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Gars and bichirs develop scales and teeth with ancient actinopterygian characteristics. Their scale surface and tooth collar are covered with enamel, also known as ganoin, whereas the tooth cap is equipped with an enamel-like tissue, acrodin. Here, we investigated the formation and mineralization of the ganoin and acrodin matrices in spotted gar, and the evolution of the scpp5, ameloblastin (ambn), and enamelin (enam) genes, which encode matrix proteins of ganoin. Results suggest that, in bichirs and gars, all these genes retain structural characteristics of their orthologs in stem actinopterygians, presumably reflecting the presence of ganoin on scales and teeth. During scale formation, Scpp5 and Enam were initially found in the incipient ganoin matrix and the underlying collagen matrix, whereas Ambn was detected mostly in a surface region of the well-developed ganoin matrix. Although collagen is the principal acrodin matrix protein, Scpp5 was detected within the matrix. Similarities in timings of mineralization and the secretion of Scpp5 suggest that acrodin evolved by the loss of the matrix secretory stage of ganoin formation: dentin formation is immediately followed by the maturation stage. The late onset of Ambn secretion during ganoin formation implies that Ambn is not essential for mineral ribbon formation, the hallmark of the enamel matrix. Furthermore, Scpp5 resembles amelogenin that is not important for the initial formation of mineral ribbons in mammals. It is thus likely that the evolution of ENAM was vital to the origin of the unique mineralization process of the enamel matrix.

Background Given the need for descriptive and increasingly mechanistic morphological analyses, contrast-enhanced microcomputed tomography (microCT) represents perhaps the best method for visualizing 3D biological soft tissues in situ. Although staining protocols using phosphotungstic acid (PTA) have been published with beautiful visualizations of soft tissue structures, these protocols are often aimed at highly specific research questions and are applicable to a limited set of model organisms, specimen ages, or tissue types. We provide detailed protocols for micro-level visualization of soft tissue structures in mice at several embryonic and early postnatal ages using PTA-enhanced microCT. Results Our protocols produce microCT scans that enable visualization and quantitative analyses of whole organisms, individual tissues, and organ systems while preserving 3D morphology and relationships with surrounding structures, with minimal soft tissue shrinkage. Of particular note, both internal and external features of the murine heart, lungs, and liver, as well as embryonic cartilage, are captured at high resolution. Conclusion These protocols have broad applicability to mouse models for a variety of diseases and conditions. Minor experimentation in the staining duration can expand this protocol to additional age groups, permitting ontogenetic studies of internal organs and soft tissue structures within their 3D in situ position.

Among extant cetaceans, mysticetes are filter feeders that do not possess teeth and use their baleen for feeding, while most odontocetes are considered suction feeders, which capture prey by suction without biting or chewing with teeth. In the present study, we address the functionality of amelogenin (AMEL) genes in cetaceans. AMEL encodes a protein that is specifically involved in dental enamel formation and is located on the sex chromosomes in eutherians. The X-copy AMELX is functional in enamel-bearing eutherians, whereas the Y-copy AMELY appears to have undergone decay and was completely lost in some species. Consistent with these premises, we detected various deleterious mutations and/or non-canonical splice junctions in AMELX of mysticetes and four suction feeding odontocetes, Delphinapterus leucas, Monodon monoceros, Kogia breviceps, and Physeter macrocephalus, and in AMELY of mysticetes and odontocetes. Regardless of the functionality, both AMELX and AMELY are equally and unusually small in cetaceans, and even their functional AMELX genes presumably encode a degenerate core region, which is thought to be essential for enamel matrix assembly and enamel crystal growth. Furthermore, our results suggest that the most recent common ancestors of extant cetaceans had functional AMELX and AMELY, both of which are similar to AMELX of Platanista minor. Similar small AMELX and AMELY in archaic cetaceans can be explained by gene conversion between AMELX and AMELY. We speculate that common ancestors of modern cetaceans employed a degenerate AMELX, transferred from a decaying AMELY by gene conversion, at an early stage of their transition to suction feeders.

The bowfin (Amia calva) is a ray-finned fish that possesses a unique suite of ancestral and derived phenotypes, which are key to understanding vertebrate evolution. The phylogenetic position of bowfin as a representative of neopterygian fishes, its archetypical body plan and its unduplicated and slowly evolving genome make bowfin a central species for the genomic exploration of ray-finned fishes. Here we present a chromosome-level genome assembly for bowfin that enables gene-order analyses, settling long-debated neopterygian phylogenetic relationships. We examine chromatin accessibility and gene expression through bowfin development to investigate the evolution of immune, scale, respiratory and fin skeletal systems and identify hundreds of gene-regulatory loci conserved across vertebrates. These resources connect developmental evolution among bony fishes, further highlighting the bowfin’s importance for illuminating vertebrate biology and diversity in the genomic era., Analysis of a chromosome-level bowfin genome assembly sheds light into neopterygian fish evolution. Chromatin accessibility and gene expression profiling provides insight into bowfin embryonic development.

Continuous fossil discoveries and recent analysis of genomic data are revealing that anatomically modern traits evolved following a complex evolutionary path. Our current understanding of human evolution considers that the skull likely evolved as a mosaic of correlated traits whose development is in turn integrated with other organs, such as the brain. However, we still know little about the complexity of the genetic networks supervising these processes and the developmental mechanisms underlying observed patterns of morphological evolution. To further understand the integrated evolution of the human skull and brain, it is crucial to characterize the gene expression patterns that provide detailed instructions to organize cell behavior, tissue growth, and organ patterning and to investigate how development translates genetic variation into phenotypic variation. We propose that developmental biology analyses using mouse models can enable the experimental test of hypotheses about how craniofacial morphological diversification occurred across the human lineage. Here, we combine molecular (Whole-Mount in situ Hybridization), 3D imaging (Optical Projection Tomography), and morphometric techniques to assess changes in the space, time, and intensity of gene expression patterns during embryonic development. We used Fgfr2+/P253R Apert syndrome mouse models, in which an Fgfr2 mutation is associated with midfacial hypoplasia, brachycephaly, and macrocephaly. These pathological phenotypes provide a practical model of processes similar to those involved in the evolution of a large globular braincase and a small, retracted face in modern humans. We compared the expression patterns of two downstream targets of Fgfr2, Dusp6 and Hand2, that are relevant for brain and craniofacial development, in 25 mice carrying the Fgfr2 mutation and 27 littermates collected between E10.5 and E11.5. Qualitative comparisons revealed that both genes are co-expressed in the brain and face with variable intensity depending on developmental time and genotype. Results suggest that the FGF signaling pathway participates in brain and face morphogenesis and that changes in the location and timing of gene expression can induce correlated changes in brain and craniofacial systems. This supports the hypothesis that facial retraction and encephalization likely evolved as direct and correlated responses to common signaling pathways. We are testing high throughput methods to produce objective quantifications of gene expression patterns that can be used in future analyses to reveal the complex genotype-phenotype correspondence in the evolution of modern human traits.

Craniofacial syndromes often involve skeletal defects of the head. Studying the development of the chondrocranium (the part of the endoskeleton that protects the brain and other sense organs) is crucial to understanding genotype-phenotype relationships and early detection of skeletal malformation. Our goal is to segment craniofacial cartilages in 3D micro-CT images of embryonic mice stained with phosphotungstic acid. However, due to high image resolution, complex object structures, and low contrast, delineating fine-grained structures in these images is very challenging, even manually. Specifically, only experts can differentiate cartilages, and it is unrealistic to manually label whole volumes for deep learning model training. We propose a new framework to progressively segment cartilages in high-resolution 3D micro-CT images using extremely sparse annotation (e.g., annotating only a few selected slices in a volume). Our model consists of a lightweight fully convolutional network (FCN) to accelerate the training speed and generate pseudo labels (PLs) for unlabeled slices. Meanwhile, we take into account the reliability of PLs using a bootstrap ensemble based uncertainty quantification method. Further, our framework gradually learns from the PLs with the guidance of the uncertainty estimation via self-training. Experiments show that our method achieves high segmentation accuracy compared to prior arts and obtains performance gains by iterative self-training.

In most modern bony vertebrates, a considerable portion of the chondrocranium remains cartilaginous only during a relatively small window of embryonic development, making it difficult to study this complex structure. Yet, the transient nature of some chondrocranial elements is precisely why it is so intriguing. Since the chondrocranium has never been lost in any vertebrate, its function is critical to craniofacial development, disease, and evolution. Experimental evidence for the various roles of the chondrocranium is limited, and though snapshots of chondrocranial development in various species at isolated time points are valuable and informative, these cannot provide the data needed to determine the functions of the chondrocranium, or its relationship to the dermatocranium in evolution, in development, or in disease. Observations of the spatiotemporal associations of chondrocranial cartilage, cartilage bone, and dermal bone over early developmental time are available for many vertebrate species and these observations represent the data from which we can build hypotheses. The testing of those hypotheses requires precise control of specific variables like developmental time and molecular signaling that can only be accomplished in a laboratory setting. Here, we employ recent advances in contrast-enhanced micro computed tomography to provide novel 3D reconstructions of the embryonic chondrocranium in relation to forming dermal and cartilage bones in laboratory mice across three embryonic days (E13.5, E14.5, and E15.5). Our observations provide support for the established hypothesis that the vertebrate dermal (exo-) skeleton and endoskeleton evolved as distinct structures and remain distinct. Additionally, we identify spatiotemporal patterning in the development of the lateral wall, roof, and braincase floor of the chondrocranium and the initial mineralization and growth of the bones associated with these cartilages that provides support for the hypothesis that the chondrocranium serves as a scaffold for developing dermatocranial bones. The experimental protocols described and data presented provide tools for further experimental work on chondrocranial development.

Secretory calcium-binding phosphoprotein (SCPP) genes are expressed in the skin and jaw during the formation of bone, teeth, and scales in osteichthyans (bony vertebrates). Among these mineralized skeletal units is the ganoid scale, found in various fossil actinopterygians (ray-finned fish) but confirmed only in Polypteriformes (bichirs, reedfish) and Lepisosteiformes (gars) among extant clades. Here, we examined SCPP genes in the genome of seven non-teleost actinopterygian species that possess or do not possess ganoid scales. As a result, 39-43 SCPP genes were identified in Polypteriformes and Lepisosteiformes, whereas 22-24 SCPP genes were found in Acipenseriformes (sturgeons, paddlefish) and Amiiformes (bowfin). Most of these genes form two clusters in the genome of Polypteriformes, Lepisosteiformes, and Amiiformes, and these two clusters are duplicated in Acipenseriformes. Despite their distant phylogenetic relationship, Polypteriformes and Lepisosteiformes retain many orthologous SCPP genes. These results imply that common ancestors of extant actinopterygians possessed a large repertoire of SCPP genes, and that many SCPP genes were lost independently in Acipenseriformes and Amiiformes. Notably, most SCPP genes originally located in one of the two SCPP gene clusters are retained in Polypteriformes and Lepisosteiformes but were secondarily lost in Acipenseriformes and Amiiformes. In Lepisosteiformes, orthologs of these lost genes show high or detectable expression levels in the skin but not in the jaw. We thus hypothesize that many SCPP genes located in this cluster are involved in the formation of ganoid scales in Polypteriformes and Lepisosteiformes, and that their orthologs and ganoid scales were convergently lost in Acipenseriformes and Amiiformes.

The bowfin fish (Amia calva) diverged before the genome duplication in teleost fishes, and its archetypical body plan and slow rate of molecular evolution make it a key species for genomic exploration as a basal representative of the neopterygian fishes. To investigate the evolution and development of ray-finned fishes, we generated a chromosome-level genome assembly for bowfin that enables gene-order analyses which settle its long-debated, phylogenetic relationship with gars. We analyze the genomic underpinnings of the bowfin’s unique combination of derived and ancestral phenotypes involving the immune system as well as scale, respiratory organ, and skeletal development. By detailing chromatin accessibility and gene expression through bowfin development, we connect developmental gene regulatory loci across vertebrates. We illustrate the utility of these genomic resources to connect developmental evolution across bony fishes, showing the importance of bowfin in understanding vertebrate biology and diversity.

Mutations of Odontogenesis-Associated Phosphoprotein (ODAPH, OMIM *614829) cause autosomal recessive amelogenesis imperfecta, however, the function of ODAPH during amelogenesis is unknown. Here we characterized normal Odaph expression by in situ hybridization, generated Odaph truncation mice using CRISPR/Cas9 to replace the TGC codon encoding Cys41 into a TGA translation termination codon, and characterized and compared molar and incisor tooth formation in Odaph+/+, Odaph+/C41*, and OdaphC41*/C41* mice. We also searched genomes to determine when Odaph first appeared phylogenetically. We determined that tooth development in Odaph+/+ and Odaph+/C41* mice was indistinguishable in all respects, so the condition in mice is inherited in a recessive pattern, as it is in humans. Odaph is specifically expressed by ameloblasts starting with the onset of post-secretory transition and continues until mid-maturation. Based upon histological and ultrastructural analyses, we determined that the secretory stage of amelogenesis is not affected in OdaphC41*/C41* mice. The enamel layer achieves a normal shape and contour, normal thickness, and normal rod decussation. The fundamental problem in OdaphC41*/C41* mice starts during post-secretory transition, which fails to generate maturation stage ameloblasts. At the onset of what should be enamel maturation, a cyst forms that separates flattened ameloblasts from the enamel surface. The maturation stage fails completely.

本稿の主たる目的は、スウェーデンのストックホルム市およびモータラ市の福祉および教育の実践を詳細に記録し、心理学的な観点から評価・検討することであった。我々は、2017年3月21日から10日間、スウェーデン王立リンショーピング大学アウトドア環境教育センターが提供するプログラムを中心として、北欧の福祉および教育の現状と課題を抽出することを目的として本研修に参加した。本稿では、（1）オンラインカウンセリングサービス、（2）ネイチャースクール、（3）小学校、（4）こども園、（5）家族センター、（6）高齢者ホーム、の6施設の視察詳細を報告することで、スウェーデンでの福祉政策と教育政策の特性をまとめ、子どもの発達や学習過程の実践的理解と心理的支援の方向性を示した。

The zygomatic bone is derived evolutionarily from the orbital series. In most modern mammals the zygomatic bone forms a large part of the face and usually serves as a bridge that connects the facial skeleton to the neurocranium. Our aim is to provide information on the contribution of the zygomatic bone to variation in midfacial protrusion using three samples; humans, domesticated dogs, and monkeys. In each case, variation in midface protrusion is a heritable trait produced by one of three classes of transmission: localized dysmorphology associated with single gene dysfunction, selective breeding, or long‐term evolution from a common ancestor. We hypothesize that the shape of the zygomatic bone reflects its role in stabilizing the connection between facial skeleton and neurocranium and consequently, changes in facial protrusion are more strongly reflected by the maxilla and premaxilla. Our geometric morphometric analyses support our hypothesis suggesting that the shape of the zygomatic bone has less to do with facial protrusion. By morphometrically dissecting the zygomatic bone we have determined a degree of modularity among parts of the midfacial skeleton suggesting that these components have the ability to vary independently and thus can evolve differentially. From these purely morphometric data, we propose that the neural crest cells that are fated to contribute to the zygomatic bone experience developmental cues that distinguish them from the maxilla and premaxilla. The spatiotemporal and molecular identity of the cues that impart zygoma progenitors with their identity remains an open question that will require alternative data sets. Anat Rec, 299:1616–1630, 2016. © 2016 The Authors The Anatomical Record Published by Wiley Periodicals, Inc.

WOS: 000458956100004 PubMed ID: 30506946 Amelogenesis imperfecta (AI) is a collection of isolated (non-syndromic) inherited diseases affecting dental enamel formation or a clinical phenotype in syndromic conditions. We characterized three consanguineous AI families with generalized irregular hypoplastic enamel with rapid attrition that perfectly segregated with homozygous defects in a novel gene: RELT that is a member of the tumor necrosis factor receptor superfamily (TNFRSF). RNAscope in situ hybridization of wild-type mouse molars and incisors showed specific Relt mRNA expression by secretory stage ameloblasts and by odontoblasts. Relt(-/-) mice generated by CRISPR/Cas9 exhibited incisor and molar enamel malformations. Relt(-/-) enamel had a rough surface and underwent rapid attrition. Normally unmineralized spaces in the deep enamel near the dentino-enamel junction (DEJ) were as highly mineralized as the adjacent enamel, which likely altered the mechanical properties of the DEJ. Phylogenetic analyses showed the existence of selective pressure on RELT gene outside of tooth development, indicating that the human condition may be syndromic, which possibly explains the history of small stature and severe childhood infections in two of the probands. Knowing a TNFRSF member is critical during the secretory stage of enamel formation advances our understanding of amelogenesis and improves our ability to diagnose human conditions featuring enamel malformations. National Institute of Dental and Craniofacial Research [DE015846DE016276]; National Research Foundation of Korea [NRF-2017R1A2A2A05069281NRF-2018R1A5A2024418] National Institute of Dental and Craniofacial Research, Grant/Award Number: DE015846DE016276; National Research Foundation of Korea, Grant/Award Number: NRF-2017R1A2A2A05069281NRF-2018R1A5A2024418

Summary We resolve debate over the evolution of vertebrate hypermineralized tissues through analyses of matrix protein-encoding secretory calcium-binding phosphoprotein (SCPP) genes and phylogenetic inference of hypermineralized tissues. Among these genes, AMBN and ENAM are found in both sarcopterygians and actinopterygians, whereas AMEL and SCPP5 are found only in sarcopterygians and actinopterygians, respectively. Actinopterygian AMBN, ENAM, and SCPP5 are expressed during the formation of hypermineralized tissues on scales and teeth: ganoin, acrodin, and collar enamel in gar, and acrodin and collar enameloid in zebrafish. Our phylogenetic analyses indicate the emergence of an ancestral enamel in stem-osteichthyans, whereas ganoin emerged in stem-actinopterygians and true enamel in stem-sarcopterygians. Thus, AMBN and ENAM originated in concert with ancestral enamel, SCPP5 evolved in association with ganoin, and AMEL evolved with true enamel. Shifts in gene expression domain and timing explain the evolution of different hypermineralized tissues. We propose that hypermineralized tissues in osteichthyans coevolved with matrix SCPP genes., Graphical Abstract, Highlights • Ganoin emerged in actinopterygians; true enamel arose in sarcopterygians • Dental enamel, acrodin, and enameloid in actinopterygians are related to ganoin • SCPP5 evolved in association with ganoin, whereas AMEL evolved with true enamel • Shifts in SCPP gene expression explain the evolution of hypermineralized tissues, Evolutionary Biology; Evolutionary Processes; Phylogenetics; Paleobiology

The earliest developmental origins of dysmorphologies are poorly understood in many congenital diseases. They often remain elusive because the first signs of genetic misregulation may initiate as subtle changes in gene expression, which are hard to detect and can be obscured later in development by secondary effects. Here, we develop a method to trace back the origins of phenotypic abnormalities by accurately quantifying the 3D spatial distribution of gene expression domains in developing organs. By applying Geometric Morphometrics to 3D gene expression data obtained by Optical Projection Tomography, we determined that our approach is sensitive enough to find regulatory abnormalities that have never been detected previously. We identified subtle but significant differences in the gene expression of a downstream target of a Fgfr2 mutation associated with Apert syndrome, demonstrating that these mouse models can further our understanding of limb defects in the human condition. Our method can be applied to different organ systems and models to investigate the etiology of malformations., eLife digest Our development in the womb is complex. Genes need to switch on and off in a precise order, controlling the activity of millions of cells as they work together to form different tissues. For everything to happen smoothly, cells must use instructions provided by each gene exactly at the correct moment and in the correct place. In this biological assembly line, the slightest change can lead to a defect. Certain genetic mutations can change when and where cells use particular genes, and this can cause errors in development. These kinds of mutations are a common cause of birth defects, but we cannot always pinpoint how they begin. For example, a single mutation in a gene called FGFR2 causes malformations in the head, the heart and the limbs in a rare disease called Apert syndrome. The first signs that development has gone wrong can be subtle changes in the use of certain genes, impossible to detect with standard methods. As development continues, other processes can mask the impact of problems with certain genes. Ultimately, changes alter the shape of the developing embryo. Genetically engineered mouse models can mimic the gene defects that cause disease in humans. But current methods are not sensitive enough to detect the very first signs of defects. Now, Martínez-Abadías et al. developed a new method to detect these subtle changes and reveal the precise moment when development starts to go wrong. In mice, a specific mutation in the FGFR2 gene affects the activity of a series of other genes. To track the levels of one of these genes, Martínez-Abadías et al. marked mouse embryos using a chemical label. Scanning the embryos then revealed the pattern of the cells using the gene during the earliest stages of development. In mice carrying a mutation in the FGFR2 gene, subtle changes in gene expression began just a few hours after their limbs start to develop. But it took another half a day to see the effects of these changes on the shape and size of the growing limbs. This approach revealed changes in gene expression before any problems with development were visible by eye. Tracking subtle changes in the way cells use genes could allow us to detect the origins of embryo malformations before they appear, pointing at the best moment to start a treatment. With further development, the model could extend to other genes, proteins, animal models and diseases.

Various secretory calcium-binding phosphoprotein (SCPP) genes are involved in the formation of the bone, dentin, enamel, and enameloid in bony vertebrates. By contrast, no SCPP gene is found in cartilaginous vertebrates. In order to explain this difference, I investigated the origin and early evolution of SCPP genes. First, I examined the phylogeny of SPARC-family genes that include evolutionary precursors of SCPP genes. Then, I analyzed the genomic arrangement of the SCPP genes and three SPARC-family genes, SPARCL1, SPARCL1L1, and SPARCR1. The results are consistent with our previous hypothesis that an SCPP gene-like structure arose in the 5′ half of SPARCL1L1 in a common ancestor of jawed vertebrates, at about the same time as the origin of mineralized skeleton. It is possible that cartilaginous vertebrates secondarily lost early SCPP genes, while bony vertebrates gained various new SCPP genes. Some of these new SCPP genes appear to have specifically involved in scale formation; however, these scale genes were lost in tetrapods.

The family of secretory calcium-binding phosphoproteins (SCPPs) have been considered vital to skeletal tissue mineralization. However, most previous SCPP studies focused on phylogenetically distant animals but not on those closely related species. Here we provide novel insights into the coevolution of SCPP genes and fish scales in 10 species from Otophysi. According to their scale phenotypes, these fishes can be divided into three groups, i.e., scaled, sparsely scaled, and scaleless. We identified homologous SCPP genes in the genomes of these species and revealed an absence of some SCPP members in some genomes, suggesting an uneven evolutionary history of SCPP genes in fishes. In addition, most of these SCPP genes, with the exception of SPP1, individually form one or two gene cluster(s) on each corresponding genome. Furthermore, we constructed phylogenetic trees using maximum likelihood method to estimate their evolution. The phylogenetic topology mostly supports two subclasses in some species, such as Cyprinus carpio, Sinocyclocheilus anshuiensis, S. grahamin, and S. rhinocerous, but not in the other examined fishes. By comparing the gene structures of recently reported candidate genes, SCPP1 and SCPP5, for determining scale phenotypes, we found that the hypothesis is suitable for Astyanax mexicanus, but denied by S. anshuiensis, even though they are both sparsely scaled for cave adaptation. Thus, we conclude that, although different fish species display similar scale phenotypes, the underlying genetic changes however might be diverse. In summary, this paper accelerates the recognition of the SCPP family in teleosts for potential scale evolution.

Nonsyndromic dentin defects classified as type II dentin dysplasia and types II and III dentinogenesis imperfecta are caused by mutations in DSPP (dentin sialophosphoprotein). Most reported disease‐causing DSPP mutations occur within the repetitive DPP (dentin phosphoprotein) coding sequence. We characterized the DPP sequences of five probands with inherited dentin defects using single molecule real‐time (SMRT) DNA sequencing. Eight of the 10 sequences matched previously reported DPP length haplotypes and two were novel. Alignment with known DPP sequences showed 32 indels arranged in 36 different patterns. Sixteen of the 32 indels were not represented in more than one haplotype. The 25 haplotypes with confirmed indels were aligned to generate a tree that describes how the length variations might have evolved. Some indels were independently generated in multiple lines. A previously reported disease‐causing DSPP mutation in Family 1 was confirmed and its position clarified (c.3135delC; p.Ser1045Argfs*269). A novel frameshift mutation (c.3504_3508dup; p.Asp1170Alafs*146) caused the dentin defects in Family 2. A COL1A2 (c.2027G>A or p.Gly676Asp) missense mutation, discovered by whole‐exome sequencing, caused the dentin defects in Family 3. We conclude that SMRT sequencing characterizes the DPP repeat region without cloning and can improve our understanding of normal and pathological length variations in DSPP alleles.

Kallikrein-related peptidase 4 (KLK4) is a secreted serine protease that degrades residual enamel proteins to facilitate their removal by ameloblasts, which increases mineralization and hardens the enamel. Mutations in human KLK4 cause hypomaturation amelogenesis imperfecta. Enamel formed by Klk4 null mice is normal in thickness and prism structure, but the enamel layer retains proteins, is hypomineralized, and undergoes rapid attrition following tooth eruption. We searched multiple databases, retrieved Klk4 and Klk5 from various mammalian genomes, and identified Klk4 in 46 boreoeutherian genomes. In non-Boreoeutheria, Klk4 was detected in only one afrotherian genome (as a pseudogene), and not in the other six afrotherian, two xenarthran, or three marsupial genomes. In contrast, Klk5 was detected in both marsupial and eutherian mammals. Our phylogenetic and mutation rate analyses support the hypothesis that Klk4 arose from Klk5 by gene duplication near the divergence of Afrotheria, Xenarthra and Boreoeutheria, and that functionally-differentiated Klk4 survived only in Boreoeutheria. Afrotherian mammals share the feature of delayed dental eruption relative to boreoeutherian mammals. KLK4 shortens the time required for enamel maturation and could have alleviated negative selection following mutations that resulted in thicker enamel or earlier tooth eruption, without reducing enamel hardness or causing dental attrition.

The coelacanth is the basal-most extant sarcopterygian that has teeth and tooth-like structures, comprising bone, dentin, and enamel or enameloid. Formation of these tissues involves many members of the secretory calcium-binding protein (SCPP) family. In tetrapods, acidic-residue-rich SCPPs are used in mineralization of bone and dentin, whereas Pro/Gln-rich SCPPs participate in enamel formation. Teleosts also employ many SCPPs for tissue mineralization. Nevertheless, the repertoire of SCPPs is largely different in teleosts and tetrapods; hence, filling this gap would be critical to elucidate early evolution of mineralized tissues in osteichthyans. In the present study, we searched for SCPP genes in the coelacanth genome and identified 11, of which two have clear orthologs in both tetrapods and teleosts, seven only in tetrapods, and two in neither of them. Given the divergence times of these vertebrate lineages, our discovery of this many SCPP genes shared between the coelacanth and tetrapods, but not with teleosts, suggests a complicated evolutionary scheme of SCPP genes in early osteichthyans. Our investigation also revealed both conserved and derived characteristics of SCPPs in the coelacanth and other vertebrates. Notably, acidic SCPPs independently evolved various acidic repeats in different lineages, while maintaining high acidity, presumably important for interactions with calcium. Furthermore, the three Pro/Gln-rich SCPP genes, required for mineralizing enamel matrix and confirmed only in tetrapods, were all identified in the coelacanth, strongly suggesting that enamel is equivalent in the coelacanth and tetrapods. This finding corroborates the previous proposition that true enamel evolved much earlier than the origin of tetrapods. J. Exp. Zool. (Mol. Dev. Evol.) 322B: 390–402, 2014. © 2013 Wiley Periodicals, Inc.

Odontogenic ameloblast-associated protein (ODAM) and amelotin (AMTN) both belong to the secretory calcium-binding phosphoprotein family, which is critical to biomineralization in vertebrates. In mammals, both ODAM and AMTN are expressed by ameloblasts in the maturation stage, when immature enamel grows into a hypermineralized inorganic tissue. At the onset of this stage, ameloblasts produce a specialized basal lamina (BL), over which both ODAM and AMTN are distributed. Enameloid is a different hypermineralized tissue that is found on the tooth surface of most ray-finned fish. Unlike amelogenesis, no such BL is produced during the maturation of enameloid. Nevertheless, ODAM is also found in ray-finned fish, and the expression of this gene has been detected in inner dental epithelial cells, which correspond to ameloblasts, after the enameloid is considerably mineralized. This specific gene expression suggests that ODAM is not a constituent of the BL but is still involved in the hypermineralization of enameloid. Both ODAM and AMTN are unusually rich in Pro and Gln, and they have 1 or 2 clusters of phospho-Ser residues. These characteristics suggest that ODAM and AMTN associate with weak interactions between relatively hydrophobic regions and further bind calcium phosphate via phospho-Ser clusters, similar to milk caseins that are evolutionary descendants of ODAM. Based on these considerations, I hypothesized that ODAM and AMTN generate and maintain the interface between unmineralized and hypermineralizing domains through weak protein–protein interactions and associations with calcium phosphate. This interface presumably facilitates hypermineralization, efficient removal of degraded proteins from the matrix, and the transfer of calcium phosphate to the matrix.