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.

Table 1. Expression of dkk1 in epithelium and mesenchyme of first-generation teeth (3V¹, 4V¹, and 5V¹) and their successors (3V², 4V², and 5V²) between 48 and 152 hpf.