Immunohistochemical and Western Blotting Analyses of Ganoine in the Ganoid Scales of Lepisosteus oculatus: an Actinopterygian Fish

Abstract

In order to compare its characteristics with those of jaw tooth collar enamel, normally developing and experimentally regenerating ganoine from ganoid scales of Lepisosteus oculatus (spotted gar), an actinopterygian fish species, was examined by Western blotting and immunohistochemistry. Amelogenin, a major enamel matrix protein (EMP), is widely found from sarcopterygian fish to mammals. Therefore, we used antimammalian amelogenin antibodies and antisera: an antibody against bovine amelogenin; antiserum against porcine amelogenin; and region-specific antibodies or antiserum against the C-terminus, middle region, or N-terminus of porcine amelogenin in this study. Positive immunoreactivity with the antibody against bovine amelogenin, antiserum against porcine amelogenin, and the middle and C-terminal region-specific antibodies was detected in both normally developing and regenerating ganoine matrix, as well as in granules found within inner ganoine epithelial cells. These immunohistochemical analyses indicated that the Lepisosteus ganoine matrix contains EMP-like proteins with epitopes similar to mammalian amelogenins. In Western blotting analyses of regenerating ganoid scales with the antibovine amelogenin antibody, two protein bands with molecular weights of approximately 78 and 65 kDa were detected, which were similar to those found in Lepisosteus tooth enamel. Our study suggests that in Lepisosteus, EMP-like proteins in the ganoine matrix corresponded to those in tooth enamel. However, it was revealed that the 78 and 65 kDa EMP-like proteins were different from 27 kDa bovine amelogenin.

Evolution of dental tissue mineralization: an analysis of the jawed vertebrate SPARC and SPARC-L families

“…This is because (i) enameloid is present at the surface of stem gnathostome odontodes, chondrichthyan teeth and dermal denticles, actinopterygian oral and pharyngeal teeth, and caudate amphibian larval teeth [ 22 – 25 ] and (ii) teleost and chondrichthyan enameloids were proposed to be homologous [ 2 , 21 ]. On the other hand, it has been inferred that the osteichthyan last common ancestor harbored enamel-covered odontodes [ 2 , 26 – 29 ]. Indeed, evidence showing that (i) EMP ( E namel M atrix P roteins) genes are specifically expressed in ameloblasts during mammalian enamel matrix secretion and maturation, [ 30 ] and (ii) EMPs are present in the gar genome [ 4 ], suggest that the surface tissue of tooth and ganoid scales of non-teleost actinopterygians (polypterids and lepisosteids, [ 26 , 27 ]) are homologous to the sarcopterygian enamel [ 4 ].…”

SCPP Genes and Their Relatives in Gar: Rapid Expansion of Mineralization Genes in Osteichthyans

“…Gars are known for their strong jaws and protective armor, equipped with various hard tissues. Their teeth consist of a body of dentin that is capped with enameloid at the apex and collared with enamel around the shaft (Prostak et al., ; Ishiyama et al., ; Ishiyama et al., ; Sasagawa and Ishiyama, ; Sasagawa et al., , ), while their scales are composed of a bony plate covered with ganoin, known as the ganoid scale of the lepidosteoid type (Williamson, ; Goodrich, ; Sire, , ; Sire et al., ; Sasagawa et al., ). Similar to their scales, ganoin is also present on the outer surface of bony fin rays (lepidotrichia) in gars (Goodrich, ; Schaeffer, ; Géraudie, ; Zylberberg et al., ).…”

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

“…In gars, scale ganoin forms on a basal bone plate (Sire et al, 2009). As IGE cells differentiate posteroanteriorly (top to bottom in Figure 3i–l), IGE cells elongate and begin to secrete the ganoin matrix on the bone matrix (thick matrix on top in Figure 3i) (Sasagawa et al, 2016; Sire, 1994). During ganoin matrix secretion, striking reactivity with the anti‐SCPP5N antibody was detected throughout the matrix (Figure 3j).…”

“…Some of these mammillary knobs subsequently aggregated, and extending mineral bundles integrated into thicker mineral bundles. We consider that these thick mineral bundles are responsible for the formation of the protoprismatic structure of ganoin (Sasagawa et al, 2016), which resembles the prismatic structure of mammalian enamel (Ørvig, 1978; Smith, 1989). Deep in the subsurface zone of the bone matrix, mineral foci coalesced into a continuous mineral layer (Figure 5e).…”

“…This result suggests that Scpp5 is secreted initially into the bone matrix in the presecretory stage earlier than Enam and that the secretion of Enam begins immediately before the onset of the secretory stage. The presecretory stage is followed by the formation of the ganoin layer through upregulation of matrix secretion (Figure 3i) (Sasagawa et al, 2016; Sire, 1994). By contrast, in the subsequent stage of acrodin formation, Scpp5 was not detected in the matrix (Figure 4d), which implies downregulation of the secretion and enzymatic processing of Scpp5.…”