Expression Levels of WNT Signaling Pathway Genes During Early Tooth Development

ABSTRACT It is known to all that Wnt signaling pathway plays an important role in the early development of tooth. Our previous research found that Wnt signaling pathway played crucial roles in dental development, and mutations in antagonist of Wnt signaling pathway may lead to the formation of supernumerary teeth. However, the expression pattern of Wnt signaling molecules in early development of tooth, especially genes with stage specificity, remains unclear. Hence, we applied RNA-seq analysis to determine the expression levels of wnt signal molecules at five different stages of rat first molar tooth germ. In addition, after literature review we summarized the function of Wnt signaling molecules during tooth development and the relationship between Wnt signaling molecules variation and tooth agenesis. Our research may have implications for exploring the role of Wnt signaling molecules in different stages of tooth development.


Introduction
The Wnt signaling pathway plays an important role in many processes of biological development, including embryogenesis, tissue homeostasis and wound repair. Several biological behaviors such as cell proliferation, differentiation, polarization, and apoptosis were regulated by the Wnt signal pathway. The Wnt signaling pathway is a complex system that consist of extracellular secreted glycoproteins (19 Wnt ligands), wnt receptors (10 Frizzled receptors, Frz and 2 LDL receptor related protein, Lrp5/6), cytoplasmic proteins (β-catenin, DVL, APC, AXIN and GSK-3β, etc.) and several Wnt-associated molecules (MSX1, DKK1, KREMEN1, and ANTXR1.1-3 According to whether it is dependent on the activation of β-catenin, Wnt signaling pathways can be divided into the canonical signaling pathway and the noncanonical signaling pathway. The Wnt ligands, the Fzl receptors and LRP form a complex that activates the downstream canonical β-catenin pathway. Moreover, Wntreceptor complexes are mediated and controlled by downstream degradation complexes composed of APC, AXIN and GSK-3β. The degradation complex leads to phosphorylation and ubiquitinationmediated degradation of β-catenin. Once a Wnt ligand binds to a Fzl receptor and LRP protein, the degeneration complex in cytoplasm is inhibited, leading to the accumulation of β-catenin.4-6 Accumulated β-catenin translocates to the nucleus, interacts with transcription factors TCF (T-cell factor)/LEF (lymphocyte-enhancer-binding factor), and promotes the transcription of Wnt target genes.7 Non-canonical Wnt signaling pathways are further divided into Wnt/planar cell polarity (PCP) signaling, Wnt/Ca2+ Pathway, Wnt RTK Pathway and Wnt Frz Pathway which are involved in the process of polarity and cell motility. 8 Tooth development is a dynamic process that includes the bud, cap and bell stages, root development and tooth eruption.9 It is universally known that Wnt signaling pathway plays an important role in tooth development. 10 The Wnt signaling molecules are spatiotemporally activated in tooth development, thereby implying its essential role in the process of odontogenesis. The mutations of Wnt signaling genes may result in tooth agenesis (TA). The genetic link between TA and the Wnt pathway was first evidenced by the identification of a mutation of the Axin2 gene in an oligodontia family.10 Mutation of Wnt10a may lead to odonto-onychodermal dysplasia, Hypohidrotic ectodermal dysplasia, Schopf-Schulz-Passarge syndrome and many non-syndromic tooth agenesis. Abnormal expression of Wnt10b is related to oligodontia, microdontia, short tooth roots, dental pulp stones, and taurodontism.11-13 Mutation of Lrp6 is also associated with oligodontia, mesiodens, fusion of teeth, odontomas, microdontia, long roots, molars with unseparated roots, and taurodontism. 14,15 Tooth development requires regulation of multiple molecules, which is the result of spatiotemporal expression of these molecules. The expression pattern of a gene or signaling are supposed to include not only the expression level but also the location at continuous stages and the study of gene expression level is an important part of learning gene expression patterns. It is known to all that Wnt signaling pathway plays an important role in tooth development. However, the expression pattern of Wnt signaling molecules in development of tooth germ are still unclear. We expect to gain a holistic understanding of the expression of the wnt signaling pathway during early tooth development and to discover genes that are specifically expressed during early tooth development. Here, we determined the expression levels of wnt signal molecules in each stage of rat first molar tooth germ development by RNA seq analysis and after literature review we summarized the function of Wnt signaling molecules during tooth development and the relationship between Wnt signaling molecules variation and tooth agenesis. Our research may have important implications for exploring the role of Wnt signaling molecules in different stages of tooth development.
It is observed that when the embryo developed to E14.5, E16.5 and E18.5, the first molar tooth germ was in bud stage, cap stage and early bell stage respectively, while the tooth germs of 1-and 7day-old mice were in late bell stage and eruption stage respectively. After sequencing, a total of 143 mRNA related to Wnt signal pathway were detected. Expression of Wnt signaling molecules were displayed in Table 1. Hierarchical clustering analysis revealed that the expression levels of these molecules showed significant time specificity Figure 1. The results showed a high degree of similarity among the tooth germ tissues at embryonic stages, and a low degree of similarity between the embryonic and postnatal tooth germs Figure 1.
Since it is difficult to completely distinguish the molecules involved in canonical Wnt signaling pathway and the noncanonical Wnt signaling pathway, therefore we classified and integrated all genes together in Table 1. Canonical Wnt signaling pathway covered 82 different genes, and the noncanonical Wnt signaling pathway covered 81 genes. There were 20 genes with overlap between those two pathways. Besides, expression levels of genes such as Fzd10, Prkaca, Fzd7, Dkk2, Cer1, Wnt10b, Ctnnbip1, Dkk1, Wnt3a, Serpinf1, Dkk3 and Wnt16 showed obvious time specificity.

Changes in DEG mRNA levels during rat molar development
We summarized the genes with special expression levels in Table 1 and used the relative expression amounts of these genes as the ordinate to draw Figure 2. The mRNA expression levels of Fzd10 and Prkaca were mainly highly expressed mainly in the E14.5 stage. ThemRNAs of Fzd7 and Dkk2 were highly expressed mainly in E16.5 phase, the mRNA ofCer1 was highly expressed in E18.5 phase, while Ctnnbip1 and Dkk1 showed high expression in P1 phase. Wnt10b and Wnt10a were highly expressed in both E18.5and P1 phases. The mRNAs of Wnt3a, Serpinf1 and Dkk3 were highly expressed at the postnatal stage compared to the embryonic stage. In contrast, Wnt16 was expressed only at E14.5 and E16.5 stages. The expression of APC in the five stages of early tooth development is higher than 8.

Discussion
We investigated five time points (E14.5, E16.5, E18.5, P1 and P7) of rat tooth germ development. Considering the small size of rat tooth germs, it is difficult to separate dental papilla and dental sac, so we took the whole tooth germs for sequencing. The sequencing results include both epithelial genes and dental papilla genes. Totally 128 mRNA related to Wnt signal pathway of rat tooth germ samples from different stages of development were detected. We observed that the expression levels of these molecules showed significant time specificity, including Fzd10, Prkaca, Fzd7, Dkk2, Cer1, Ctnnbip1, Dkk1, Wnt10b, Wnt3a, Serpinf1, Dkk3, Wnt16. Wnt10b, Dkk1 and Serpinf1 have been found to be associated with tooth agenesis, but little research has been done on the relationship between the other genes mentioned above and tooth germ development. We obtained a relatively complete gene expression profile related to Wnt signaling pathway through RNA sequencing. Our study enriches the understanding of Wnt signal pathways and provides a lot of useful information for subsequent research.

Wnt signaling molecules and tooth agenesis
Gene mutation may disrupt specific signaling networks and cause a wide variety of selective tooth agenesis patterns. Tooth agenesis can either occur as an isolated condition (non-syndromic tooth agenesis) or can occur as part of a genetic syndrome (syndromic tooth agenesis). Some genes are responsible for syndromic tooth agenesis, including Robinow syndrome and Gardner syndrome. The mutations of APC may cause Gardner syndrome, besides gene mutations of Ror2, Dvl1, Dvl3 and Wnt5a are associated with Robinow syndrome. Genes responsible for tooth agenesis were displayed in Table 2. By searching for HGMD, we found that there are as many as 2037 different mutation forms in APC (Table 2).

Apc
Adenomatous polyposis coli (APC) gene encodes a tumor suppressor protein that acts as an antagonist of the Wnt signaling pathway. It is also involved in cell migration and adhesion, transcriptional activation, and apoptosis. 61 Mutations in Apc may cause familial adenomatous polyposis (FAP), an autosomal dominant pre-malignant disease that usually progresses to malignancy. 62 Besides, other diseases such as Gardner syndrome, which is characterized by the presence of multiple intracolonic polyps and extracolonic tumors, can also be caused by Apc mutation. Our previous study found that patients with Gardner syndrome also had multiple impacted and supernumerary teeth. 16 We found that Apc was expressed at E14.5-P7 and there are up to 2037 different mutation forms in Apc, suggesting that Apc played an important role in all stages of early tooth development.

Wnt10b
Wingless-Type MMTVIntegration Site Family, Member 10B (Wnt10b) is a ligand of Wnt signaling pathway and has been implicated in regulation of   embryogenesis. Multiple variants of Wnt10b were found by mutational analysis in patients with nonsyndromic tooth agenesis. [11][12][13] Besides, mutations of Wnt10b are associated not only with oligodontia and tooth agenesis, but also with microdontia, short tooth roots, dental pulp stones, and taurodontism. 11 The mutations of Wnt10b decreased Wnt signaling in dental pulp stem cells, affecting the development of permanent dentition, particularly the lateral incisors. 13 Wnt10b mutation allele-transfected into stem cells from human exfoliated deciduous teeth resulted in decreased gene transcription, while Wnt10b protein increased with the increase of mutant alleles, indicating that Wnt10b variants have functional effects on gene regulation and reveal that these variants may affect tooth development leading to tooth agenesis. 63 A. Nadiri et al. observed that the relative amount of Wnt10b was maximal at E14 stage during the development of rat molar tooth germs by western blotting. 64 At the late bell stage, Wnt10b was mainly detected in the inner dental epithelium, indicating that Wnt10b may be involved in cell proliferation. 64 Unlike other studies, our study found that Wnt10b was significantly expressed at E18.5 (early bell stage) and P1 (late bell stage) stages. Therefore, the expression of Wnt10b during the tooth germ development needs to be further verified and explored.

Wnt10a
Wingless-Type MMTV Integration Site Family, Member 10A (Wnt10a) is another important ligand of Wnt signaling pathway, which appears to have specific relevance to skin, its appendages and teeth. Wnt10a is associated with both syndromic and non-syndromic tooth agenesis. Schöpf-Schulz-Passarge syndrome (SSPS) is an autosomal recessive form of ectodermal dysplasia resulting from mutations in Wnt10a. 65 Tooth agenesis in SSPS patients can be characterized by conical primary teeth, agenesis of permanent teeth and hypodontia. 24 Odonto-onycho-dermal dysplasia (OODD) is a rare autosomal recessive syndrome mainly characterized by dry hair, anodontia of permanent teeth, smooth tongue, keratoderma, and hyperhidrosis. 26 Recently, Wnt10a mutations were found in patients with nonsyndromic tooth agenesis as well, mainly characterized by oligodontia and agenesis of the maxillary permanent canines. Upper and lower premolars were the most affected missing teeth. 12,21 In our research, Wnt10a was significantly expressed in E18.5 (early bell stage) and P1 (late bell stage) stages and the expression level decreased in P7 phase. Our results implied that Wnt10a played an important role in the formation of tooth hard tissue.

Dkk1
Dickkopf-related protein 1(Dkk1) is a typical antagonist of Wnt/β-catenin signaling by competing for the Wnt receptor LRP5/6. The mutation of Dkk1 may cause oligodontia and short root anomaly. 27,66 Researchers observed that strong expression of Dkk1 was localized in preodontoblasts on the labial side of the incisors. 67 At postnatal day 2, Dkk1 is prominently expressed in the preodontoblasts and odontoblasts in mouse molar germs. 68 In Dkk1 transgenic mice, overexpression of Dkk1 in pulp and odontoblast cells delayed the maturation of dentinogenesis during post-natal development. 69 The dental crown begins to form in the late bell stage (P1). In this stage, peripheral cells of the dental papilla differentiate into odontoblasts that secrete dentin. Our results demonstrated that Dkk1 was significantly expressed in the P1 phase, which indicated that Dkk1 played an important role in the formation of dentin.

Serpinf1
Serpin family F member 1 (Serpinf1) encodes the 50-kDa secreted glycoprotein pigment epitheliumderived factor (PEDF), which inhibits Wnt/β-catenin signaling pathway. Mutation of Serpinf1 may cause a unique autosomal recessive disease, Osteogenesis imperfecta (OI) type VI (MIM #610968). OI is characterized by recurrent fractures, progressive skeletal deformities, and growth deficiency, blue or gray discoloration of the sclera and dentinogenesis imperfecta. 70 Mutations in Serpinf1 have been reported in Chinese individuals with OI. 46 Our findingSerpinf1 mainly high expressed during postnatal stages, indicated that Serpinf1 played an important role in the formation and mineralization of tooth hard tissues.

Other important genes
Fzd10 is one of the FZD family receptors which act through canonical Wnt signaling. 42 Fzd10regulates cell proliferation and mediates Wnt1-induced neurogenesis in the developing spinal cord. 71 Investigations have demonstrated that mutations in the gene of Prkaca result in the development of adrenocortical adenomas associated with Cushing's syndrome. 72 Fzd7 plays a significant role in the regulation of multipotentiality of human pluripotent stem cells and its down-regulation accompanies differentiation and exit from the pluripotent stem cell state Dkk2 plays an important role in ovarian cancer. 73,74 It has been well documented that Cer1 plays dual roles in neural induction and suppression of mesodermal or endodermal lineages. 40 We observed that the mRNA expression levels of Fzd10 and Prkaca were mainly high during bud stage (E14.5), Fzd7 and Dkk2 were mainly highly expressed during cap stage (E16.5), and Cer1 was high during early bell stage (E18.5), indicating that they may play important roles in the early stage of tooth germ development. The expression level of Ctnnnbip1 was mainly high during P1 stage (late bell stage), enlightening that Ctnnbipmay be related to the formation and mineralization of enamel and dentin. Compared to the embryonic stages, the mRNA expression levels of Wnt3a and Dkk3 were highly expressed during postnatal stages, which implied that once significant high expression of Wnt3a and Dkk3 were found, it indicated that tooth germ has developed to the stage of hard tissue formation and mineralization. By contrary, Wnt16 was expressed only during E14.5 and E16.5, suggesting that Wnt16 was one of the important markers of early development of tooth germ. In summary, we determined the expression levels of Wnt signaling molecules at each stage of rat first molar tooth germ development by RNAseq analysis and after literature review we summarized the relationship between Wnt signaling molecules variants and tooth agenesis. Our data provided important information about novel genes in tooth germ development. Our findings may help to elucidate the molecular mechanisms of tooth germ development and provide a theoretical basis for further studies on the expression and function of genes involved in human tooth development and regeneration.

Tissue acquisition and preparation
Specific pathogen-free (SPF)-grade Sprague Dawley (SD) rats (Department of Laboratory Animal Science, Fudan University, Shanghai, China) were used in our study. The day when the vaginal plug was formed as a reference of embryonic day 0 (E0), embryos at E14.5, E16.5 and E18.5 stages were used. One-day-old (P1) and seven-dayold (P7) rats were used as well. We separated the embryos from the uterus of pregnant female rats. Each group has 8 mouse embryos, and the sex of the embryos was randomized to both males and females. The first mandibular molar tooth germs were isolated from these embryos and rats using a zoom stereo microscope (Olympus SZ51, Tokyo, Japan) and prepared for subsequent experiments. All molars were dissected by one same researcher. The rats were euthanized with phenobarbital sodium (i.p., 50 mg/kg) anesthesia. All animal experiments were approved by the Institutional Animal Care and Use Committee of Fudan University.

RNA-seq
Total RNA was obtained from the tooth germ cells in the embryonic (E14.5, E16.5, and E18.5) and postnatal (P1 and P7) phases using TRI reagent (Invitrogen, Carlsbad, CA, USA). More than eight tooth germs were used in each phase. An RNA-seq library was constructed following the manufacturer's instructions. Then, next-generation sequencing was performed using an Illumina NovaSeq platform (IGA, Udine, Italy). mRNAs related to wnt signal pathway were selected and hierarchical clustering analysis was performed.

Database search and literature review
We queried the Human Gene Mutation Database (HGMD ® ) (https://www.hgmd.cf.ac.uk/ac/index. php) for all variants of Wnt signaling genes. A comprehensive review of the PubMed database was undertaken for reports in the English language from 1January 2012 to 1 October 2022. The following search terms were used: (tooth [MeSHTerms]) AND (((Wnt Pathway [MeSH Terms]) OR Wnt Pathway, Canonical) OR Wntbeta-Catenin Signaling Pathway). Titles and abstracts were reviewed, and references and citations of relevant studies were also examined.