
==== Front
iScience
iScience
iScience
2589-0042
Elsevier

S2589-0042(24)02010-8
10.1016/j.isci.2024.110785
110785
Article
Follistatin controls the number of murine teeth by limiting TGF-β signaling
Zhu Shicheng 13
Huo Suman 13
Wang Zhongzheng 13
Huang Caiyan 1
Li Chuanxu 1
Song Hanjing 1
Yang Xueqin 1
He Rui 2
Ding Cheng 2
Qiu Mengsheng 1
Zhu Xiao-Jing xiao_jingzhu@hznu.edu.cn
14∗
1 College of Life and Environmental Sciences, Zhejiang Key Laboratory of Organ Development and Regeneration, Hangzhou Normal University, Hangzhou, Zhejiang 311121, China
2 The Affiliated Hospital, Hangzhou Normal University, Hangzhou, Zhejiang 310015, China
∗ Corresponding author xiao_jingzhu@hznu.edu.cn
3 These authors contributed equally

4 Lead contact

22 8 2024
20 9 2024
22 8 2024
27 9 11078524 4 2024
24 6 2024
19 8 2024
© 2024 The Author(s)
2024
https://creativecommons.org/licenses/by-nc/4.0/ This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-nc/4.0/).
Summary

Supernumerary teeth are common developmental anomalies of dentition. However, the factors and mechanisms driving their formation remain largely unknown. Here, we report that conditional knockout of Fst, encoding an antagonist for the transforming growth factor β (TGF-β) signaling pathway, in both oral epithelium and mesenchyme of mice (FstCKO) led to supernumerary upper incisor teeth, arising from the lingual dental epithelium of the native teeth and preceded by an enlarged and split lingual cervical loop. Fst-deficiency greatly activated TGF-β signaling in developing maxillary incisor teeth, associated with increased epithelium cell proliferation. Moreover, FstCKO teeth exhibited increased expression of Tbx1, Sp6, and Sox2, which were identified as direct targets of TGF-β/SMAD2 signaling. Finally, we show that upregulation of Tbx1 in response to Fst-deficiency was largely responsible for the formation of extra teeth in FstCKO mice. Taken together, our investigation indicates a novel role for Fst in controlling murine tooth number by restricting TGF-β signaling.

Graphical abstract

Highlights

• Conditional knockout of Fst leads to supernumerary upper incisor teeth in mice

• Fst-deficiency activates TGF-β signaling and leads to increased cell proliferation

• Tbx1, Sp6, and Sox2 are identified as direct targets of TGF-β/SMAD2 signaling

• Upregulation of Tbx1 contributes to the formation of extra teeth in FstCKO

Biological sciences; Genetics; Animal Physiology

Subject areas

Biological sciences
Genetics
Animal Physiology
Published: August 22, 2024
==== Body
pmcIntroduction

Supernumerary teeth, defined as teeth that exceed the normal dental formula, are common dental anomalies in humans. Supernumerary teeth can manifest as a single tooth or multiple teeth, in a unilateral or bilateral fashion, and in any region of the dentition.1,2 The most frequent site for an extra tooth is the anterior maxillary region. Supernumerary teeth can occur sporadically or are associated with syndromic diseases, such as amelogenesis imperfecta, Bloch-Sulzberger syndrome, craniosynostosis, cleidocranial dysplasia, and familial adenomatous polyposis.1,2 The etiologies of tooth number abnormality are associated with the mechanisms that regulate tooth development, which have been extensively investigated using mouse models.2 However, due to the variety and complexity of supernumerary tooth formation, more studies are required to explore the underlying mechanism.

Mammalian tooth development is tightly regulated by sequential and reciprocal interactions between the oral epithelium and the underlying cranial neural crest-derived mesenchyme. These interactions are mediated by multiple signaling pathways, including the wingless/integrated (WNT), bone morphogenetic protein (BMP), sonic hedgehog (SHH), and fibroblast growth factor (FGF) pathways.3 Genetic disruption of these pathways often leads to dental anomalies, including the formation of supernumerary teeth.2 β-Catenin is the core component of the Wnt signaling pathway. Mesenchymal ablation of β-catenin results in the formation of double incisors in mice,4 whereas constitutive activation of β-catenin or conditional deletion of Apc, an inhibitor of canonical Wnt signaling, in the oral and dental epithelium leads to the formation of supernumerary teeth.5,6 Sostdc1 (ectodin, USAG-1, Wise) is a secreted inhibitor of WNT and BMP pathways. Sostdc1-deficient mice display one extra tooth bud that develops from the lingual dental epithelium of the mandible incisor.7 It has been suggested that enhanced BMP signaling can account for supernumerary tooth formation in Sostdc1 knockout mice.8 Ectopic/expanded epithelial expression of Shh has been found in many mouse models with excess teeth.4,5,7,9 Deletion of Gas1, which encodes an SHH antagonist in diastema mesenchyme, causes ectopic diastema teeth accompanied by increased SHH signaling activity.10 The sprouty genes, which encode intracellular antagonists of FGF signaling, also play an essential role in regulating tooth number in mice.9,11 One plausible explanation for supernumerary tooth formation is the revitalization of replacement teeth.7

Follistatin (FST) is an extracellular glycoprotein that binds to various ligands of the transforming growth factor β (TGF-β) superfamily, such as activins and BMPs, to limit their signaling potential. Fst knockout mice die within hours of birth, displaying developmental defects in the whiskers, teeth and muscles, and skeletal defects of the hard palate and the thirteenth pair of ribs.12 Mouse incisor growth is fine-tuned by a balance between tooth wear and growth, which is underpinned by the renewal and differentiation of stem cells in both epithelial and mesenchymal stem cell niches.13 The epithelial stem cells that give rise to enamel-producing ameloblasts are resident in a niche called the labial cervical loop (laCL) at the proximal end of the incisor. The other epithelial stem cells, which do not generate ameloblasts, are fewer and are located on the lingual side (liCL). In conventional Fst knockout mice, the liCL is enlarged and ameloblasts differentiate ectopically on the lingual surface, while epithelial Fst overexpression leads to severely hypoplastic cervical loops followed by inhibited ameloblast differentiation and loss of the enamel layer.14

To further decipher the regulatory mechanism of FST in tooth development, we created a mouse model (FstCKO) in which Fst is simultaneously knocked out in oral and dental epithelium and in the underlying mesenchyme. Intriguingly, we found that FstCKO mice displayed enamel-free supernumerary maxillary incisors. In addition, Fst is required to limit dental TGF-β signaling activity and to regulate the expression of a variety of genes essential for tooth development. Fst-deficiency also led to increased cell proliferation in upper teeth. Furthermore, we showed that several key genes in incisor development, including Tbx1, Sp6, and Sox2, are probably direct targets of TGF-β signaling. Finally, we demonstrated that upregulation of Tbx1 in response to Fst-deficiency contributed to the generation of extra teeth in FstCKO mice.

Results

Lack of Fst expression results in supernumerary upper incisor formation

Fst null mutant mice die shortly after birth12; therefore, we used conditional knockout mice of Fst to investigate the consequence of Fst-deficiency in postnatal development with a homozygous FstFlox allele.15 Fst is expressed in the oral and dental epithelium and mesenchyme of all the tooth germs since E11.5.14,16,17 To enable conditional inactivation of Fst, we generated homozygous FstFlox mice carrying the K14-Cre transgenic allele or Wnt1-Cre allele to delete Fst in oral and dental epithelium or in the underlying mesenchyme. However, Fst knockout mice driven by either Cre did not display defective tooth development (Figure S1). As FST is a secreted protein, it is possible that FST from the adjacent tissue can compensate for the FST-deficiency in the knockout tissue. Thus, we generated homozygous FstFlox mice carrying both K14-Cre and Wnt1-Cre (FstCKO) to enable deletion of Fst from both epithelium and the underlying mesenchyme of the developing teeth (Figures 1 and 2). Morphological observation revealed that control mice exhibited two maxillary incisors (Figure 1A, the left panels). Remarkably, FstCKO mice displayed supernumerary incisors adjacent to the native upper incisors on the lingual side (Figure 1A, the right panels; Figures S1B and S2C). Three-dimensional reconstruction of micro-computed tomography (CT) images of mouse skull also demonstrated the presence of extra upper incisors in FstCKO mice (Figure 1B). Micro-CT analysis further revealed that the extra incisors in FstCKO were enamel-free (Figure 1C, arrowheads). The native upper incisors of both control and FstCKO mice exhibited normal enamel deposition (Figure 1C, arrows). Among all the postnatal mice observed, the native upper incisors developed normally (n > 10). Lower incisors and molars also developed normally in FstCKO (Figure S3).Figure 1 Fst-deficiency leads to extra maxillary incisors in mice

(A) Control mice display normal upper incisors. In contrast, FstCKO mice exhibit two extra maxillary incisors (arrowheads).

(B) Micro-CT reconstructions of skulls from P36 control and FstCKO mice. Molars are indicated by green arrows. Extra upper incisors in FstCKO mice are indicated by a red arrowhead.

(C) The left and the middle panels are three-dimensional images of micro-CT for upper incisors. The right panels are transverse micro-CT sections. The dashed lines represent the position of the right panel sections in the incisor. Enamel is indicated by arrows. Note the absence of enamel in the extra upper incisors of FstCKO mice (arrowheads). En, enamel; de, dentin; si, supernumerary incisor. Scale bars: 1 mm.

Figure 2 Histological analyses of upper incisor sagittal sections from control and FstCKO mice

(A) HE staining of upper incisor sagittal sections from control and FstCKO mice at E14.5 and E16.5. The liCL is enlarged and splits in FstCKO mice (arrows).

(B) Masson’s trichrome staining showing normal native incisor development in control and FstCKO mice at P10 (the upper panels). Excess incisors are obvious in Fst-deficient mice at P10 (the lower right panel). Cl, cervical loop; ek, enamel knot; la, labial; li, lingual; ni, native incisor; si, supernumerary incisor. Scale bars: (A), 100 μm; (B), 500 μm.

To investigate the timing of the first appearance of excess maxillary incisors, we examined histological sections from control and Fst-deficient mice during tooth development. A clear sign of supernumerary upper incisor formation in FstCKO mice was observed at E14.5 as a lingual enlargement and branching of the native tooth germ, which became more obvious at E16.5 (Figure 2A, arrows). At P10, the native upper incisors displayed normal enamel formation in both control and FstCKO mice (Figure 2B, the upper panels). By contrast, enamel was absent in the extra teeth in FstCKO mice at P10 (Figure 2B, the right lower panel). Postnatal FstCKO mice, of both sexes, developed supernumerary incisor teeth with 100% penetration (n > 10). Additionally, all FstCKO embryos examined show sign of supernumerary upper incisor teeth (n > 30). Taken together, these results showed that loss of Fst leads to generation of enamel-free supernumerary upper incisors in mice.

Fst deficiency activates TGF-β signaling, upregulates SOX2 level, and leads to increased cell proliferation in the maxillary incisor

FST is an endogenous blocker of the TGF-β signaling pathway by neutralizing its ligands, such as activin.18 We, therefore, first investigated the effect of Fst-deficiency on activin signaling in developing upper incisors at E14.5. Immunohistochemical staining of maxillary incisor sections showed that phosphorylated SMAD2 (p-SMAD2) was barely detected in the control mice. By contrast, positive p-SMAD2 immunoreactivity was observed throughout the upper incisor tooth germ, the adjacent mesenchyme, and oral epithelium in FstCKO mice (Figure 3A, the upper panel; Figure 3B), indicating that activin signaling was significantly increased in response to Fst-deficiency.Figure 3 Fst deficiency activates TGF-β signaling, upregulates SOX2 level, and leads to increased cell proliferation in the maxillary incisor

(A) Immunofluorescence staining of sagittal sections showing increased p-SMAD2 and SOX2 levels in Fst-deficient upper incisors at E14.5. The magnified images of the regions within the dotted boxes (a' and a'') are shown to the right.

(B) Comparison of p-SMAD2 intensity in the designated areas of upper incisors in control and FstCKO.

(C) Immunofluorescence staining showing Cyclin D1 in sagittal sections of control and FstCKO upper incisors at E14.5 and E16.5.

(D) Statistical analysis revealing a significant increase in the number of cyclin D1-positive cells in the designated area of FstCKO upper incisors as compared with the control. The dental epithelium in the lingual side of the dashed line is used for analysis. Data are represented as mean ± SEM. ∗∗ Student’s t test, p < 0.01. Scale bars, a' and a'', 50 μm; others, 100 μm.

SOX2 plays essential roles in the maintenance of stem cell pluripotency and is required for dental stem cell proliferation and incisor development in mice.13 SOX2 was expressed in the laCL of both control and FstCKO incisors at E14.5 (Figure 3A, the lower panel, arrowheads). In control incisors, SOX2 immunoreactivity was also found in the lingual dental and oral epithelium. Notably, increased and expanded SOX2 expression was found in the presumptive location of supernumerary teeth in Fst-deficient mice (Figure 3A, the lower panel, arrow), indicating the ectopic generation of epithelium stem cells for extra tooth formation in FstCKO mice.

Next, we investigated whether cell proliferation was altered in developing FstCKO upper incisors. We found that the number of cyclin D1-positive cells was significantly increased in the lingual dental epithelium of upper incisors deficient in Fst at E14.5 and E16.5 (Figures 3C and 3D). These observations demonstrated that Fst suppresses the proliferation of lingual dental epithelial cells in normal upper incisor development.

Fst-deficiency results in aberrant expression of key tooth development genes

To further elucidate the mechanism underlying incisor development mediated by FST, we investigated gene expression in maxillary incisors of FstCKO and control mice at E14.0 by RNA sequencing (RNA-seq). We identified 551 differentially expressed genes (DEGs) in upper incisors between FstCKO and control mice (Table S1). As shown in the volcano plots, the expression of several key genes for tooth development, including Osr1, Bmp3, Sp6, Tbx1, Irx1/2, Fgf15, and Odam, was significantly changed in the mutant (Figure 4A). Among these genes, the expression of Irx1 has been reported to be absent in all tooth germs of activinβA mutant embryos,19 indicating that the TGF-β/activin signaling pathway is crucial for its expression. Therefore, the upregulation of Irx1 in FstCKO teeth suggests that TGF-β/activin signaling activity is increased. Expression of several DEGs was validated by quantitative reverse transcription PCR (qRT-PCR) analysis (Figure 4B). Next, we performed enrichment analysis using Enrichr, a suite of gene set enrichment analysis tools,20 with upregulated DEGs in FstCKO. Analysis against the “TF perturbations followed by expression table” revealed that the upregulated DEGs were mostly enriched with genes that were downregulated in Smad3 knockout tissues (Figure S4). Given that activin signaling acts through SMAD2/3, this result also supports the notion that Fst-deficiency increases TGF-β signaling activity.Figure 4 Differential gene expression in maxillary incisors lacking Fst

(A) Volcano plots of RNA-seq data showing gene expression differences between upper incisors from FstCKO mice and their control littermates at E14.0. Annotated dots indicate representative DEGs.

(B) Validation of RNA-seq data by qRT-PCR.

(C and D) In situ hybridization of sagittal sections showing the expression of Sp6 and Tbx1 in developing upper incisors. Ectopic and increased expression of Sp6 and Tbx1 in FstCKO upper incisors is marked by arrows. Data are represented as mean ± SEM. ∗∗ Student’s t test, p < 0.01, ∗∗∗ Student’s t test, p < 0.001. Scale bars, 200 μm.

To further validate the RNA-seq data, we conducted in situ hybridization experiments involving Sp6 and Tbx1. Sp6 is essential for tooth development, as its absence leads to an overabundance of teeth, along with their malformed structures.21 Conversely, transgenic mice exhibiting epithelial Sp6-overexpression develop fewer molars in the mandible.22 In situ hybridization showed that Sp6 transcripts were primarily located in the inner enamel epithelium (IEE) in both control and FstCKO upper incisors at E14.5; however, the area of Sp6 expression was greatly expanded in FstCKO incisors (arrow, Figure 4C). At E16.5, Sp6 was mainly expressed in the labial IEE and weakly expressed in the lingual IEE in control teeth (Figure 4C). In incisors lacking Fst, Sp6 expression was detected in both the labial IEE and the branched IEE at the liCL (arrows, Figure 4C). Tbx1 plays an important role in regulating incisor development. Tbx1 knockout in mice leads to hypoplastic incisors lacking enamel.23 In situ hybridization showed that Tbx1 was mainly expressed in the dental epithelium near to the dental papilla mesenchyme in control upper incisors at E14.5 (Figure 4D). However, its expression in the lingual dental epithelium was significantly upregulated in FstCKO upper incisors (arrows, Figure 4D). At E16.5, Tbx1 was mainly expressed in the inner dental epithelium on the labial side of the upper incisor and weakly expressed in the liCL (Figure 4D). In FstCKO mice, Tbx1 transcript levels were greatly increased in the split liCL (arrows, Figure 4D). Overall, these results indicate that FST plays an important role in regulating the expression of key molecules for tooth development.

TGF-β signaling directly targets Tbx1, Sp6, and Sox2 during tooth development

Due to the increased activity of the TGF-β signaling pathway resulting from the Fst knockout, we subsequently examined whether the crucial genes that are upregulated in FstCKO incisors are directly regulated by this pathway. Activation of activin/TGF-β signaling involves phosphorylation of SMAD2/3, which form heteromeric complexes with SMAD4. These then translocate to the nucleus and regulate target gene expression.24 The presence of consensus p-SMAD2/3-binding sites, AGACWB,25 were observed within regulatory regions of Tbx1, Sp6, Sox2, and Ascl5 genome. We then used cleavage under targets and tagmentation (CUT&Tag) assays and p-SMAD2 antibody to examine whether these genes are TGF-β target genes in E14.0 upper incisor tooth samples. RT-PCR analysis of immunoprecipitated DNA showed that there was specific enrichment of p-SMAD2 to a DNA fragment that corresponds to one of potential sites from Tbx1, Sp6, and Sox2 promoters with antibodies against p-SMAD2 (Figure 5A, arrows), suggesting that the expression of Tbx1, Sp6, and Sox2 is probably directly regulated by TGF-β signaling in developing upper incisor. By contrast, the p-SMAD2 antibody did not enrich DNA fragments from the Ascl5 promoter region (Figure 5A).Figure 5 Activin/TGF-β signaling directly regulates key genes for incisor development and increased Tbx1 expression accounts for supernumerary tooth formation in FstCKO mice

(A) CUT&Tag analysis showing the enrichment of DNA fragments containing p-SMAD2-binding sites (S1−3) in Tbx1, Sp6, and Sox2, but not in Ascl5. CUT&Tag analysis was performed with p-SMAD2 antibody and IgG control in mouse upper incisor tooth germs at around E14. Immunoprecipitated DNA was amplified by RT-PCR and analyzed by agarose gel electrophoresis. Fragments amplified using genomic DNA were used as positive controls for each primer set. Specific enrichment of p-SMAD2 to a DNA fragment containing potential p-SMAD2-binding sites is indicated by an arrow.

(B) CCK8 assay shows that knockdown of TBX1 expression in HEK293T cells impairs cell proliferation.

(C) Rescue of Fst-deficiency-induced extra upper incisor formation by Tbx1 knockdown in tooth explants. Maxillary incisors were dissected, transfected with indicated siRNAs, and cultured for 3 days after transfection. Data are represented as mean ± SEM. ∗∗∗ Student’s t test, p < 0.001.

FST regulates the number of maxillary incisor teeth by upregulating Tbx1

Tbx1 plays critical roles in dental epithelial cell proliferation and its expression is upregulated in FstCKO incisors. Furthermore, Tbx1 potentially contributes to the maintenance of epithelial stem cells through the regulation of Sox2.26 Consequently, we investigated whether the upregulation of Tbx1 is the primary driver behind the excessive teeth formation observed in FstCKO mice. Consistent with previous reports showing that Tbx1 positively regulates cell proliferation,23,27,28 our Cell Counting Kit 8 (CCK8) assay showed that knockdown of TBX1 in HEK293T cells dramatically decreased cell proliferation (Figure 5B). Subsequently, we conducted a rescue experiment using organ culture to investigate whether the downregulation of Tbx1 expression could potentially reverse the supernumerary tooth formation observed in Fst-deficient incisor teeth. Maxillary incisors were dissected, transfected with indicated siRNAs, and cultured in a Trowell-type organ culture system. Knockdown of Fst caused supernumerary tooth formation in the tooth explants. In contrast, knockdown of Tbx1 led to hypoplastic incisor development (Figure 5C). Notably, supernumerary tooth formation in Fst-knockdown incisor explants was significantly rescued by knockdown of Tbx1 (Figure 5C). Taken together, these results indicate that overexpression of Tbx1 caused by Fst-deficiency can account for supernumerary incisor formation in FstCKO mice. Given that Fst-deficiency augments TGF-β signaling activity and Tbx1 is the direct target of TGF-β signaling, these findings suggest that FST modulates tooth number through the regulation of TGF-β-mediated activation of Tbx1.

Discussion

Supernumerary teeth are a common developmental anomaly of dentition. Here, we showed that Fst deficiency led to supernumerary upper incisor tooth formation in mice. The generation of extra teeth was associated with enhanced TGF-β signaling and increased cell proliferation. Furthermore, we showed that Tbx1, Sp6, and Sox2 are probably direct targets of p-SMAD2 and that knockdown of Tbx1 expression prevented extra tooth formation in Fst-deficient in tooth explants. Taken together, our investigation provides a novel mechanism at the cellular level for Fst controlling tooth number in mice.

Mouse incisors grow continuously throughout life because they have differential proximal niches that contain epithelial and mesenchymal stem cells respectively. Fst plays a critical role in regulating the proliferation of dental epithelial stem cells and transit amplifying cells.17 Intriguingly, FstCKO mice exhibit supernumerary upper incisor tooth formation, rather than the generation of lingual enamel as in Fst−/− mice.14 Wnt1-Cre line exhibits Cre recombination activity in cranial neural crest cell-derived mesenchymal cells at around E8.0.29,30 K14-Cre becomes active in oral and dental epithelium from E11.5 and enables elimination of gene product at E12.5.29 By that time, Fst has already been deleted from all tissues of Fst−/−. Therefore, it is possible that the timing of losing Fst controls the cell fate of dental epithelial stem cells, resulting in different phenotypes in FstCKO and Fst−/−.

Moreover, unlike Fst−/− mice, native upper incisors of FstCKO mice are covered by enamel only on the labial side. The liCL of the Fst-deficient native incisor is split in two; therefore, it is reasonable that the remaining portion of the liCL has insufficient epithelial stem cells for producing enamel. Interestingly, the supernumerary upper incisors in FstCKO mice are enamel-free, which may also be caused by insufficient epithelial stem cells. Another possibility is that differentiation of the epithelial stem cells in the extra tooth is impaired.

Fst−/− mice exhibit an enlarged stem cell niche in the upper incisor. Excessive generation of SOX2-positive cells are present in FstCKO upper incisors, indicating that expansion of the CL area in Fst−/− teeth is probably associated with an increased number of SOX2-positive cells. These results, together with ectopic development of Sox2-expressing taste progenitors in the Fst−/− tongue,31 support Fst as negatively regulating Sox2 expression in various tissues. The small supernumerary teeth in FstCKO mice are located lingually to the native incisor, similar to the arrangement of rabbit upper incisor teeth. Studies in vertebrate with replacement tooth formation reveal the successional tooth forming on the lingual side of the deciduous tooth from the successional dental lamina, which displays Sox2 expression.32 In mice, a transient rudimentary successional dental lamina also forms and houses Sox2-positive cells.32,33 Similarly, expanded SOX2 expression is observed in lingual dental epithelium of native upper incisors in FstCKO mice. Taken together, these results suggest that the formation of supernumerary teeth in FstCKO mice could be a result of revitalization of the rudimentary successional tooth germ.

TBX1 is the major genetic determinant of DiGeorge syndrome in humans. Mice deficient of Tbx1 exhibit features of DiGeorge syndrome, including cardiac, craniofacial, and dental anomalies.34 In mice, Tbx1 plays a pivotal role in amelogenesis and in the maintenance of epithelial stem cells because Tbx1 knockout leads to severely reduced or completely missing CL and hypoplastic and enamel-free incisors.23 As in Fst−/−mice, Spry2+/−;Spry4−/− mutant lower incisors display enamel on both labial and lingual sides.35 Loss of asymmetry in Spry2+/−;Spry4−/− mice is associated with ectopically expressed Tbx1 in the enlarged liCL.23 Tbx1 over-expression leads to increased laCL size and dental stem cell proliferation.28 Together with the finding that Tbx1 expression is increased in the liCL of FstCKO mice, these results indicate that the enlarged liCL of Fst−/− mice may also be associated with increased Tbx1 expression in the liCL. We also showed that upregulation of Tbx1 is required for extra teeth formation in FstCKO mice, which may also be attributed to its positive role in regulating epithelial stem cell proliferation. Intriguingly, it is reported that Tbx1 possibly acts upstream of Sox2.26 Together with the fact that Sox2 is essential for epithelial stem cell proliferation and is upregulated in FstCKO incisor germs, these results indicate that Tbx1 could regulate dental epithelial stem cell proliferation by regulating Sox2. However, more experiments are required to illustrate the regulatory relationship of Tbx1 and Sox2 in further studies.

In this report, we show that FST acts upstream of multiple key tooth development genes and is involved in determining tooth number. These findings, together with other advances in the molecular mechanisms underlying supernumerary tooth formation and in stem cell biology, will help reveal the etiology of supernumerary teeth in humans and will be informative for tooth regeneration and tooth engineering.

Limitations of the study

Our study shows that FST-deficiency leads to supernumerary incisor teeth in mice, suggesting that FST can be used as a novel target for tooth regeneration, however, more experiments should be conducted to prove this possibility in other animal models in future studies.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to the lead contact, Xiao-Jing Zhu (xiao_jingzhu@hznu.edu.cn).

Materials availability

This study did not generate new unique materials or reagents.

Data and code availability

• The RNA-seq data have been deposited in the Genome Sequence Archive of the National Genomics Data Center, China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences, and is publicly accessible as of the date of publication. Accession number is listed in the key resources table.

• This paper does not report original code.

• Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Acknowledgments

The authors thank Dr. Martin Matzuk from Baylor College of Medicine for kindly providing FstCKO mice for the study. We thank all members of the Zhang laboratory at Zhejiang Key Laboratory of Organ Development and Regeneration, 10.13039/501100007820 Hangzhou Normal University for helpful discussions.

Author contributions

S.Z., S.H., and Z.W.: contributed to data acquisition, analysis, and interpretation and critically revised the manuscript. C.H., C.L., H.S., and X.Y.: contributed to data acquisition and analysis and critically revised the manuscript. R.H., C.D., and M.Q.: contributed to data analysis and critically revised the manuscript. X.J.Z.: contributed to design, data interpretation, drafted and critically revised the manuscript. All authors gave their final approval and agree to be accountable for all aspects of the work.

Declaration of interests

The authors declare no competing interests.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER	
Antibodies	
	
phospho-SMAD2	Thermo Fisher Scientific	Cat#44-244G, RRID:AB_2533614	
SOX2	Abcam	Cat#ab97959, RRID:AB_2341193	
Cyclin D1	Abcam	Cat#ab134175, RRID:AB_2750906	
	
Critical commercial assays	
	
Masson's Trichrome stain kit	Solarbio life sciences	Cat#G1340	
RevertAid™ Master Mix	Thermo Fisher Scientific	Cat# M1632	
UltraSYBR mixture	CWBIO	Cat#E606335	
Trizol	Thermo Fisher Scientific	Cat# 15596026CN	
T7 RNA polymerase	Promega	Cat# P2075	
Sp6 RNA polymerase	Promega	Cat# P1085	
DIG RNA Labeling Mix	Roche	Cat#11277073910	
Anti-DIG-AP Fab fragments	Roche	Cat#11093274910	
BM purple	Roche	Cat#11442074001	
Hyperactive In-Situ ChIP Library Prep Kit for Illumina (pA-Tn5)	Vazyme	Cat#TD902	
CCK8	Sangon	Cat#E606335	
Lipofectamine® 3000	Thermo Fisher Scientific	Cat# L3000015	
	
Deposited data	
	
RNA-Seq data of E14.0 upper incisors	This paper	GSA:CRA007363	
	
Experimental models: Cell lines	
	
HEK293T	ATCC	Cat#CRL-3216	
	
Experimental models: Organisms/strains	
	
Mouse: FstFlox/Flox	From Dr. Martin Matzuk	Jorgez et al.15	
Mouse: Wnt1-Cre	The Jackson Laboratory	JAX: 004782	
Mouse: K14-Cre	The Jackson Laboratory	JAX: 007807	
	
Oligonucleotides	
	
Genotyping, QPCR and amplification primers for probe construction: see Table S2	This paper	N/A	
RT-PCR primers: see Table S3	This paper	N/A	
TBX1 siRNA: UGACCAAUAACCUGCUGGA	This paper	N/A	
Tbx1 siRNA: UGACCAAUAACCUGCUGGA	This paper	N/A	
Fst siRNA: GGAUGUGAACGACAAUACU	This paper	N/A	
	
Recombinant DNA	
	
pGEM®-T Easy Vector	Promega	Cat#A1360	
pGEM®-T-Fst	This paper	N/A	
pGEM®-T-Sp6	This paper	N/A	
pGEM®-T-Tbx1	This paper	N/A	
	
Software and algorithms	
	
GraphPad Prism 9.5	Graphpad Software	http://www.graphpad.com/	
Photoshop	Photoshop Software	https://www.adobe.com	

Experimental model and study participant details

Ethics statement

All animal experiments were approved by the Animal Users Committee of Hangzhou Normal University and carried out in strict accordance with the Guide for the Care and Use of Laboratory Animals at Hangzhou Normal University.

Animals

FstFlox/Flox mice have been described previously.15 Wnt1-Cre and K14-Cre mouse lines were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). The mice are on the C57BL/6 background and were reared in specific pathogen free facilities. FstFlox/Flox mice were crossed with the mice carrying both Wnt1-Cre and K14-Cre to obtain FstFlox/+;Wnt1-Cre;K14-Cre mice, which were back-crossed with FstFlox/Flox to produce FstFlox/Flox;Wnt1-Cre;K14-Cre (FstCKO). FstFlox/Flox, and Cre− or Cre+;FstFlox/+ littermates were used as control mice. Primer sequences for genotyping are shown in Table S2. The morning of vaginal plug appearance was determined as embryonic day 0.5. The experiment was conducted using mouse embryos at E14.0, E14.5, and E16.5, as well as postnatal mice at P0, P36, and four months of age. Gender does not affect the experimental results, as both female and male FstCKO mice exhibit the hyperdontia phenotype. All of the available FstCKO mice, both male and female, were arbitrarily used for experiments.

Method details

MicroCT, histology, immunohistochemistry, and in situ hybridization

For microCT analysis, post-natal day (P)36 mice were euthanized and dissected tissues were fixed in 4% paraformaldehyde and processed according to standard protocols. Embryos were dissected and fixed in 4% PFA (Sangon, Shanghai, China) overnight at 4°C. Samples were dehydrated through an ethanol series and embedded in paraffin. After deparaffinization and hydration, 7 μm sections were stained with hematoxylin and eosin (HE) following standard protocols. Masson trichrome staining was performed using a Masson's Trichrome stain kit (Solarbio life sciences, Beijing, China) according to the manufacturer’s instructions. Standard hematoxylin/eosin (HE) staining was performed following standard protocols. Immunostaining was performed on 5-μm-thick paraffin sections using antibodies against phospho-SMAD2 (Thermo Fisher, 44-244G, 1:200), SOX2 (Abcam, ab97959, 1:200), and Cyclin D1 (Abcam, ab134175, 1:200) according to the manufacturer’s instructions. For in situ hybridization, primers were synthesized and used to amplify cDNA fragments prepared from embryonic mouse incisors. The amplified fragments were cloned into a pGEM®-T Easy vector (Promega), and the resulting plasmids were linearized to serve as templates for probe synthesis. Digoxigenin-labeled riboprobes were transcribed from these templates using T7 or SP6 RNA polymerase (Promega) in the presence of DIG RNA Labeling Mix (Roche). These probes were applied to 12-μm-thick paraffin sections, following standard deparaffinization, rehydration, proteinase K treatment, and hybridization procedures as described previously.36 Gene expression was visualized by sequential incubation with Anti-DIG-AP Fab fragments (Roche) and BM purple (Roche). Primer sequences are provided in Table S2.

RNA-Seq

Upper incisors were dissected from E14.0 embryos. Total RNA was isolated and subjected to RNA-Seq analysis by BGI (Wuhan, China). For each genotype, two independent samples were sequenced. The genes for which the average FPKM (fragments per kilobase of transcript per million fragments mapped) value was above 1 in WT or FstCKO mice were selected for further analysis. Genes with fold change (FC) ≥ 1.5 and Qvalue < 0.001 were considered differentially expressed (Table S1). The advanced volcano plot was generated using OmicStudio tools. Enrichment analysis of RNA-Seq data was performed using Enrichr, which is a comprehensive resource for curated gene sets.37,38

Quantitative reverse transcription PCR (qRT-PCR)

qRT-PCR was performed using UltraSYBR mixture (CWBIO, Beijing, China) with the StepOnePlus™ Real-Time PCR System. Upper incisor tooth germs were dissected from E14.0 embryos and total RNA was isolated using Trizol (Thermo Fisher). cDNA was synthesized using RevertAid™ Master Mix (Thermo Fisher, M16325) and used as template in a 20 μl qRT-PCR reaction system. Actb was used as a reference gene. To investigate whether Fst was successfully deleted in the dental epithelium of FstK14-Cre mice, cervical loop and ameloblast cells derived from dental epithelium were isolated from the upper incisors of 4-month-old control and FstK14-Cre mice, and were used for qRT-PCR analysis. To investigate whether Fst was deleted in the dental mesenchyme of FstWnt1-Cre mice, the dental mesenchyme was isolated from the upper incisors of 4-month-old control and FstWnt14-Cre mice and subjected to qRT-PCR. Gapdh was used as the reference gene. Primer sequences were from PrimerBank39 and are shown in Table S2. Data were analyzed using the 2-ΔΔ CT method and are represented as the mean ± SEM. A p-value < 0.05 was considered statistically significant.

CUT&Tag

Upper incisors were dissected from E14.0 embryos and subjected to CUT&Tag assays using the Hyperactive In-Situ ChIP Library Prep Kit for Illumina (pA-Tn5) (Vazyme, TD902). Briefly, tooth cells were digested, harvested, mixed with the ConA beads, permeabilized, and incubated with the phospho-SMAD2 antibody or normal rabbit IgG, and the secondary antibody. Subsequently, cells were mixed with the pA-Tn5 Transposon in the tagmentation buffer for targeted tagmentation. DNA was then extracted using phenol/chloroform and precipitated with ethanol, amplified, and used as template for RT-PCR analysis. RT-PCR Primers were designed to amplify the DNA fragments containing potential p-SMAD2/3-binding sites in the gene regulatory regions (Table S3). Fragments amplified using genomic DNA were used as positive controls for each primer set.

CCK8 assays

Forty-eight hours after transfection with TBX1 or Scrambled siRNA, HEK293T cells were seeded in DMEM containing 10% FBS at approximately 3000 cells/well into 96-well plates. CCK8 (Sangon, Shanghai, China) solution was added to each well and incubated for 1 h at 37°C. Absorbance at 450 nm was then measured in a microplate reader. The sense sequence of the siRNA for TBX1 was 5′-GCAAAGAUAGCGAGAAAUA-3′.

Organ culture

Upper incisor tooth germs with some surrounding tissues were dissected from the maxillae of embryos at approximately E14.5 and cultured using a Trowell type organ culture system.7 Explants were subjected to organ culture and transfected with indicated siRNAs using Lipofectamine® 3000 (Thermo Fisher) on day one. Photographs were taken under light microscopy. The siRNA sense sequences for Fst and Tbx1 were 5′-GGAUGUGAACGACAAUACU-3′ and 5′-UGACCAAUAACCUGCUGGA-3′, respectively. At least six tooth germs were used for each condition in one experiment. The experiments were repeated independently for at least two times.

Quantification and statistical analysis

For quantification of proliferation, Cyclin D1-positive cells within a defined area were counted. The intensity of pSMAD2-staining within a defined area was analyzed by the Photoshop Software. Three independent embryonic samples for each genotype were used for statistical analysis. Data was analyzed by GraphPad. Statistical significance was calculated using Student’s t-test. A p-value < 0.05 was considered statistically significant.

Supplemental information

Document S1. Figures S1–S4 and Tables S2 and S3

Table S1. Differentially expressed genes in FstCKO and control incisors, related to Figure 4

Supplemental information can be found online at https://doi.org/10.1016/j.isci.2024.110785.
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