Wnt signaling during tooth replacement in zebrafish (Danio rerio): pitfalls and perspectives

Abstract

The canonical (β-catenin dependent) Wnt signaling pathway has emerged as a likely candidate for regulating tooth replacement in continuously renewing dentitions. So far, the involvement of canonical Wnt signaling has been experimentally demonstrated predominantly in amniotes. These studies tend to show stimulation of tooth formation by activation of the Wnt pathway, and inhibition of tooth formation when blocking the pathway. Here, we report a strong and dynamic expression of the soluble Wnt inhibitor dickkopf1 (dkk1) in developing zebrafish (Danio rerio) tooth germs, suggesting an active repression of Wnt signaling during morphogenesis and cytodifferentiation of a tooth, and derepression of Wnt signaling during start of replacement tooth formation. To further analyse the role of Wnt signaling, we used different gain-of-function approaches. These yielded disjunct results, yet none of them indicating enhanced tooth replacement. Thus, masterblind (mbl) mutants, defective in axin1, mimic overexpression of Wnt, but display a normally patterned dentition in which teeth are replaced at the appropriate times and positions. Activating the pathway with LiCl had variable outcomes, either resulting in the absence, or the delayed formation, of first-generation teeth, or yielding a regular dentition with normal replacement, but no supernumerary teeth or accelerated tooth replacement. The failure so far to influence tooth replacement in the zebrafish by perturbing Wnt signaling is discussed in the light of (i) potential technical pitfalls related to dose- or time-dependency, (ii) the complexity of the canonical Wnt pathway, and (iii) species-specific differences in the nature and activity of pathway components. Finally, we emphasize the importance of in-depth knowledge of the wild-type pattern for reliable interpretations. It is hoped that our analysis can be inspiring to critically assess and elucidate the role of Wnt signaling in tooth development in polyphyodonts.

Keywords: APC; LiCl; Wnt; axin; dickkopf; polyphyodont; tooth replacement; zebrafish.

Figure 1

Simplified scheme of canonical Wnt signaling. In the absence of a ligand (A), or in the presence of an inhibitor, β-catenin accumulates in the cytoplasm and is targeted for destruction. In the presence of a ligand (B), the destruction complex is disassembled, β-catenin accumulates and translocates to the nucleus, where it associates with transcription factors of the LEF/TCF family to regulate transcription of target genes. Abbreviations: APC, Adenomatous Polyposis Coli; β-ctn, β-catenin; β-TrCP, β-transducin-repeat-containing protein; CK1α, casein kinase 1α; Dsh, Disheveled; Fz, frizzled; GSK-3β, glycogen synthase kinase 3β; LRP, low density lipoprotein receptor related protein; P, phosphorylated; PM, plasma membrane; TCF, T Cell Factor; Ub, Ubiquitin. Adapted from Denayer (2006).

Figure 2

Simplified scheme of tooth development in zebrafish, as exemplified for tooth 4V¹. Tooth development proceeds through five successive, and partially overlapping stages, initiation, morphogenesis, early cytodifferentiation, late cytodifferentiation, and attachment. Only the epithelium is color-coded. Abbreviations: dp, dental papilla; eo, enamel organ; ide, inner dental epithelium; ode, outer dental epithelium; pc, pulp cavity; pe, pharyngeal epithelium; tm, tooth matrix.

Figure 3

Expression of dkk1 in different stages of development of first- and second-generation teeth. (A) 60 hpf: weak mesenchymal expression in tooth 4V¹, epithelial expression starts to be upregulated in 3V¹; (B) 72 hpf: strong epithelial expression in 3V¹ and 5V¹, but loss of signal in 4V¹; (C) 80 hpf: start of formation of 4V² showing no signal yet; epithelial signal is weakening in 3V¹ and 5V¹; (D,E) 100 hpf; dkk1 is now downregulated in all first-generation teeth [3V¹ and 4V¹ in (D), 5V¹ in (E)], whilst the gene is upregulated in the first replacement tooth (4V²); (F) 144 hpf: expression in the two next second-generation teeth, 3V² and 5V². Dark-colored arrowheads indicate specific first-generation teeth and point downwards: dark green, 4V¹, red, 3V¹, dark blue, 5V¹; light-colored arrowheads indicate second-generation teeth and point upwards: light green, 4V², pink, 3V², light blue, 5V². Note that epithelium and mesenchyme of a tooth germ are not necessarily visible on a single section through the germ. Sections may also be slightly oblique and different sets of teeth may be visible on either body side. Insets show higher magnification of the teeth on the left side in each micrograph. Scale bars = 50 μm.

Figure 4

Dentition in mbl and apc mutant zebrafish. Dentition in WT (A) and mbl mutants (B) at 5dpf, and WT (C) and apc mutants (D) at 3 dpf. Insets: WT (A) and mbl mutant (B) at 6 dpf, and WT (C) and apc mutant (D) at 2 dpf. Color codes for arrowheads as in Figure 3. Note the complete similarity in tooth pattern between 5 dpf WT (A) and mbl mutants (B), and the substantial developmental delay in 3 dpf apc mutants (D) compared to WT fish (C), as can be observed from the large amount of yolk (y) still present, the thin endodermal layer covering the yolk (black arrowhead), and the absence of cartilage in the neurocranial base flanking the notochord (nc). Ov, otic vesicle. Note that sections are slightly oblique and different sets of teeth are visible on either body side. Scale bars = 50 μm.

Figure 5

Enhanced Wnt signaling through LiCl treatment. Dentition of control zebrafish at start of treatment at 45 hpf (A), and treated with 300 mM LiCl for 1 h (B, fixed at 60 hpf and C, fixed at 112 hpf). In (B,C) only tooth 4V¹ is present. Dentition of control zebrafish at start of treatment at 72 hpf (D), and treated with 300 mM LiCl for 1 h (E, fixed at 92 hpf), or treated with 300 mM LiCl for 10 min (F, fixed at 112 hpf). (E) All three first-generation teeth (3V¹, 4V¹, and 5V¹) have formed and replacement tooth 4V² is in morphogenesis stage (note that the section is slightly oblique and different sets of teeth are visible on either body side); (F) several tooth buds appear to form from a single enamel organ (black arrowhead, inset). Color codes for arrowheads as in Figure 3. Scale bars = 50 μm for A–F and 20 μm for inset.