An in situ hybridization study of Hyaluronan synthase (Has) mRNA in developing mouse molar and incisor tooth germs

Highlights

• We examine expression of three Hyaluronan synthase in developing mice tooth development.

• Dental epithelial and mesenchymal cells differ from Hyaluronan synthase during tooth morphogenesis.

• Has3 is the main factor for HA synthesis by epithelium, and may in part regulate crown and root formation of the tooth germ.

Abstract

Hyaluronan (HA) is a major constituent molecule in most extracellular matrices and is synthesized by Hyaluronan synthase (Has). In the present study, we examined expression patterns of Has1, -2, -3 mRNA in developing mouse molar and incisor tooth germs from embryonic day (E) 11.5 to postnatal day (P) 7, focusing on Hertwig’s epithelial root sheath (HERS) and the apical bud in particular. Has1 mRNA expression was not detected in all tooth germs examined. Has2 mRNA was expressed in the surrounding mesenchyme from E12.0 to 18.0 in both molar and incisor tooth germs, but disappeared after birth. Meanwhile, Has3 mRNA was exclusively expressed within the enamel organ, especially in the inner enamel epithelium (IEE), stellate reticulum (SR), and stratum intermedium (SI) until the early bell stage at E16.0. Has3 mRNA disappeared as IEE differentiated into differentiating ameloblasts (dABs), but remained in SI until the root developmental stage of the molar tooth germ at P7. Has3 mRNA was also expressed in HERS until P7. In incisors, Has3 mRNA was expressed in the apical bud, especially in the transit-amplifying (TA) cell region from E16.0 to P7, and in the papillary layer (PL) adjacent to the mature enamel. These gene expression patterns suggested that Has3 is the main control factor for prenatal and postnatal HA synthesis of the tooth germ, and may in part regulate crown and root formation of the tooth germ, maintenance of stem cell niches in the apical bud as well as mineral transport in PL.

Introduction

Hyaluronan (HA) is a major constituent molecule in most extracellular matrices (ECM), and consists of a linear polysaccharide composed of repeating disaccharides of D-glucuronic acid and N-acetylglucosamine (Laurent and Fraser, 1992). HA shows physical properties such as hydrophilicity, viscoelasticity and extensibility, and is thought to contribute to maintaining physiological and physical properties and homeostasis (Laurent and Fraser, 1992). Meanwhile, HA plays an important role in cell behavior such as cell proliferation, differentiation, and migration (Toole, 2001, Spicer and Tien, 2004). HA also exerts physiological functions when bound to proteoglycans such as aggrecan, versican, brevican, and neurocan in the form of large aggregates (Ruoslahti, 1996, Watanabe et al., 1997, Shibata et al., 2000). Despite its simple chemical structure, HA is involved in many physiological functions.

Hyaluronan synthase (Has) is a glycosyltransferases enzyme involved in HA synthesis, and three types of Has (Has1, Has2 and Has3) have been cloned (Spicer et al., 1997, Itano et al., 1999). In contrast to typical glycosyltransferases, Has family is localized on the plasma membrane. Has isozymes have similar amino acid sequences and molecular structural characteristics. Each Has mRNA, however, shows distinct expression patterns in Xenopus laevis (Nardini et al., 2004) and mice (Tien and Spicer, 2005), and possesses different enzymatic stabilities for HA (Itano et al., 1999). The molecular size of HA products vary among the three isozymes. HA synthesized by Has2 (average molecular mass of >2 × 10⁶ Da) is much larger than those of Has1 and Has3 (peaks with molecular masses of 2 × 10⁵ to 2 × 10⁶ Da) (Itano et al., 1999). Both Has1-and Has3-deficient mice show no gross abnormalities (Bai et al., 2005, Kobayashi et al., 2010, Kessler et al., 2015), but in a dextran sodium sulfate (DSS)-induced colitis model, the former exhibits a dramatic increase in submucosal microvasculature, hyaluronan deposition, and leukocyte infiltration (Kessler et al., 2015). Has1 and Has3 double gene-deficient mice are also phenotypically normal (Kessler et al., 2015). Meanwhile, Has2-deficient mice result in embryonic lethality with severe cardiac and vascular malformations (Camenisch et al., 2000). Thus, these results suggest the different physiological roles of individual Has isozymes.

Tooth morphogenesis (tooth germ formation) is regulated by the sequential and reciprocal interaction between the dental epithelium and mesenchymal tissue, and consequently produces various kinds of teeth of specific shapes and sizes within the jaw (Piesco and Avery, 2002). Tooth morphogenesis is a continuous process involving the bud, cap, bell, and apposition stages according to the shape of the epithelial portion of the tooth germ, which then shifts to the root developmental stage. In addition, Hertwig’s epithelial root sheath (HERS) is known to be involved in root formation (Piesco and Avery, 2002, Nanci, 2013).

The rodent incisor grows continuously throughout the lifetime of the animal, and the apical end of the epithelium in the incisor has a special epithelial structure considered to be a stem cell niche (Harada et al., 1999). The term “cervical loop” is applied to the reflection zone in molars, where the inner enamel epithelium (IEE) and the outer enamel epithelium (OEE) meet at the rim of the enamel organ (Nanci, 2013). The apical epithelial structure in the incisor is also called the cervical loop (Tummers and Thesleff, 2003), but due to its special structural features, Harada and Ohshima (2004) proposed that it should be termed the “apical bud”, consisting of the stellate reticulum (SR) surrounded concentrically by the basal epithelium (BE), IEE, and OEE. Thus, we used the term “apical bud” in the present study.

Several signaling pathways and transcription factors are known to contribute to interactions between epithelial and mesenchymal tissues during tooth morphogenesis (Jussila and Thesleff, 2012). In addition, the involvement of proteoglycans including syndecan, versican, perlecan, and HA has been suggested in developing tooth morphogenesis (Vainio et al., 1989, Shibata et al., 2002, Ida-Yonemochi et al., 2005, Jiang et al., 2010). Based on the results described above, we hypothesized that Has family is involved in tooth morphogenesis via HA synthesis.

Related to tooth morphogenesis, Tien and Spicer (2005) reported that Has2 and Has3 mRNA was expressed in the dental lamina up to the molar cap stage, but detailed gene expression patterns of Has mRNA remain unclear, especially during the bell stage to postnatal root developmental stage in molars and in developing incisors. Thus, the purpose of the present in situ hybridization study was to examine the expression patterns of Has1, -2, and -3 mRNA in developing mouse molars and incisors, focusing on HERS and the apical buds in particular.