Angled Growth of the Dental Lamina Is Accompanied by Asymmetrical Expression of the WNT Pathway Receptor Frizzled 6

Abstract

Frizzled 6 (FZD6) belongs to a family of proteins that serve as receptors in the WNT signaling pathway. FZD6 plays an important role in the establishment of planar cell polarity in many embryonic processes such as convergent extension during gastrulation, neural tube closure, or hair patterning. Based on its role during hair development, we hypothesized that FZD6 may have similar expression pattern and function in the dental lamina, which is also a distinct epithelial protrusion growing characteristically angled into the mesenchyme. Diphyodont minipig was selected as a model species because its dentition closely resemble human ones with successional generation of teeth initiated from the dental lamina. We revealed asymmetrical expression of FZD6 in the dental lamina of early as well as late stages during its regression with stronger expression located on the labial side of the dental lamina. During lamina regression, FZD6-positive cells were found in its superficial part and the signal coincided with the upregulation of molecules involved in epithelial-mesenchymal transition and increased migratory potential of epithelial cells. FZD6-expression was also turned on during differentiation of cells producing hard tissues, in which mature odontoblasts, ameloblasts, or surrounding osteoblasts were FZD6-positive. On the other hand, the tip of successional lamina and its lingual part, in which progenitor cells are located, exhibited FZD6-negativity. In conclusion, asymmetrical expression of FZD6 correlates with the growth directionality and side-specific morphological differences in the dental lamina of diphyodont species. Based on observed expression pattern, we propose that the dental lamina is other epithelial tissue, where planar cell polarity signaling is involved during its asymmetrical growth.

Keywords: FZD6; WNT signaling; ameloblast; epithelial remnants; odontoblast; osteoblast; planar cell polarity (PCP); successional dental lamina.

Figure 1

FZD6 expression is asymmetrical in the dental lamina. (A) At epithelial thickening stage, the oral epithelium and surrounding mesenchyme are FZD6-positive. (B,C,C′ in detail) Later, FZD6-positive signal is located on the labial side (arrow) of the protruding dental lamina. (D,D′ in detail) At dental bud stage, stronger FZD6-immunopositivity (arrow) was detected on the labial side of dental germ. (E,F) At bell stage, the labial cervical loop is more positive that the lingual one. Almost no signal was visible in the stellate reticulum. The dental papilla and surrounding mesenchyme were FZD6-negative. b, bone; de, dental epithelium; dl, dental lamina; dp, dental papilla; oe, oral epithelium; th, tooth. Scale bar = 100 μm.

Figure 2

The tip of growing successional dental lamina is FZD6-negative. (A) In the most superficial area, flat cells connecting the lamina to the oral epithelium were FZD6-positive. (B) Cells on the labial side separating from the lamina were also FZD-positive similarly to the stalk of very flat cells connecting the dental lamina to the tooth. However, the apical tip of the successional lamina was FZD6-negative. (C) When dental lamina disconnected from the oral epithelium, the stronger positivity of Fzd6 was evident in the superficial fragments and epithelial clusters of cells. (D) Rudimental interdental lamina exhibited only weak FZD6 expression. dl, dental lamina; oe, oral epithelium; sdl, successional dental lamina; th, tooth; red arrow, epithelial remnants. Scale bar = 100 μm.

Figure 3

Details of FZD6 expression in the dental lamina. (A,A′,B,B′) Stronger expression of FZD6 was observed in the superficial area of the dental lamina, which was still connected to the oral epithelium. (C,C′) No FZD6 expression was found in deeper successional dental lamina while spotted pattern of expression was observed in surrounding mesenchymal cells. (D,D′) Later in development, superficial part was disconnected from the oral epithelium and cells exhibited strong FZD6-positivity. (E,E′) Expression was gradually lost in the central part of the lamina. (F,F′) The tip of the successional lamina was negative similar to younger stage, only few positive cells were found in more superficial position. dl, dental lamina; ep, epithelial pearl; oe, oral epitheliuml; sdl, successional dental lamina. Scale bar = 25 μm.

Figure 4

FZD6 expression in epithelial remnants and pearls. (A,B) Localized and cytoplasmatic expression of FZD6 was found in epithelial remnants. (C) The expression was found in the epithelial clusters during process of pearl formation as well as in the basal layer of already formed pearls (D), while their central area was negative. ep, epithelial pearl; red arrow, epithelial remnants. Scale bar = 100 μm.

Figure 5

FZD6 in differentiated ameloblasts and odontoblasts. (A,A′) Differentiated and secretory ameloblasts were FZD6-positive while non-differentiated cells of the inner enamel epithelium were negative. Similar pattern was observed in the dental papilla where differentiated odontoblasts exhibiting dentin production were FZD6-positive. FZD6 signal was localized at different stages of ameloblasts differentiation after their elongation. (B,B′) FZD6 signal was located also in the structures surrounding teeth such as alveolar bone where osteoblasts were positive and secretory cells of salivary gland or their ducts (C,C′). am, ameloblast; dp, dental papilla; od, odontoblast; oee, outer enamel epithelium; sg, salivary gland. Scale bar = 100 μm.

Figure 6

Schema of FZD6 protein expression through odontogenesis. (A) At the epithelial thickening stage, a strong FZD6 signal was apparent especially in basal layers of the oral and future dental epithelium. (B) Later, FZD6-positivity was detected in the dental lamina with stronger expression on the labial side. (C) At the bell stage, distinct expression was found throughout the oral epithelium and in the labial cervical loop area. Almost no signal was visible in the stellate reticulum. (D) The tip of successional dental lamina was FZD6-negative while cells in the superficial part of the lamina were FZD6-positive. Nuclei are labeled by blue and FZD6 expression in green through the epithelial tissues. de, dental epithelium; dl, dental lamina; oe, oral epithelium; th, tooth.