FIGURE 1. Tooth attachment in actinopterygians. (A) Schematic representation of type 1 and type 2 tooth attachment modes according to Fink (1981). In type 1 (e.g., zebrafish), a cylindrical collar of tissue, hitherto called “bone of attachment” (“attachment bone” in the terminology of Fink, 1981), firmly ankyloses the tooth to the supporting bone, resulting in continuous mineralization between the tooth base and the supporting (dentigerous) bone. In type 2, the cylindrical collar of “bone of attachment” is connected to the tooth by a ligament. The collar is positioned either on top, or inserted into the supporting bone. Note that in the latter case, Berkovitz and Shellis (2016) label the “bone of attachment” as “pedicel”, reserving the term “bone of attachment” for the tissue serving to attach the pedicels. Note that the term “bone of attachment” is used between quotation marks, pending a more appropriate term based on the findings in this study. (B) and (C) Toluidine blue-stained semi-thin sections of a zebrafish initiator tooth at 6dph (B) and an adult tooth (C), both attached, as in type 1 attachment. Note multiple osteocytes in the adult supporting bone, a single one in the “bone of attachment” (encircled). Inset: enlargement of “bone of attachment” (dotted rectangle) with cell prolongations (open arrowhead). (D) Scanning electron micrograph of attached adult zebrafish teeth. (E) Overview transmission electron microscopy (TEM) picture of the base of a zebrafish first-generation tooth at the level of the cervical loop. The dentin, “bone of attachment” and supporting bone form a continuous mineralized tissue, covered along the pulpal side with scleroblasts that appear to be involved in the deposition of more than one matrix. cb, ceratobranchial cartilage; cl, cervical loop; d, dentin; dp, dental pulp; sb, supporting bone; asterisks, “bone of attachment”; arrows, scleroblasts forming “bone of attachment”; black arrowheads, odontoblasts. Scale bar (B)=10μm, (C)=50μm, (D)=100μm, and (E)=5μm.

Figure 2. Expression of secretory calcium-binding phosphoprotein 5 (scpp5) during zebrafish tooth development. Transverse sections of zebrafish postembryonic stages (72–120hpf – A–E) and adults (F–J) in the region of the pharyngeal jaws, with explanatory schemes for postembryonic stages. At 72hpf (A) the initiator tooth (4V¹) at LC stage expresses scpp5 in both ameloblasts and odontoblasts, whereas no expression is detected for the first-generation teeth 3V¹ and 5V¹, still at EC stage. Between 80 (B) and 96hpf (C) tooth 4V¹, now attached, shows no expression of scpp5, while 3V¹ and 5V¹ at LC stages start to express scpp5 in both ameloblasts and odontoblasts. Between 96 (D) and 120hpf (E) the second-generation teeth (4V², 3V², and 5V²) fail to express scpp5 during EC but upregulate expression in both ameloblasts and odontoblasts at LC. During the development of adult teeth, the expression of scpp5 is detected in both ameloblasts and odontoblasts at EC (F), LC (G) and when the tooth starts to attach to bone (H). Expression is restricted to odontoblasts when the tooth is completely attached [(I) and (J)]. Tooth developmental stages: A, phase of attachment; EC, early cytodifferentiation; LC, late cytodifferentiation. Br, brain; cl, cervical loop; Nt, notochord; sb, supporting bone; Y, yolk; asterisks, “bone of attachment”; arrows, cells forming “bone of attachment”; white arrowheads, ameloblasts; black arrowheads, odontoblasts. Scale bar (A-E)=10μm, (F–G)=50μm, (H–J)=100μm.

FIGURE 3. Zns-5 immunodetection during adult zebrafish tooth development. Transverse sections of adult zebrafish in the region of the pharyngeal jaws, used for Zns-5 immunohistochemistry (A–C) or stained with azan (A’–C’). During EC (A,A’) and LC (B,B’), Zns-5 is localized in ameloblasts, with no signal detected in odontoblasts. When teeth are attached (C,C’) ameloblasts cease to stain for Zns-5 and the antigen is only detected in the cells lining the “bone of attachment” and the osteoblasts lining the supporting bone. Note osteocytes in the supporting bone, but their absence in the “bone of attachment” (C’). cl, cervical loop; sb, supporting bone; asterisks, “bone of attachment”; black arrowheads, odontoblasts; black arrows, cells forming “bone of attachment”; white arrowheads, ameloblasts; white arrows, osteoblasts. Scale bar (A–D, C,’D’)=100μm, (A’,B’)=50μm.

Figure 4. Interpretative scheme showing localization of scpp5 transcripts and Zns-5 detection during zebrafish tooth development. Panel (A) shows a representation of scpp5 expression during first and second generation and adult tooth formation, from the stage of initiation and morphogenesis to tooth attachment, and of Zns-5 detection in adult teeth, at the same developmental stages. Panel (B) shows the comparison between the expression of scpp5 and the localization of Zns-5, the former being exclusively expressed by ameloblasts and odontoblasts and the latter by cells forming the pedicel and by osteoblasts. In both panels, the adult tooth is depicted in a similar way as the first/second-generation teeth, although the tooth is larger, with thicker walls, and is attached to bone with all cartilage having been resorbed in this area. cb, ceratobranchial cartilage; cl, cervical loop; and dp, dental pulp.