To connect human biology to fish biomedical models, we sequenced the genome of spotted gar (Lepisosteus oculatus), whose lineage diverged from teleosts before the teleost genome duplication (TGD). The slowly evolving gar genome conserved in content and size many entire chromosomes from bony vertebrate ancestors. Gar bridges teleosts to tetrapods by illuminating the evolution of immunity, mineralization, and development (e.g., Hox, ParaHox, and miRNA genes). Numerous conserved non-coding elements (CNEs, often cis-regulatory) undetectable in direct human-teleost comparisons become apparent using gar: functional studies uncovered conserved roles of such cryptic CNEs, facilitating annotation of sequences identified in human genome-wide association studies. Transcriptomic analyses revealed that the sum of expression domains and levels from duplicated teleost genes often approximate patterns and levels of gar genes, consistent with subfunctionalization. The gar genome provides a resource for understanding evolution after genome duplication, the origin of vertebrate genomes, and the function of human regulatory sequences.

Gar is an actinopterygian that has bone, dentin, enameloid, and ganoin (enamel) in teeth and/or scales. Mineralization of these tissues involves genes encoding various secretory calcium-binding phosphoproteins (SCPPs) in osteichthyans, but no SCPP genes have been identified in chondrichthyans to date. In the gar genome, we identified 38 SCPP genes, seven of which encode "acidic-residue-rich" proteins and 31 encode "Pro/Gln (P/Q) rich" proteins. These gar SCPP genes constitute the largest known repertoire, including many newly identified P/Q-rich genes expressed in teeth and/or scales. Among gar SCPP genes, six acidic and three P/Q-rich genes were identified as orthologs of sarcopterygian genes. The sarcopterygian orthologs of most of these acidic genes are involved in bone and/or dentin formation, and sarcopterygian orthologs of all three P/Q-rich genes participate in enamel formation. The finding of these genes in gar suggests that an elaborate SCPP gene-based genetic system for tissue mineralization was already present in stem osteichthyans. While SCPP genes have been thought to originate from ancient SPARCL1, SPARCL1L1 appears to be more closely related to these genes, because it established a structure similar to acidic SCPP genes probably in stem gnathostomes, perhaps at about the same time with the origin of tissue mineralization. Assuming enamel evolved in stem osteichthyans, all P/Q-rich SCPP genes likely arose within the osteichthyan lineage. Furthermore, the absence of acidic SCPP genes in chondrichthyans might be explained by the secondary loss of earliest acidic genes. It appears that many SCPP genes expanded rapidly in stem osteichthyans and in basal actinopterygians.

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The morphological features of tooth enamel and enameloid in actinopterygian fish are reviewed to provide basic data concerning the biomineralization of teeth in lower vertebrates. Enameloid, which covers the tooth surface, is a unique well-mineralized tissue and usually has the same functions as mammalian tooth enamel. However, the development of enameloid is different from that of the enamel produced by dental epithelial cells. Enameloid is made by a combination of odontoblasts and dental epithelial cells. An organic matrix that contains collagen is provided by odontoblasts, and then dental epithelial cells dissolve the degenerate matrix and supply inorganic ions during advanced crystal growth in enameloid. It is likely that enameloid is a good model for studying the growth of well-mineralized hard tissues in vertebrates. Some actinopterygian fish possess a collar enamel layer that is situated at the surface of the tooth shaft, indicating that the origin of tooth enamel is found in fish. Collar enamel is thought to be a precursor of mammalian enamel, although it is thin and not well mineralized in comparison with enameloid. In Lepisosteus and Polypterus, both of which are living actinopterygians, both enameloid and enamel are found in the same tooth. Therefore, they are suitable materials for examining the developmental processes of enameloid and enamel and the relationship among them.

We used the evolutionary analysis of amelogenin (AMEL) in 80 amniotes (52 mammalian and 28 reptilian sequences) to aid in the genetic diagnosis of X-linked amelogenesis imperfecta (AIH1). Out of 191 residues, 77 were found to be unchanged in mammals, and only 34 in amniotes. The latter are considered crucial residues for enamel formation, while the 43 residues conserved only in mammals could indicate that they play new, important roles for enamel formation in this lineage. The 5 substitutions leading to AIH1 were validated when the mammalian dataset was used, and 4 of them with the amniote dataset. These 2 sequence datasets will facilitate the validation of any human AMEL mutation suspected of involvement in AIH1. This evolutionary analysis also revealed numerous residues that appeared to be important for correct AMEL function, but their role remains to be elucidated.

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Histologically the outer layer of the collar enameloid obviously differs from the inner layer, and it has a degree of mineralization nearly as high as the cap enameloid which has the highest. In the stage of matrix formation, the organic matrix of the collar enameloid contains a number of collagen fibers, and odontoblasts display features suggesting that these cells actively synthesized and secreted collagen. A number of cell processes, matrix vesicles and some cell debris which were probably derived from the odontoblasts were observed in the organic matrix of the collar enameloid. We consider that the majority of the organic matrix in collar enameloid originates from the odontoblasts. In the stage of maturation, collagen fibers were not observed in the outer layer of the collar enameloid in demineralized specimens. In the IDE cells during this stage, the complex infoldings of cell membranes developed in the distal portion, and several lysosomal granules and irregular-shaped granules containing many tubular structures, were observed in the distal cytoplasm. In the ODE cells, abundant labyrinthine canals appeared in the cytoplasm, and capillary vessels were found close to the outer surface of the ODE cells. We assume that the higher mineralized outer layer of the collar enameloid is made possible by the absorptive and transport functions of the epithelial cells during the stage of maturation. It is considered that the collar enameloid in this study was initially produced by the odontoblasts and then reconstructed by the epithelial cells, so that the collar enameloid differs from true enamel.

Previous studies have demonstrated the morphological similarity of the enamel-like layer found in the teeth of the coelacanth, lungfish and gar-pike to the enamel of tetrapods. In order to clarify the phylogenetic continuity between both structures, tooth germs of the gar-pike were immunocytochemically studied using an anti-bovine amelogenin polyclonal antibody. Intense immunoreaction was shown over the enamel-like matrix layer. Certain cell organelles associated with the secretory pathway of the ameloblasts were recognized as immunoreactive. These results indicate that the enamel-like layer of the gar-pike is a tissue homologous with the mammalian enamel because both possess a common, amelogenin-like substance.