Beta-Catenin and Plakoglobin Expression during Zebrafish Tooth Development and Replacement

Abstract

We analyzed the protein distribution of two cadherin-associated molecules, plakoglobin and β-catenin, during the different stages of tooth development and tooth replacement in zebrafish. Plakoglobin was detected at the plasma membrane already at the onset of tooth development in the epithelial cells of the tooth. This pattern remained unaltered during further tooth development. The mesenchymal cells only showed plakoglobin from cytodifferentiation onwards. Plakoglobin 1a morpholino-injected embryos showed normal tooth development with proper initiation and differentiation. Although plakoglobin is clearly present during normal odontogenesis, the loss of plakoglobin 1a does not influence tooth development. β-catenin was found at the cell borders of all cells of the successional lamina but also in the nuclei of surrounding mesenchymal cells. Only membranous, not nuclear, β-catenin, was found during morphogenesis stage. However, during cytodifferentiation stage, both nuclear and membrane-bound β-catenin was detected in the layers of the enamel organ as well as in the differentiating odontoblasts. Nuclear β-catenin is an indication of an activated Wnt pathway, therefore suggesting a possible role for Wnt signalling during zebrafish tooth development and replacement.

Fig 1. Plakoglobin distribution during the development of first-generation teeth.

Cross-sections through the pharyngeal region of a 48 hpf (A), 72 hpf (C), 96 hpf (E) and 100 hpf (G) zebrafish embryo and corresponding schematic drawings in B, D, F and H. A,B: Initiation stage of tooth 4V¹; the pharyngeal epithelium (ph.e.) expresses plakoglobin, in contrast to the mesenchymal cells. C,D: Morphogenesis stage of tooth 4V¹; plakoglobin is clearly expressed at the cell borders of the epithelial-derived tissue (arrowhead). The keratinized pad (k.p.) opposite the developing teeth, strongly expresses plakoglobin. Boxed area in C is magnified in C’. E,F: Tooth 4V¹ in late cytodifferentiation stage; teeth 3V¹ and 5V¹ in early cytodifferentiation stage. All teeth present display plakoglobin expression in epithelial-derived cell layers. G,H: Initiation of the first replacement tooth (arrow), 4V². The epithelial outgrowth shows plakoglobin expression while the condensed mesenchyme is negative. Boxed area in G is magnified in G’. Diagrams: blue patch: placode; green line: contour of the tooth; orange: tooth matrix. Orientation: dorsal to the top, ventral to the bottom of each figure; dashed line indicates mediosagittal plane. Additional abbreviations: ph.c: pharyngeal cavity; pl: placode; *: dental papilla. Scale bars = 20 μm.

Fig 2. Plakoglobin distribution in adult replacement teeth.

Cross-sections through one of the paired fifth branchial arches of an adult zebrafish. A: In contrast to the surrounding mesenchyme, all the cells constituting the successional lamina (s.l.) express plakoglobin at their cell membrane. Also the cells of the crypt epithelium express plakoglobin. B: Morphogenesis stage; the condensed mesenchyme constituting the dental papilla (black asterisk) is plakoglobin-negative. The enamel organ (arrowhead), including cervical loops (white asterisk), is strongly expressing plakoglobin. C: During early cytodifferentiation, the expression of plakoglobin remains limited to the inner and outer dental epithelium (arrowhead). D: Tooth in late cytodifferentiation stage, showing plakoglobin expression in the enamel organ (arrowhead) and in the differentiating odontoblasts lining the dental papilla (asterisk). E: Erupted functional tooth (FT) showing that even in a fully developed tooth plakoglobin expression persists in the reduced enamel organ (arrow). Orientation: dorsal to the top, ventral to the bottom, medial to the right and lateral to the left of the figure. Additional abbreviations: c: crypt surrounding the tip of the functional predecessor. Scale bars = 50 μm.

Fig 3. Comparing tooth development in control-injected and plakoglobin morpholino-injected zebrafish.

A,C,E: cross-sections through the pharyngeal cavity (ph.c.) of control-injected zebrafish at 72, 80 and 96 hpf, respectively; B,D,F: cross-sections at approximately the same level in plakoglobin morphant (MO) zebrafish at 72, 80 and 96 hpf, respectively. A,B: In both control-injected and morphant embryos the first tooth to develop, 4V¹ is in early cytodifferentiation stage. C,D: Tooth 4V¹ has continued to develop in control-injected and morphant embryos. Moreover, tooth 5V¹ is also visible in both dentitions. E,F: The first three tooth positions develop normally in both control-injected and in morphant zebrafish. Orientation: dorsal to the top, ventral to the bottom of each figure; dashed line indicates mediosagittal plane. Additional abbreviations: ph.e: pharyneal epithelium, line: contour of the developing tooth. Scale bars = 20 μm.

Fig 4. β-catenin distribution during the development of adult replacement teeth.

A,A’: The successional lamina (s.l.) shows expression of β-catenin at the cell membrane and in some nuclei. The mesenchyme also displays nuclear β-catenin. B: β-catenin is detected only at the plasma membrane of the epithelial cells during morphogenesis stage (arrowhead). C: In the enamel organ (arrowhead) β-catenin is expressed at the plasma membrane of cells of both inner and outer dental epithelium. C’: The dental papilla (asterisk) shows odontoblasts with membrane-bound and nuclear β-catenin expression. D: During late cytodifferentiation stage, β-catenin remains expressed in both the inner and outer dental epithelium as well as in the polarized odontoblasts (arrow) adjoining the tooth matrix. Orientation: dorsal to the top, ventral to the bottom, medial to the right and lateral to the left of the figure. Additional abbreviations: c: crypt slightly posterior to the tip of the functional predecessor; FT: functional tooth. Scale bars = 50 µm.