13.5 to E16.5 in a lingual-to-buccal gradient pattern (Fig. 6B–F) indicated that there might be differential regulation mechanisms between the lingual and buccal side of dental enamel organ during mouse molar morphogenesis.
The role of HERS cells in root formation is widely accepted, the precise function of these cells remains unclear (Thomas, 1995; Tummers and Thesleff, 2003). Our result showed that Sox2 was expressed in the apical end of HERS cells (Figs. 4F, G and 7E, F, H). A recent study has also shown the expression profile of stem cell markers in human Hertwig’s epithelial root sheath/Epithelial rests of Malassez cells, such as Bmi-1, Oct-3/4, Nanog, and SSEA-4 (Nam et al., 2011). Taken together, the Sox2 positive cells in the HERS apical end of our results indicated Sox2 might be involved in maintaining the stem cell characteristics of HERS cells during root formation. We also found Sox2 positive cells around some blood vessels (Figs. 2S, 4G–J and 7H–L and data not shown at other stages). Previous study has demonstrated that stem cells reside in the perivascular niche of dental pulp (Shi and Gronthos, 2003). Therefore, these Sox2 positive cells observed in the dental pulp might be some stem cells/progenitors within the perivascular region. In addition, we observed some differences in the expression patterns between Sox2 mRNA and Sox2 protein. The Sox2 mRNA exhibited wider expression than that of Sox2 protein: (1) Sox2 mRNA could be broadly detected in the dental mesenchymal cells, while little Sox2 protein was examined in limited number of cells; (2) Sox2 mRNA could be observed in differentiated odontoblasts and ameloblasts, while Sox2 protein was not detected; (3) Sox2 mRNA was detected in the lingual cervical loop, while Sox2 protein was not. The expression of Sox2 protein was only restricted to some specific stem cell like or progenitor cell lineages. To ensure the in situ hybridization result of Sox2, two probes targeting different region of the transcript of Sox2 were developed (refer to Section 3.3.1. Probe constructs). Same results were achieved using the two probes (results of probe 2 not shown). Moreover, we ob-
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Fig. 5. Localization of Sox2 protein during mouse incisor development from E11.5 to PN20. (A) At E11.5, Sox2 was expressed in the oral epithelium (black arrow) and in dental epithelium (red arrow). (B and C) At E13.5, Sox2 was evidently localized in the dental epithelium and vestibular lamina. (D–F) At E14.5, Sox2 was stained in the dental epithelium with abundant signal in the labial cervical loop area (C and E, blue arrow), and the vestibular lamina was also stained (C and F, red arrow). A limited number of mesenchymal cells adjacent to the epithelium were also found to express Sox2 protein (A–E, red arrowheads). (G–U) Sagittal sections from E16.5 to PN20 showed that Sox2 signal was evidently and specifically restricted in the labial cervical loop area, including the basal epithelium, stellate reticulum, and outer enamel epithelium. The transitamplifying (TA) cells (red arrows) adjacent to the apical bud and the stratum intermedium (blue arrows) layer also exhibited expression of Sox2 protein. Sox2 protein signal was also found in some mesenchymal cells adjacent to the labial cervical loop (A, C, D, F, G, I, J, L, M, O, red arrowheads). (C, E, F, H, I, K, L, N, O, Q, R, T, U) Higher-magnification images of black, red and blue boxes in panels B, D, G, J, M, P, S, respectively. oe, Oral epithelium; de, dental epithelium; cm, condensed mesenchyme; vl, vestibular lamina; laCL, labial cervical loop; BE, basal epithelium; OEE, outer dental epithelium; SR, stellate reticulum; SI, stratum intermedium and TA, transit-amplifying cells. Scale bars: A, C, E, F, H, I, K, L, N, O, Q, R, T, U: 50 lm; B and D: 100 lm; G, J, M, P, S: 200 lm.
served similar cases in the dermal papillae of hair follicle. Sox2 mRNA could be detected in most part of the dermal papillae, while the expression of Sox2 protein was confined to a limited subset of cells localized in the tip of the dermal papillae (Fig. S1C and F). Similarly, the germ stem cells have been reported to express little Sox2 protein in spite of the high RNA expression levels (Arnold et al., 2011; Imamura et al., 2006).
An open question is what mechanisms lead to Sox2 mRNA and protein expression discrepancy. A potential explanation for the expression discrepancies observed between Sox2 mRNA and protein could be ascribed to the post-transcriptional regulation. MicroRNAs are endogenously expressed non-coding RNA molecules that affect protein synthesis by post-transcriptional mechanisms (Bartel, 2004; Chekulaeva and Filipowicz, 2009; Fabian et al., 2010). Sox2
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Fig. 6. Localization of Sox2 protein during mouse molar development from E11.5 to E18.5. (A) At E11.5, Sox2 was expressed in the oral epithelium (black arrow) and dental epithelium (red arrow). (B) At E13.5, Strong expression of Sox2 was observed in the lingual side of the dental epithelium; only weak signal of Sox2 was found in the buccal side of the dental epithelium. (C) At E14.5, Sox2 started to be expressed in the stellate reticulum. Similar to E13.5, asymmetric distribution of Sox2 signal was found, with stronger staining in the lingual side of the tooth germ. No staining was found in the primary enamel knot. (D–F) At E16.5, preferential expression of Sox2 was also observed in the lingual side of the outer enamel epithelium, the cervical loop, and the inner enamel epithelium between secondary enamel knots. (G–I) At E18.5, Sox2 signal was maintained in the stratum intermedium (SI), the stellate reticulum adjacent to SI layer, and the inner enamel epithelium, especially the cervical loop area. (E, F, H, I) Highermagnification images of black and red boxes in panel D and G, respectively. oe, oral epithelium; de, dental epithelium; dm, dental mesenchyme, cm, condensed mesenchyme; dp, dental papilla; pek, primary enamel knot; T, tongue; CL, cervical loop; IEE, inner enamel epithelium; SR, stellate reticulum; SI: stratum intermedium and sek, secondary enamel knot. Scale bars: A, B, C, E, F, H, I: 50 lm; D: 100 lm; G: 200 lm.
are reported to be regulated by MicroRNAs (Marson et al., 2008; Tay et al., 2008; Xu et al., 2009). Recently, microRNAs are also found in the stem cell niche of the adult mouse incisor (Cao et al., 2010; Jheon et al., 2011). Further study needs to elucidate how Sox2 was regulated at post-transcription level during tooth development. In conclusion, the prominent and persistent Sox2 mRNA and protein expression profile in the labial apical bud of the continuously growing incisor and in the dental epithelium during mouse molar formation indicated that (1) Sox2 was involved in mouse odontogenesis, (2) Sox2 might function in maintaining the specific epithelial stem cell niche of mouse incisor and in maintaining the epithelium progenitors during molar development, (3) there might be some other unknown mechanisms to regulate the Sox2 mRNA and protein expression in a more accurate manner. However, the exact function of Sox2 in tooth development is still unknown. Therefore, our future study is to explore the function of Sox2 in tooth development by using specific conditional knockout mouse targeting Sox2 in tooth structure. 3. Experimental procedures All experiments were performed according to the guideline of Animal Welfare Committee of the School and Hospital of Stomatology at Wuhan University. 3.1. Animals The adult ICR mice were mated overnight. E0.5 was designated as the day on which the presence of a vaginal plug was confirmed.
At least three embryos and postnatal mice at each developmental stage (E11.5, E13.5, E14.5, E16.5, E18.5, PN2, PN6, PN10, PN14 and PN20) were used in this study.
3.2. Real-time PCR Total RNA was isolated from the whole tooth germs (E11.5 to PN20, as shown in Fig. 1) or from the tissue of apical bud and anterior part of mouse incisor separately (PN2 to PN20, as shown in Fig. S2) using Trizol reagent (Invitrogen) according to the manufacturer’s protocol. A standard reverse transcript reaction (RT) was used to synthesize cDNA using RevertAid™ M-MuLV Reverse Transcriptase (Fermentas, EU). And 1ug of total RNA was used for RT reactions. Real-time PCR was performed with SYBR Premix Ex TaqTM (Takara Bio Inc., Shiga, Japan) according to manufacturer’s instructions. Signals were detected in the ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA). The cycle thresholds were calculated for further statistical analysis. As a control, the level of GAPDH mRNA was determined in the real-time PCR assay of each RNA sample and was used to correct for experimental variation. Primers are as follows: for Sox2 amplification, (forward) 50 -GTTCTAGTGGTACGTTAGGCGCTTC-30 , (reverse) 50 -TCGCCCGGAG TCTAGCTCTAAATA-30 ; for GAPDH control amplification, (forward) 50 -TGTGTCCGTCGTGGATCTGA-30 and (reverse) 50 -TTGCTGTTGAA GTCGCAGGAG-30 .Quantification of the relative expression levels of the Sox2 was achieved by normalizing for the endogenous GAPDH using the 44CT method. The gene expression ratio was shown as mean ± standard deviation from three independent experiments.
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Fig. 7. Localization of Sox2 protein during mouse molar development from PN2 to PN20. (A–C) At PN2, Sox2 was restricted in the stratum intermedium (SI) (black arrows), stellate reticulum (red arrows) adjacent to SI layer, and some inner enamel epithelium cells (blue arrow) within the cervical loop area. (D–H) At PN6 and PN14, Sox2 protein was detected in the apical end of Hertwig’s epithelium root sheath (HERS) (blue arrows). Sox2 signal was also detected in the cells at the perivascular region of the dental pulp at PN14 and PN20 (G–L, red asterisks). The blood vessels were marked by CD31 within the dental pulp (I, L). (B, C, E, F, H, K) Higher-magnification images of red and black boxes in panel A, D, G and J, respectively. SR: stellate reticulum; SI, stratum intermedium; HERS, Hertwig’s epithelium root sheath; BV, blood vessel. Scale bars: A, D, G: 200 lm; B, C, E, F, H, I, K, L: 50 lm; J: 100 lm.
3.3. In situ hybridization 3.3.1. Probe constructs Total RNA was extracted from the brain of ICR mouse embryo using TRIZol Reagent (Invitrogen, USA) following the manufacture’s instruction. RNA sample was then subjected to SuperScriptÒ III First-Strand Synthesis System (Invitrogen, USA) to generated Sox2 cDNA fragment. Two probes targeting different region of the transcript of Sox2 were developed. The 415-bp product for sox2 (Probe 1) was amplified using the primers (forward: 50 -CGCAAGCTTAAACC GTGATGCCGACTA-30 , reverse: 50 -TCTGGATCCATCCGAATAAACTCCT TCCTTG-30 , bases underlined indicate the restriction enzyme sites); the 515-bp product for sox2 (Probe 2) was amplified using the primers (forward: 50 -CGCAAGCTTAACGCCTTCATGGTATGGTC-30 , reverse: 50 -TCTGGATCCATGTAGGTCTGCGAGCTGGT-30 , bases underlined indicate the restriction enzyme sites). The PCR products were confirmed by DNA sequencing (Invitrogen, CA), and purified using QIAquickÒ Gel Extraction Kit (QIAGEN, Germany) and inserted into pSPT18 (Roche, Germany). The single-strand DNA was generated using restriction enzyme digestion and purified with QIAquickÒ Gel Extraction Kit (QIAGEN, Germany). Sense and antisense digoxigenin-labeled RNA probes were generated using DIG RNA Labeling Kit (SP6/T7) according to the manufacture’s instruction
(Roche, Germany). The yield of DIG-labeling was estimated in a spot test with DIG Nucleic Acid Detection Kit (Roche). 3.3.2. Section in situ hybridization For in situ hybridization, mandibles of each stage were fixed with 4% paraformaldehyde (PFA) in 0.01 M phosphate-buffered saline (PBS, pH 7.4) overnight at 4 °C and demineralized with 10% ethylenediamine tetra-acetic acid (pH 7.4) for two days to one month depending on stages at 4 °C. After having been embedded in paraffin, these samples were sectioned at a thickness of 7 lm. In situ hybridization on wax sections was performed as previously described (Hosoya et al., 2008). Sections were baked at 60 °C, de-waxed in xylene, rehydrated through a graded series of alcohol and post-fixed in 4% PFA. Sections were pre-hybridized in a humid chamber containing 50% formamide in 2xSSC, at 55 °C, for 30 min. Digoxigenin (DIG) labeled RNA probes were prewarmed at 85 °C and hybridized to sections overnight at 64 °C. Sense probes were used as negative control (Figs. 2F and 3I). 3.4. Immunohistochemistry For immunohistochemistry, specimens of each stage were sectioned at a thickness of 5 lm. Sections were de-waxed and re-hy-
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drated, incubated in 3% hydrogen peroxide for 15 min, and then were boiled in 10 mM citrate buffer (pH 6.0) for 5 min (121 °C) followed by cooling down at room temperature for 20 min. Before incubation with primary antibody, 2.5% BSA (Roche) was used for blocking. The slides were incubated with antibodies against Sox2 (1:250, rabbit monoclonal antibody, 2683-1, Epitomics), and CD31 (1:100, goat polyclonal antibody, AF3628, R&D, USA) respectively, at 4 °C overnight. After washed with PBS, the specimens were made to react with polymer Helper (Zhong Shan Golden Bridge Biotechnology) and poly-HRP-anti-Rabbit IgG, poly-HRPanti-Goat IgG (Zhong Shan Golden Bridge Biotechnology) respectively at room temperature for 15 min each. Finally, the specimens were visualized using a diaminobenzidine (DAB) reagent kit (Maixin. Bio). Then the immunostained sections were counterstained with hematoxylin. Acknowledgements This study was supported by Grants from the Natural Science Foundation of China (NSFC) (No. 30872880) and National 973 project of China (No. 2010CB534915). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.gep.2012.07.001. References Arnold, K., Sarkar, A., Yram, M.A., Polo, J.M., Bronson, R., Sengupta, S., Seandel, M., Geijsen, N., Hochedlinger, K., 2011. Sox2(+) adult stem and progenitor cells are important for tissue regeneration and survival of mice. Cell Stem Cell 9, 317– 329. Avilion, A.A., Nicolis, S.K., Pevny, L.H., Perez, L., Vivian, N., Lovell-Badge, R., 2003. Multipotent cell lineages in early mouse development depend on SOX2 function. Genes Dev 17, 126–140. Bartel, D.P., 2004. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281–297. Brazel, C.Y., Limke, T.L., Osborne, J.K., Miura, T., Cai, J., Pevny, L., Rao, M.S., 2005. Sox2 expression defines a heterogeneous population of neurosphere-forming cells in the adult murine brain. Aging Cell 4, 197–207. Cao, H., Wang, J., Li, X., Florez, S., Huang, Z., Venugopalan, S.R., Elangovan, S., Skobe, Z., Margolis, H.C., Martin, J.F., Amendt, B.A., 2010. MicroRNAs play a critical role in tooth development. J Dent Res 89, 779–784. Chekulaeva, M., Filipowicz, W., 2009. Mechanisms of miRNA-mediated posttranscriptional regulation in animal cells. Curr Opin Cell Biol 21, 452–460. Chuong, C.M., Noveen, A., 1999. Phenotypic determination of epithelial appendages: genes, developmental pathways, and evolution. J Investig Dermatol Symp Proc 4, 307–311. Driskell, R.R., Giangreco, A., Jensen, K.B., Mulder, K.W., Watt, F.M., 2009. Sox2positive dermal papilla cells specify hair follicle type in mammalian epidermis. Development 136, 2815–2823. Ellis, P., Fagan, B.M., Magness, S.T., Hutton, S., Taranova, O., Hayashi, S., McMahon, A., Rao, M., Pevny, L., 2004. SOX2, a persistent marker for multipotential neural stem cells derived from embryonic stem cells, the embryo or the adult. Dev Neurosci 26, 148–165. Fabian, M.R., Sonenberg, N., Filipowicz, W., 2010. Regulation of mRNA translation and stability by microRNAs. Annu Rev Biochem 79, 351–379. Gee, S.T., Milgram, S.L., Kramer, K.L., Conlon, F.L., Moody, S.A., 2011. Yes-associated protein 65 (YAP) expands neural progenitors and regulates Pax3 expression in the neural plate border zone. PLoS One 6, e20309. Harada, H., Ohshima, H., 2004. New perspectives on tooth development and the dental stem cell niche. Arch Histol Cytol 67, 1–11.
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Expression of MicroRNAs in the stem cell niche of the adult mouse incisor. PLoS One 6, e24536. Kamachi, Y., Uchikawa, M., Kondoh, H., 2000. Pairing SOX off: with partners in the regulation of embryonic development. Trends Genet 16, 182–187. Lefebvre, V., Dumitriu, B., Penzo-Mendez, A., Han, Y., Pallavi, B., 2007. Control of cell fate and differentiation by Sry-related high-mobility-group box (Sox) transcription factors. Int J Biochem Cell Biol 39, 2195–2214. Li, L., Kwon, H.J., Harada, H., Ohshima, H., Cho, S.W., Jung, H.S., 2011. Expression patterns of ABCG2, Bmi-1, Oct-3/4, and Yap in the developing mouse incisor. Gene Expr Patterns 11, 163–170. Marson, A., Levine, S.S., Cole, M.F., Frampton, G.M., Brambrink, T., Johnstone, S., Guenther, M.G., Johnston, W.K., Wernig, M., Newman, J., Calabrese, J.M., Dennis, L.M., Volkert, T.L., Gupta, S., Love, J., Hannett, N., Sharp, P.A., Bartel, D.P., Jaenisch, R., Young, R.A., 2008. Connecting microRNA genes to the core transcriptional regulatory circuitry of embryonic stem cells. Cell 134, 521–533. Masui, S., Nakatake, Y., Toyooka, Y., Shimosato, D., Yagi, R., Takahashi, K., Okochi, H., Okuda, A., Matoba, R., Sharov, A.A., Ko, M.S., Niwa, H., 2007. Pluripotency governed by Sox2 via regulation of Oct3/4 expression in mouse embryonic stem cells. Nat Cell Biol 9, 625–635. Nam, H., Kim, J., Park, J., Park, J.C., Kim, J.W., Seo, B.M., Lee, J.C., Lee, G., 2011. Expression profile of the stem cell markers in human Hertwig’s epithelial root sheath/epithelial rests of Malassez cells. Mol Cells 31, 355–360. Ohshima, H., Nakasone, N., Hashimoto, E., Sakai, H., Nakakura-Ohshima, K., Harada, H., 2005. The eternal tooth germ is formed at the apical end of continuously growing teeth. Arch Oral Biol 50, 153–157. Okubo, T., Clark, C., Hogan, B.L., 2009. Cell lineage mapping of taste bud cells and keratinocytes in the mouse tongue and soft palate. Stem Cells 27, 442–450. 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Gene Expression Patterns journal homepage: www.elsevier.com/locate/gep
Expression pattern of Sox2 during mouse tooth development Li Zhang 1, Guohua Yuan 1, Huan Liu, Heng Lin, Chunyan Wan, Zhi Chen ⇑ State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory for Oral Biomedicine of Ministry of Education (KLOBM), School and Hospital of Stomatology, Wuhan University, Wuhan 430079, China
a r t i c l e
i n f o
Article history: Received 26 December 2011 Received in revised form 4 July 2012 Accepted 14 July 2012 Available online 24 July 2012 Keywords: Sox2 Epithelium stem cell Apical bud Mouse odontogenesis
a b s t r a c t The transcription factor Sox2 plays important roles in maintaining the pluripotency of embryonic stem cells and adult progenitors. However, whether Sox2 is involved in odontogenesis has not been reported. In this study, we examined the expression pattern of Sox2 during mouse incisor and molar development using real-time PCR, in situ hybridization and immunohistochemistry. Sox2 mRNA was expressed in the dental epithelium and mesenchyme while Sox2 protein was mainly detected in the epithelium from embryonic day (E) 11.5 to postnatal (PN) day 20. In the case of incisor, Sox2 mRNA and protein were expressed in most of dental epithelial cells from E11.5 to E14.5, and they were both highly expressed in the labial cervical loop area from E16.5 to PN20. During molar development, we observed an asymmetrical distribution of Sox2 protein in the epithelium from E13.5 to E16.5, with stronger signals in the lingual side. From E18.5 to PN2, Sox2 was expressed within the cervical loop area, and the stellate intermediate layer. From PN6 to PN14, Sox2 expression was confined mainly to the apical end of hertwig’s epithelium root sheath (HERS) cells. Sox2 was also detected within the perivascular region of the dental pulp at PN14 and PN20. Our results suggested that: (1) Sox2 was involved in mouse odontogenesis, and (2) it might participate in maintaining the pluripotency of the epithelial stem cells of labial cervical loop in mouse incisor development and the epithelium progenitors during molar development, (3) Sox2 might be regulated at post-transcription level during mouse odontogenesis. Ó 2012 Elsevier B.V. All rights reserved.
Odontogenesis is regulated by the complex interactions between an ectoderm, the dental epithelium and the cranial neural crest-derived mesenchyme (Slavkin et al., 1984; Thesleff et al., 1995; Vainio and Thesleff, 1992). Tooth morphogenesis goes through initiation stage, bud stage, cap stage and bell stage, and this process exhibits many morphological and molecular similarities with other developing epithelial appendages, such as hair follicles, salivary glands, lungs, mammary glands and kidneys (Chuong and Noveen, 1999; Iseki et al., 1996; Pispa and Thesleff, 2003; Thesleff et al., 1995). Sox2 belongs to the family of SRY (sex-determining region on the Y chromosome)-related HMG (high-mobility-group) transcription factors which are important for cell fate and differentiation in multiple developmental and physiological processes (Kamachi et al., 2000; Lefebvre et al., 2007). During embryogenesis, Sox2 mRNA is initially expressed in morula cells at embryonic day (E) 2.5, then specifically within the inner cell mass (ICM) at E3.5 (Avilion et al., 2003). Embryos deficient for Sox2 lack a pluripotent ICM and fail to survive shortly after implantation (Avilion et al., 2003; Masui et al., 2007). Sox2 is also widely expressed in adult tis-
⇑ Corresponding author. Tel.: +86 27 87686198; fax: +86 27 87873849. 1
E-mail address: [email protected] (Z. Chen). Both authors contributed equally to this paper.
1567-133X/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gep.2012.07.001
sues, such as the progenitors in brain (Brazel et al., 2005; Ellis et al., 2004), tongue epithelium (Okubo et al., 2009), neural retina (Taranova et al., 2006), dermal papilla of the hair follicle (Driskell et al., 2009), glandular stomach, cervix and tests (Arnold et al., 2011). Recently, it has been reported that forced expression of Sox2, together with Oct-3/4, c-myc and klf4, can endow adult mouse fibroblasts with pluripotency, giving rise to induced pluripotent stem cells (Takahashi and Yamanaka, 2006). However, whether Sox2 is involved in tooth development is unknown. In this study, we for the first time examined detailed Sox2 expression profile during mouse incisor and molar development by real-time PCR, in situ hybridization and immunohistochemistry.
1. Results 1.1. Sox2 mRNA expressed in mouse tooth germs To examine a possible involvement of Sox2 during tooth development, Sox2 mRNA expression was first confirmed in the tissues harvested from the whole incisor or molar germs (Fig. 1A, cartoon depiction of the mouse molar (M1) and incisor (dashed line) at different stages from embryonic day (E) 11.5 to postnatal day (PN) 20 by real-time PCR. The result showed that Sox2 was expressed at all these stages investigated (Fig. 1B and C). The expression level of
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Fig. 1. Real-time PCR analysis of Sox2 mRNA expression in mouse incisors and molars. (A) Cartoon depiction of the mouse molar (M1) and incisor (dashed line) Sox2 transcript was detected from the tissues taken from the whole mandibular mouse incisors from E11.5 to PN20. (B) Sox2 transcript was expressed in the tissues taken from first mandibular molars (M1) from E11.5 to PN20. E, embryonic day and PN, postnatal day. All error bars represent mean ± SD.
Sox2 mRNA during incisor development was gradually down-regulated with age (E11.5 to PN20), and maintained in a certain moderate level at postnatal stages (PN2 to PN20) (Fig. 1B). During molar development, the expression level of Sox2 mRNA was upregulated from E11.5 to E13.5, and then down-regulated from E13.5 to E18.5, and after postnatal life (PN2 to PN20), Sox2 mRNA expression was reduced to a much lower level compared with embryonic stages (Fig. 1C).
1.2. Expression pattern of Sox2 mRNA during mouse incisor development To further characterize in situ expression of Sox2 transcript, we examined the expression of Sox2 mRNA during mouse incisor development using in situ hybridization from E11.5 to PN20. At the lamina stage (E11.5), the late bud stage (E13.5) and the cap stage (E14.5), Sox2 mRNA was broadly detected both in the dental epithelium and mesenchyme (Fig. 2A–E). And the signal in the epithelium was slightly stronger than that in the dental mesenchyme. Sox2 mRNA was highly expressed within the labial cervical loop area of dental epithelium at E14.5 (Fig. 2D and E). From the early bell stage (E16.5) to PN20, the signal of Sox2 mRNA was much stronger in the dental epithelium than that in the dental mesenchyme. The labial side of the dental epithelium, especially the labial cervical loop area (laCL), including the basal epithelium (BE), the stellate reticulum (SR) and the outer enamel epithelium (OEE) showed strong Sox2 mRNA signal (Fig. 2G, H, J, K, M, N, Q and R). The transit-amplifying (TA) cells of the inner enamel epithelium adjacent to the apical bud and the stellate intermediate (SI) also exhibited expression of Sox2 mRNA (Fig. 2G, H, J, K, M, N, P, Q and R). In addition, we found Sox2 mRNA in the epithelium cells of lingual cervical loop (liCL) (Fig. 2G, I, J, L, M and O), as well as in the pre-ameloblasts and ameloblasts (Fig. 2P and U, red arrows). We also observed Sox2 mRNA expression in the odontoblasts (Fig. 2T and U, black arrows) and the cells in the perivascular region (Fig. 2S, red asterisks). The eye, brain and dermal papillae of hair follicle were used as positive control (Fig. S1A–C). Strong signals of Sox2 mRNA were observed in these tissues. Sense probe was used as negative control (Fig. 2F) and no positive signal was observed.
1.3. Expression pattern of Sox2 mRNA during mouse molar development In situ hybridization was performed to further investigate the in situ expression of Sox2 mRNA during the mouse molar development from E11.5 to PN20. At the lamina stage (E11.5), Sox2 mRNA was expressed both in the epithelium (Fig. 3A, black arrow) including the dental lamina (Fig. 3A, red arrow), and in the underlying mesenchyme. At the bud stage (E13.5) and the cap stage (E14.5), the expression of Sox2 in the dental epithelium of the enamel organ was stronger than that in the underlying mesenchyme. At the early bell stage (E16.5), the inner enamel epithelium showed strong signal of Sox2 mRNA (Fig. 3D and E). At the late bell stage (E18.5) and PN2, Sox2 was primarily localized in the inner enamel epithelium, especially in the cervical loop area, the stellate intermediate (SI) layer and the stellate reticulum that adjacent to the SI layer (Figs. 3F–H; 4A–C). From PN6 to PN14, the cells of the hertwig’s epithelium root sheath (HERS) showed positive expression of Sox2 (Fig. 4D, F and G, blue arrows). Sox2 mRNA was also detected in the cells around blood vessels marked by CD31 within the dental pulp at PN14 and PN20 (Fig. 4G–J, red asterisks). In addition, we also found Sox2 mRNA signal in the odontoblasts and ameloblasts (Fig. 4B and E, black and red asterisks, respectively). Sense probe was used as negative control (Fig. 3I) and no positive signal was observed.
1.4. Localization of Sox2 protein during mouse incisor development Based on the expression pattern of Sox2 mRNA, immunohistochemistry was performed to further elucidate the translation expression of Sox2 during mouse incisor development. Interestingly, Sox2 protein was mainly detected in the dental epithelium. At the lamina stage (E11.5), the late bud stage (E13.5) and the cap stage (E14.5), Sox2 protein was highly observed in most of the cells within the dental epithelium (Fig. 5A–C, red arrows), while the protein signal was only observed in a limited number of mesenchymal cells adjacent to the epithelium (Fig. 5A–E, red arrowheads). At E14.5, Sox2 protein was strongly expressed within the labial cervical loop area of dental epithelium (Fig. 5D and E,
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Fig. 2. Expression of Sox2 mRNA during mouse incisor development from E11.5 to PN20. The samples are sagittal sections. At E11.5, E13.5 and E14.5, Sox2 mRNA was broadly detected both in the dental epithelium and mesenchyme (A–E). The expression of Sox2 in the epithelium was slightly stronger than that in the mesenchyme. At E14.5, Sox2 signal was strongly expressed within the labial cervical loop area of dental epithelium (D and E). (F) Sense probe was used as negative control and no positive signal was observed. (G–U) From E16.5 to PN20, Sox2 mRNA signal was much stronger in the dental epithelium than in the dental mesenchyme. The labial side of the dental epithelium, especially the labial cervical loop area (laCL), including the basal epithelium (BE), the stellate reticulum (SR) and the outer enamel epithelium (OEE) (G, H, J, K, M, N, P–R) showed strong Sox2 mRNA expression. The transit-amplifying (TA) cells and the stellate intermediate (SI) also expressed Sox2 mRNA (G, H, J, K, M, N, P–R). In addition, Sox2 mRNA was found in the epithelium cells of lingual cervical loop (liCL) (G, I, J, L, M, O), as well as in the pre-ameloblasts and ameloblasts (P and U, red arrows). Sox2 mRNA expression was also expressed in the odontoblasts (T and U, black arrows) and the cells in the perivascular region (S, red asterisks). (C, E, H, I, K, L, N, O) Higher-magnification images of black and red boxes in panels B, D, G, J, M, respectively. Black and red dashed line outlined the dental epithelium and blood vessel, respectively. de, Dental epithelium; dm, dental mesenchyme; cm, condensed mesenchyme; vl, vestibular lamina; liCL, lingual cervical loop; laCL, labial cervical loop; BE, basal epithelium; OEE, outer dental epithelium; SR, stellate reticulum; SI, stratum intermedium and TA, transit-amplifying cells. Od, odontoblast; Am, ameloblast; pre-Am, pre-ameloblast; dp, dental pulp and BV, blood vessel. Scale bars: A, C, E, H, I, K, L, N–U: 50 lm; B and D: 100 lm; F, G, J, M: 200 lm.
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Fig. 3. Expression of Sox2 mRNA during mouse molar development from E11.5 to E18.5. The samples are frontal sections from E11.5 to E16.5, and sagittal sections at E18.5. At E11.5, Sox2 mRNA was expressed both in the epithelium (A, black arrow) including the dental lamina (A, red arrow) and in the underlying mesenchyme (dm). At E13.5 and E14.5, the expression of Sox2 in the dental epithelium was stronger than that in the underlying mesenchyme (B and C). At E16.5, the inner enamel epithelium showed strong signal of Sox2 (D–F). At E18.5, Sox2 was primarily localized in the stellate intermediate (SI) layer, the stellate reticulum adjacent to SI layer, and the inner enamel epithelium, especially in the cervical loop area (G and H). (I) Sense probe was used as negative control (F) and no positive signal was observed. Dashed line outlined the dental epithelium. (E, G and H) Higher-magnification images in panel D and F, respectively. oe, oral epithelium; de, dental epithelium; dm, dental mesenchyme; cm, condensed mesenchyme; dp, dental papilla; pek, primary enamel knot; T, tongue; CL, cervical loop; IEE, inner enamel epithelium; SR: stellate reticulum and SI: stratum intermedium. Scale bars: A, B, C, E, G, H: 50 lm; D and I: 100 lm; F: 200 lm.
blue arrow).The vestibular lamina also showed strong signal of Sox2 (Fig. 5D and F, red arrow). At the early bell stage (E16.5), the expression of Sox2 was restricted to the labial side of the dental epithelium, especially the labial cervical loop area, including the basal epithelium (BE), the stellate reticulum (SR) and the outer enamel epithelium (OEE) (Fig. 5G and I). The transit-amplifying (TA) cells of the inner enamel epithelium adjacent to the apical bud and the stellate intermediate (SI) also exhibited expression of Sox2 (Fig. 5G–I, red arrows and blue arrow, respectively). From the middle bell stage (E18.5) to PN20, the expression of Sox2 was primarily and obviously maintained in the cells within the labial cervical loop (Fig. 5J–U), the TA cells adjacent to the labial cervical loop (Fig. 5J–U, red arrows) and the SI layer (Fig. 5G, H, J, K, M, N, P, Q, S and T, blue arrows). Some mesenchymal cells adjacent to the labial cervical loop were also found to express weak Sox2 protein signal (Fig. 5G, I, J, L, M, O, P, R, S and U, red arrowheads).
1.5. Localization of Sox2 protein during mouse molar development At the lamina stage (E11.5), Sox2 was highly expressed in the epithelium (Fig. 6A, black arrow) including the dental lamina (Fig. 6A, red arrow). At the bud stage (E13.5), Sox2 protein strongly expressed in the lingual side of the dental epithelium, only weak signal of Sox2 was found in the buccal side of the dental epithelium (Fig. 6B). From the cap stage (E14.5), Sox2 started to be expressed in the stellate reticulum (Fig. 6C). Similar to E13.5, asymmetric distribution of Sox2 signal was found, with stronger staining in the lingual side than the buccal side of the tooth germ (Fig. 6C). No staining was found in the cells of the primary enamel knot. We also
found some small number of mesenchymal cells adjacent to the epithelium expressed Sox2 protein at E11.5, E13.5 and E14.5 (Fig. 6A–E, red arrowheads). At the early bell stage (E16.5), preferential expression of Sox2 was also observed in the lingual side of the outer enamel epithelium, the cervical loop, and the inner enamel epithelium between secondary enamel knots (Fig. 6D–F). At the late bell stage (E18.5), Sox2 was primarily localized in the inner enamel epithelium, especially the cervical loop area, the stellate intermediate (SI) layer and the stellate reticulum adjacent to SI layer (Fig. 6G–I). At PN2, Sox2 was localized in the stellate intermediate layer (Fig. 7A–C, black arrows), and the stellate reticulum adjacent to the SI layer (Fig. 7A–C, red arrows). Besides, a few cells within the cervical loop were stained by Sox2 (Fig. 7C, blue arrow). At PN6, the cells of the hertwig’s epithelium root sheath (HERS) showed nuclei expression of Sox2 (Fig. 7D–F, blue arrows). At PN14, Sox2 expression was maintained in the cells within the apical end of HERS (Fig. 7F and G, blue arrows). Sox2 signal was detected in the cells at the perivascular region of the dental pulp at PN14 and PN20 (Fig. 7G–L, red asterisks).We could sometimes found Sox2 positive cells within the perivascular region as early as at E18.5 (data not shown). The blood vessels were marked by CD31 within the dental pulp (Fig. 7I and L). We could never found the expression of Sox2 protein in odontoblasts or ameloblasts.
2. Discussion In our present study, we clearly showed the spatial and temporal expression pattern of Sox2 mRNA and protein during mouse incisor and molar development. Sox2 mRNA was expressed at both
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Fig. 4. Expression of Sox2 mRNA during mouse molar development from PN2 to PN20. At PN2, Sox2 mRNA was primarily localized in the stellate intermediate (SI) layer, the stellate reticulum adjacent to SI layer and the inner enamel epithelium, especially in the cervical loop area (A–C, black, red and blue arrows, respectively). From PN6 to PN14, the cells of the hertwig’s epithelium root sheath (HERS) showed positive expression of Sox2 (D, F and G, blue arrows). Sox2 mRNA was also detected in the cells around blood vessels (G–J, red asterisks) marked by CD31 (H and J) within the dental pulp at PN14 and PN20. In addition, Sox2 mRNA signal was observed in the odontoblasts and ameloblasts (B, D and E, black and red asterisks, respectively). Dashed line outlined the structure of HERS. (B, C, E and F) Higher-magnification images of boxes in panel A and D, respectively. SR, stellate reticulum; SI, stratum intermedium; Am, ameloblasts; Od, odontoblasts; HERS, Hertwig’s epithelium root sheath; PN, postnatal day. Scale bars: A and D: 200 lm; B, C, F–J: 50 lm.
dental epithelium and mesenchyme, while Sox2 protein was mainly expressed in the dental epithelial cells. Both Sox2 mRNA and protein were highly expressed in the labial cervical loop area of the mouse incisor and the dental epithelium during molar development. Sox2 has been reported to play important roles in maintaining the pluripotency of embryonic stem cells and adult progenitors (Arnold et al., 2011; Avilion et al., 2003; Masui et al., 2007). Mouse incisors are continuously growing teeth throughout life. The labial cervical loop area is believed to contain stem cells that are responsible for the continuous growth (Harada et al., 1999; Harada and Ohshima, 2004; Ohshima et al., 2005). Some stem cell related markers, such as Oct-3/4, Bmi-1, and Yap have been reported to be localized in the apical bud of developing mouse incisor (Li et al., 2011). And it is also well known that Sox2 associates with Oct-3/4 to maintain self-renewal of ES cells (Masui et al., 2007; Okumura-Nakanishi et al., 2005; Rodda et al., 2005). Combined with our results, the persistent expression of Sox2 within the apical bud of mouse incisor (Figs. 2H, K, N, Q, R and 5G–U) suggested that Sox2 might be responsible for maintaining the stem cell niche in this specific epithelium structure, either by itself or by interactive cooperation with other stem cell molecules, such as Oct-3/4, Yap and Nanog (Gee et al., 2011; Masui et al., 2007; Rodda et al., 2005). Compared with the continuously growing incisor, mouse molar has limited growth ability. Our results suggested Sox2 was also involved in molar development. The asymmetric expression of Sox2 protein in molar germs from E13.5 to E16.5 in a lingual-to-buccal gradient pattern (Fig. 6B–F) indicated that there might be differential regulation mechanisms between the lingual and buccal side of dental enamel organ during mouse molar morphogenesis.
The role of HERS cells in root formation is widely accepted, the precise function of these cells remains unclear (Thomas, 1995; Tummers and Thesleff, 2003). Our result showed that Sox2 was expressed in the apical end of HERS cells (Figs. 4F, G and 7E, F, H). A recent study has also shown the expression profile of stem cell markers in human Hertwig’s epithelial root sheath/Epithelial rests of Malassez cells, such as Bmi-1, Oct-3/4, Nanog, and SSEA-4 (Nam et al., 2011). Taken together, the Sox2 positive cells in the HERS apical end of our results indicated Sox2 might be involved in maintaining the stem cell characteristics of HERS cells during root formation. We also found Sox2 positive cells around some blood vessels (Figs. 2S, 4G–J and 7H–L and data not shown at other stages). Previous study has demonstrated that stem cells reside in the perivascular niche of dental pulp (Shi and Gronthos, 2003). Therefore, these Sox2 positive cells observed in the dental pulp might be some stem cells/progenitors within the perivascular region. In addition, we observed some differences in the expression patterns between Sox2 mRNA and Sox2 protein. The Sox2 mRNA exhibited wider expression than that of Sox2 protein: (1) Sox2 mRNA could be broadly detected in the dental mesenchymal cells, while little Sox2 protein was examined in limited number of cells; (2) Sox2 mRNA could be observed in differentiated odontoblasts and ameloblasts, while Sox2 protein was not detected; (3) Sox2 mRNA was detected in the lingual cervical loop, while Sox2 protein was not. The expression of Sox2 protein was only restricted to some specific stem cell like or progenitor cell lineages. To ensure the in situ hybridization result of Sox2, two probes targeting different region of the transcript of Sox2 were developed (refer to Section 3.3.1. Probe constructs). Same results were achieved using the two probes (results of probe 2 not shown). Moreover, we ob-
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Fig. 5. Localization of Sox2 protein during mouse incisor development from E11.5 to PN20. (A) At E11.5, Sox2 was expressed in the oral epithelium (black arrow) and in dental epithelium (red arrow). (B and C) At E13.5, Sox2 was evidently localized in the dental epithelium and vestibular lamina. (D–F) At E14.5, Sox2 was stained in the dental epithelium with abundant signal in the labial cervical loop area (C and E, blue arrow), and the vestibular lamina was also stained (C and F, red arrow). A limited number of mesenchymal cells adjacent to the epithelium were also found to express Sox2 protein (A–E, red arrowheads). (G–U) Sagittal sections from E16.5 to PN20 showed that Sox2 signal was evidently and specifically restricted in the labial cervical loop area, including the basal epithelium, stellate reticulum, and outer enamel epithelium. The transitamplifying (TA) cells (red arrows) adjacent to the apical bud and the stratum intermedium (blue arrows) layer also exhibited expression of Sox2 protein. Sox2 protein signal was also found in some mesenchymal cells adjacent to the labial cervical loop (A, C, D, F, G, I, J, L, M, O, red arrowheads). (C, E, F, H, I, K, L, N, O, Q, R, T, U) Higher-magnification images of black, red and blue boxes in panels B, D, G, J, M, P, S, respectively. oe, Oral epithelium; de, dental epithelium; cm, condensed mesenchyme; vl, vestibular lamina; laCL, labial cervical loop; BE, basal epithelium; OEE, outer dental epithelium; SR, stellate reticulum; SI, stratum intermedium and TA, transit-amplifying cells. Scale bars: A, C, E, F, H, I, K, L, N, O, Q, R, T, U: 50 lm; B and D: 100 lm; G, J, M, P, S: 200 lm.
served similar cases in the dermal papillae of hair follicle. Sox2 mRNA could be detected in most part of the dermal papillae, while the expression of Sox2 protein was confined to a limited subset of cells localized in the tip of the dermal papillae (Fig. S1C and F). Similarly, the germ stem cells have been reported to express little Sox2 protein in spite of the high RNA expression levels (Arnold et al., 2011; Imamura et al., 2006).
An open question is what mechanisms lead to Sox2 mRNA and protein expression discrepancy. A potential explanation for the expression discrepancies observed between Sox2 mRNA and protein could be ascribed to the post-transcriptional regulation. MicroRNAs are endogenously expressed non-coding RNA molecules that affect protein synthesis by post-transcriptional mechanisms (Bartel, 2004; Chekulaeva and Filipowicz, 2009; Fabian et al., 2010). Sox2
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Fig. 6. Localization of Sox2 protein during mouse molar development from E11.5 to E18.5. (A) At E11.5, Sox2 was expressed in the oral epithelium (black arrow) and dental epithelium (red arrow). (B) At E13.5, Strong expression of Sox2 was observed in the lingual side of the dental epithelium; only weak signal of Sox2 was found in the buccal side of the dental epithelium. (C) At E14.5, Sox2 started to be expressed in the stellate reticulum. Similar to E13.5, asymmetric distribution of Sox2 signal was found, with stronger staining in the lingual side of the tooth germ. No staining was found in the primary enamel knot. (D–F) At E16.5, preferential expression of Sox2 was also observed in the lingual side of the outer enamel epithelium, the cervical loop, and the inner enamel epithelium between secondary enamel knots. (G–I) At E18.5, Sox2 signal was maintained in the stratum intermedium (SI), the stellate reticulum adjacent to SI layer, and the inner enamel epithelium, especially the cervical loop area. (E, F, H, I) Highermagnification images of black and red boxes in panel D and G, respectively. oe, oral epithelium; de, dental epithelium; dm, dental mesenchyme, cm, condensed mesenchyme; dp, dental papilla; pek, primary enamel knot; T, tongue; CL, cervical loop; IEE, inner enamel epithelium; SR, stellate reticulum; SI: stratum intermedium and sek, secondary enamel knot. Scale bars: A, B, C, E, F, H, I: 50 lm; D: 100 lm; G: 200 lm.
are reported to be regulated by MicroRNAs (Marson et al., 2008; Tay et al., 2008; Xu et al., 2009). Recently, microRNAs are also found in the stem cell niche of the adult mouse incisor (Cao et al., 2010; Jheon et al., 2011). Further study needs to elucidate how Sox2 was regulated at post-transcription level during tooth development. In conclusion, the prominent and persistent Sox2 mRNA and protein expression profile in the labial apical bud of the continuously growing incisor and in the dental epithelium during mouse molar formation indicated that (1) Sox2 was involved in mouse odontogenesis, (2) Sox2 might function in maintaining the specific epithelial stem cell niche of mouse incisor and in maintaining the epithelium progenitors during molar development, (3) there might be some other unknown mechanisms to regulate the Sox2 mRNA and protein expression in a more accurate manner. However, the exact function of Sox2 in tooth development is still unknown. Therefore, our future study is to explore the function of Sox2 in tooth development by using specific conditional knockout mouse targeting Sox2 in tooth structure. 3. Experimental procedures All experiments were performed according to the guideline of Animal Welfare Committee of the School and Hospital of Stomatology at Wuhan University. 3.1. Animals The adult ICR mice were mated overnight. E0.5 was designated as the day on which the presence of a vaginal plug was confirmed.
At least three embryos and postnatal mice at each developmental stage (E11.5, E13.5, E14.5, E16.5, E18.5, PN2, PN6, PN10, PN14 and PN20) were used in this study.
3.2. Real-time PCR Total RNA was isolated from the whole tooth germs (E11.5 to PN20, as shown in Fig. 1) or from the tissue of apical bud and anterior part of mouse incisor separately (PN2 to PN20, as shown in Fig. S2) using Trizol reagent (Invitrogen) according to the manufacturer’s protocol. A standard reverse transcript reaction (RT) was used to synthesize cDNA using RevertAid™ M-MuLV Reverse Transcriptase (Fermentas, EU). And 1ug of total RNA was used for RT reactions. Real-time PCR was performed with SYBR Premix Ex TaqTM (Takara Bio Inc., Shiga, Japan) according to manufacturer’s instructions. Signals were detected in the ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA). The cycle thresholds were calculated for further statistical analysis. As a control, the level of GAPDH mRNA was determined in the real-time PCR assay of each RNA sample and was used to correct for experimental variation. Primers are as follows: for Sox2 amplification, (forward) 50 -GTTCTAGTGGTACGTTAGGCGCTTC-30 , (reverse) 50 -TCGCCCGGAG TCTAGCTCTAAATA-30 ; for GAPDH control amplification, (forward) 50 -TGTGTCCGTCGTGGATCTGA-30 and (reverse) 50 -TTGCTGTTGAA GTCGCAGGAG-30 .Quantification of the relative expression levels of the Sox2 was achieved by normalizing for the endogenous GAPDH using the 44CT method. The gene expression ratio was shown as mean ± standard deviation from three independent experiments.
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Fig. 7. Localization of Sox2 protein during mouse molar development from PN2 to PN20. (A–C) At PN2, Sox2 was restricted in the stratum intermedium (SI) (black arrows), stellate reticulum (red arrows) adjacent to SI layer, and some inner enamel epithelium cells (blue arrow) within the cervical loop area. (D–H) At PN6 and PN14, Sox2 protein was detected in the apical end of Hertwig’s epithelium root sheath (HERS) (blue arrows). Sox2 signal was also detected in the cells at the perivascular region of the dental pulp at PN14 and PN20 (G–L, red asterisks). The blood vessels were marked by CD31 within the dental pulp (I, L). (B, C, E, F, H, K) Higher-magnification images of red and black boxes in panel A, D, G and J, respectively. SR: stellate reticulum; SI, stratum intermedium; HERS, Hertwig’s epithelium root sheath; BV, blood vessel. Scale bars: A, D, G: 200 lm; B, C, E, F, H, I, K, L: 50 lm; J: 100 lm.
3.3. In situ hybridization 3.3.1. Probe constructs Total RNA was extracted from the brain of ICR mouse embryo using TRIZol Reagent (Invitrogen, USA) following the manufacture’s instruction. RNA sample was then subjected to SuperScriptÒ III First-Strand Synthesis System (Invitrogen, USA) to generated Sox2 cDNA fragment. Two probes targeting different region of the transcript of Sox2 were developed. The 415-bp product for sox2 (Probe 1) was amplified using the primers (forward: 50 -CGCAAGCTTAAACC GTGATGCCGACTA-30 , reverse: 50 -TCTGGATCCATCCGAATAAACTCCT TCCTTG-30 , bases underlined indicate the restriction enzyme sites); the 515-bp product for sox2 (Probe 2) was amplified using the primers (forward: 50 -CGCAAGCTTAACGCCTTCATGGTATGGTC-30 , reverse: 50 -TCTGGATCCATGTAGGTCTGCGAGCTGGT-30 , bases underlined indicate the restriction enzyme sites). The PCR products were confirmed by DNA sequencing (Invitrogen, CA), and purified using QIAquickÒ Gel Extraction Kit (QIAGEN, Germany) and inserted into pSPT18 (Roche, Germany). The single-strand DNA was generated using restriction enzyme digestion and purified with QIAquickÒ Gel Extraction Kit (QIAGEN, Germany). Sense and antisense digoxigenin-labeled RNA probes were generated using DIG RNA Labeling Kit (SP6/T7) according to the manufacture’s instruction
(Roche, Germany). The yield of DIG-labeling was estimated in a spot test with DIG Nucleic Acid Detection Kit (Roche). 3.3.2. Section in situ hybridization For in situ hybridization, mandibles of each stage were fixed with 4% paraformaldehyde (PFA) in 0.01 M phosphate-buffered saline (PBS, pH 7.4) overnight at 4 °C and demineralized with 10% ethylenediamine tetra-acetic acid (pH 7.4) for two days to one month depending on stages at 4 °C. After having been embedded in paraffin, these samples were sectioned at a thickness of 7 lm. In situ hybridization on wax sections was performed as previously described (Hosoya et al., 2008). Sections were baked at 60 °C, de-waxed in xylene, rehydrated through a graded series of alcohol and post-fixed in 4% PFA. Sections were pre-hybridized in a humid chamber containing 50% formamide in 2xSSC, at 55 °C, for 30 min. Digoxigenin (DIG) labeled RNA probes were prewarmed at 85 °C and hybridized to sections overnight at 64 °C. Sense probes were used as negative control (Figs. 2F and 3I). 3.4. Immunohistochemistry For immunohistochemistry, specimens of each stage were sectioned at a thickness of 5 lm. Sections were de-waxed and re-hy-
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drated, incubated in 3% hydrogen peroxide for 15 min, and then were boiled in 10 mM citrate buffer (pH 6.0) for 5 min (121 °C) followed by cooling down at room temperature for 20 min. Before incubation with primary antibody, 2.5% BSA (Roche) was used for blocking. The slides were incubated with antibodies against Sox2 (1:250, rabbit monoclonal antibody, 2683-1, Epitomics), and CD31 (1:100, goat polyclonal antibody, AF3628, R&D, USA) respectively, at 4 °C overnight. After washed with PBS, the specimens were made to react with polymer Helper (Zhong Shan Golden Bridge Biotechnology) and poly-HRP-anti-Rabbit IgG, poly-HRPanti-Goat IgG (Zhong Shan Golden Bridge Biotechnology) respectively at room temperature for 15 min each. Finally, the specimens were visualized using a diaminobenzidine (DAB) reagent kit (Maixin. Bio). Then the immunostained sections were counterstained with hematoxylin. Acknowledgements This study was supported by Grants from the Natural Science Foundation of China (NSFC) (No. 30872880) and National 973 project of China (No. 2010CB534915). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.gep.2012.07.001. References Arnold, K., Sarkar, A., Yram, M.A., Polo, J.M., Bronson, R., Sengupta, S., Seandel, M., Geijsen, N., Hochedlinger, K., 2011. Sox2(+) adult stem and progenitor cells are important for tissue regeneration and survival of mice. Cell Stem Cell 9, 317– 329. Avilion, A.A., Nicolis, S.K., Pevny, L.H., Perez, L., Vivian, N., Lovell-Badge, R., 2003. Multipotent cell lineages in early mouse development depend on SOX2 function. Genes Dev 17, 126–140. Bartel, D.P., 2004. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281–297. Brazel, C.Y., Limke, T.L., Osborne, J.K., Miura, T., Cai, J., Pevny, L., Rao, M.S., 2005. Sox2 expression defines a heterogeneous population of neurosphere-forming cells in the adult murine brain. Aging Cell 4, 197–207. Cao, H., Wang, J., Li, X., Florez, S., Huang, Z., Venugopalan, S.R., Elangovan, S., Skobe, Z., Margolis, H.C., Martin, J.F., Amendt, B.A., 2010. MicroRNAs play a critical role in tooth development. J Dent Res 89, 779–784. Chekulaeva, M., Filipowicz, W., 2009. Mechanisms of miRNA-mediated posttranscriptional regulation in animal cells. Curr Opin Cell Biol 21, 452–460. Chuong, C.M., Noveen, A., 1999. Phenotypic determination of epithelial appendages: genes, developmental pathways, and evolution. J Investig Dermatol Symp Proc 4, 307–311. Driskell, R.R., Giangreco, A., Jensen, K.B., Mulder, K.W., Watt, F.M., 2009. Sox2positive dermal papilla cells specify hair follicle type in mammalian epidermis. Development 136, 2815–2823. Ellis, P., Fagan, B.M., Magness, S.T., Hutton, S., Taranova, O., Hayashi, S., McMahon, A., Rao, M., Pevny, L., 2004. SOX2, a persistent marker for multipotential neural stem cells derived from embryonic stem cells, the embryo or the adult. Dev Neurosci 26, 148–165. Fabian, M.R., Sonenberg, N., Filipowicz, W., 2010. Regulation of mRNA translation and stability by microRNAs. Annu Rev Biochem 79, 351–379. Gee, S.T., Milgram, S.L., Kramer, K.L., Conlon, F.L., Moody, S.A., 2011. Yes-associated protein 65 (YAP) expands neural progenitors and regulates Pax3 expression in the neural plate border zone. PLoS One 6, e20309. Harada, H., Ohshima, H., 2004. New perspectives on tooth development and the dental stem cell niche. Arch Histol Cytol 67, 1–11.
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