Immunolocalization of enamel matrix protein-like proteins in the tooth enameloid of spotted gar, Lepisosteus oculatus, an actinopterygian bony fish

Abstract

Enameloid is a well-mineralized tissue covering the tooth surface in fish and it corresponds to the outer-most layer of dentin. It was reported that both dental epithelial cells and odontoblasts are involved in the formation of enameloid. Nevertheless, the localization and timing of secretion of ectodermal enamel matrix proteins (EMPs) in enameloid are unclear. In the present study, the enameloid matrix during the stages of enameloid formation in spotted gar, Lepisosteus oculatus, an actinopterygian, was examined mainly by transmission electron microscopy-based immunohistochemistry using an anti-mammalian amelogenin (AMEL) antibody and antiserum. Positive immunoreactivity with the antibody and antiserum was found in enameloid from the surface to the dentin-enameloid junction just before the formation of crystallites. This immunoreactivity disappeared rapidly before the full appearance of crystallites in the enameloid during the stage of mineralization. Immunolabelling was usually found along the collagen fibrils but was not seen on the electron-dense fibrous structures, which were probably derived from matrix vesicles in the previous stage. In inner dental epithelial (IDE) cells, the granules in the distal cytoplasm often showed positive immunoreactivity, suggesting that the EMP-like proteins originated from IDE cells. EMP-like proteins in the enameloid matrix might be common to the EMP-like proteins previously reported in the collar enamel of teeth and ganoine of ganoid scales, because they exhibited marked immunoreactivity with the same anti-mammalian AMEL antibodies. It is likely that EMP-like proteins are involved in the formation of crystallites along collagen fibrils in enameloid.

Ganoin and acrodin formation on scales and teeth in spotted gar: A vital role of enamelin in the unique process of enamel mineralization

“…Dentin forms next, growing away from acrodin, and then collar ganoin develops on the surface of dentin. The formation of acrodin progresses through matrix formation, mineralization, and maturation (Sasagawa & Ishiyama, 2005a; Sasagawa et al, 2019). Using LM, reactivity with the anti‐SCPP5N antibody was initially detected within the acrodin matrix in the mineralization stage after the body of the matrix was formed.…”

“…While the acrodin matrix undergoes maturation, IDE cells begin to secrete the collar ganoin matrix (Sasagawa & Ishiyama, 2005a; Sasagawa et al, 2019). In the secretory stage of collar ganoin formation, the anti‐SCPP5N antibody showed marked reactivity across the entire thickness of the collar ganoin matrix (Figure 3e,f).…”

“…In the late mineralization stage, fine crystallites were found in a narrow surface region of the acrodin matrix (Figure 4f). These fine crystallites subsequently fused with more massive slender crystallites that grew from the junction with dentin (Sasagawa et al, 2019). Investigation using sequential sections revealed that the distribution of these fine crystallites (Figure 4f) overlaps with the surface region showing reactivity with the anti‐AMBN antibody (Figure 4g).…”

“…Despite these similarities, however, the collar ganoin matrix initiates mineralization after the matrix forms an unmineralized layer (Sasagawa et al, 2008; Sasagawa et al, 2019). This process significantly differs from the formation of scale ganoin and mammalian enamel, in which mineralization begins concurrently with the initial formation of the matrix layer (Figure 5a–d) (Rönnholm, 1962a, 1962b; Smith et al, 2016).…”

The conundrum of pharyngeal teeth origin: the role of germ layers, pouches, and gill slits

“…While the development of the teeth in basal (non‐teleost) actinopterygians has been studied in some detail [e.g. bichir, Polypterus senegalus (Sire et al ., 2002 ); bowfin, Amia calva (Degener, 1924 ); gar, Lepisosteus oculatus (Sasagawa et al ., 2019 ); Siberian sturgeon, Acipenser baeri (Gisbert et al ., 1998 )], the germ layer origin of the enamel organ has usually not been addressed. Still, endoderm forms substantial parts of the orofacial epithelia in these taxa, and that includes contributions to the teeth (Minarik et al ., 2017 ).…”