Continuous tooth replacement: what can teleost fish teach us?

Most tooth-bearing non-mammalian vertebrates have the capacity to replace their teeth throughout life. This capacity was lost in mammals, which replace their teeth only once at most. Not surprisingly, continuous tooth replacement has attracted much attention. Classical morphological studies (e.g. to analyse patterns of replacement) are now being complemented by molecular studies that investigate the expression of genes involved in tooth formation. This review focuses on ray-finned fish (actinopterygians), which have teeth often distributed throughout the mouth and pharynx, and more specifically on teleost fish, the largest group of extant vertebrates. First we highlight the diversity in tooth distribution and in tooth replacement patterns. Replacement tooth formation can start from a distinct (usually discontinuous and transient) dental lamina, but also in the absence of a successional lamina, e.g. from the surface epithelium of the oropharynx or from the outer dental epithelium of a predecessor tooth. The relationship of a replacement tooth to its predecessor is closely related to whether replacement is the result of a prepattern or occurs on demand. As replacement teeth do not necessarily have the same molecular signature as first-generation teeth, the question of the actual trigger for tooth replacement is discussed. Much emphasis has been laid in the past on the potential role of epithelial stem cells in initiating tooth replacement. The outcome of such studies has been equivocal, possibly related to the taxa investigated, and the permanent or transient nature of the dental lamina. Alternatively, replacement may result from local proliferation of undifferentiated progenitors, stimulated by hitherto unknown, perhaps mesenchymal, factors. So far, the role of the neurovascular link in continuous tooth replacement has been poorly investigated, despite the presence of a rich vascularisation surrounding actinopterygian (as well as chondrichthyan) teeth and despite a complete arrest of tooth replacement after nerve resection. Lastly, tooth replacement is possibly co-opted as a process to expand the number of teeth in a dentition ontogenetically whilst conserving features of the primary dentition. That neither a dental lamina, nor stem cells appear to be required for tooth replacement places teleosts in an advantageous position as models for tooth regeneration in humans, where the dental lamina regresses and epithelial stem cells are considered lost.

Bone Formation in Zebrafish: The Significance of DAF-FM DA Staining for Nitric Oxide Detection.

DAF-FM DA is widely used as a live staining compound to show the presence of nitric oxide (NO) in cells. Applying this stain to live zebrafish embryos is known to indicate early centers of bone formation, but the precise (cellular) location of the signal has hitherto not been revealed. Using sections of zebrafish embryos live-stained with DAF-FM DA, we could confirm that the fluorescent signals were predominantly located in areas of ongoing bone formation. Signals were observed in the bone and tooth matrix, in the notochord sheath, as well as in the bulbus arteriosus. Surprisingly, however, they were exclusively extracellular, even after very short staining times. Von Kossa and Alizarin red S staining to reveal mineral deposits showed that DAF-FM DA stains both the mineralized and non-mineralized bone matrix (osteoid), excluding that DAF-FM DA binds non-specifically to calcified structures. The importance of NO in bone formation by osteoblasts is nevertheless undisputed, as shown by the absence of bone structures after the inhibition of NOS enzymes that catalyze the formation of NO. In conclusion, in zebrafish skeletal biology, DAF-FM DA is appropriate to reveal bone formation in vivo, independent of mineralization of the bone matrix, but it does not demonstrate intracellular NO.

Deformity or variation? Phenotypic diversity in the zebrafish vertebral column.

Vertebral bodies are composed of two types of metameric elements, centra and arches, each of which is considered as a developmental module. Most parts of the teleost vertebral column have a one-to-one relationship between centra and arches, although, in all teleosts, this one-to-one relationship is lost in the caudal fin endoskeleton. Deviation from the one-to-one relationship occurs in most vertebrates, related to changes in the number of vertebral centra or to a change in the number of arches. In zebrafish, deviations also occur predominantly in the caudal region of the vertebral column. In-depth phenotypic analysis of wild-type zebrafish was performed using whole-mount stained samples, histological analyses and synchrotron radiation X-ray tomographic microscopy 3D reconstructions. Three deviant centra phenotypes were observed: (i) fusion of two vertebral centra, (ii) wedge-shaped hemivertebrae and (iii) centra with reduced length. Neural and haemal arches and their spines displayed bilateral and unilateral variations that resemble vertebral column phenotypes of stem-ward actinopterygians or other gnathostomes as well as pathological conditions in extant species. Whether it is possible to distinguish variations from pathological alterations and whether alterations resemble ancestral conditions is discussed in the context of centra and arch variations in other vertebrate groups and basal actinopterygian species.

Single-cell transcriptomes and runx2b-/- mutants reveal the genetic signatures of intermuscular bone formation in zebrafish.

Intermuscular bones (IBs) are mineralized spicules, present in the myosepta of many, but not all, teleost species. IBs are often small and sharp, and they consequently limit how the fish can be processed; the IBs may cause injury or trauma if lodged in consumers' throats or mouths, and therefore affect the appeal of the fish to many consumers. The development of IBs in teleosts is still not fully understood and the molecular basis of IB development remains to be established. Here, the characteristics of IB tissue are evaluated based on single-cell transcriptomics in wild-type zebrafish. The analysis defined 18 distinct cell types. Differentiation trajectories showed that IBs are derived from tendons and that a core tendon-osteoblast cell lineage is related to IB formation. In particular, the functions of 10 candidate genes were evaluated via CRISPR-Cas9 mutants. Among those, runx2b-/- mutants completely lost IBs, while swimming performance, growth and bone mineral density were not significantly different from runx2b+/+ zebrafish. Comparative single-cell RNA sequencing (scRNA-seq) analysis in runx2b-/- and runx2b+/+ zebrafish revealed the role of osteoblasts in IB formation. In addition, differentially expressed genes were enriched in the transforming growth factor ß/bone morphogenetic protein (TGF-ß/BMP) pathway after runx2b deletion. This study provides evidence for the crucial role of runx2b regulation in IB formation. Genetic breeding can target runx2b regulation and generate strains of commercial fish species without IBs, which can improve the safe consumption and economic value of many farmed fish species.

A Baseline for Skeletal Investigations in Medaka (Oryzias latipes): The Effects of Rearing Density on the Postcranial Phenotype.

Oryzias latipes is increasingly used as a model in biomedical skeletal research. The standard approach is to generate genetic variants with particular skeletal phenotypes which resemble skeletal diseases in humans. The proper diagnosis of skeletal variation is key for this type of research. However, even laboratory rearing conditions can alter skeletal phenotypes. The subject of this study is the link between skeletal phenotypes and rearing conditions. Thus, wildtype medaka were reared from hatching to an early juvenile stage at low (LD: 5 individuals/L), medium (MD: 15 individuals/L), and high (HD: 45 individuals/L) densities. The objectives of the study are: (I) provide a comprehensive overview of the postcranial skeletal elements in medaka; (II) evaluate the effects of rearing density on specific meristic counts and on the variability in type and incidence of skeletal anomalies; (III) define the best laboratory settings to obtain a skeletal reference for a sound evaluation of future experimental conditions; (IV) contribute to elucidating the structural and cellular changes related to the onset of skeletal anomalies. The results from this study reveal that rearing densities greater than 5 medaka/L reduce the animals' growth. This reduction is related to decreased mineralization of dermal (fin rays) and perichondral (fin supporting elements) bone. Furthermore, high density increases anomalies affecting the caudal fin endoskeleton and dermal rays, and the preural vertebral centra. A series of static observations on Alizarin red S whole mount-stained preural fusions provide insights into the etiology of centra fusion. The fusion of preural centra involves the ectopic formation of bony bridges over the intact intervertebral ligament. An apparent consequence is the degradation of the intervertebral ligaments and the remodeling and reshaping of the fused vertebral centra into a biconoid-shaped centrum. From this study it can be concluded that it is paramount to take into account the rearing conditions, natural variability, skeletal phenotypic plasticity, and the genetic background along with species-specific peculiarities when screening for skeletal phenotypes of mutant or wildtype medaka.

Compression Fractures and Partial Phenotype Rescue With a Low Phosphorus Diet in the Chihuahua Zebrafish Osteogenesis Imperfecta Model.

Osteogenesis imperfecta (OI) is a group of heritable disorders affecting bone and other connective tissues. Dominant OI forms are mainly caused by mutations in collagen type I. Patients suffer from skeletal deformities, fractures of long bones and vertebral compression fractures from early childhood onward. Altered collagen structure and excess mineralisation are the main causes for the bone phenotype. The Chihuahua (Chi/+) zebrafish has become an important model for OI. Given that reduced dietary phosphorus (P) intake reduces the bone mineral content and promotes bone matrix formation in teleosts, including zebrafish, we tested whether a low dietary P (LP) intake mitigates the OI phenotype in the Chi/+ model. To answer this question, we characterised the Chi/+ vertebral column phenotype at a morphological, cellular and subcellular level. We present the first description of vertebral compression fractures in Chi/+ and assess the effects of LP diet on the Chi/+ phenotype (Chi/+LP). Compared to untreated Chi/+, two months of LP dietary treatment decreases vertebral deformities in the abdominal region and reduces shape variation of caudal vertebral bodies to a condition more similar to wild type (WT). At the histological level, the osteoid layer, covering the bone at the vertebral body endplates in WT zebrafish, is absent in Chi/+, but it is partially restored with the LP diet. Whole mount-stained specimens and histological sections show various stages of vertebral compression fractures in Chi/+ and Chi/+LP animals. Both Chi/+ and Chi/+LP show abundant osteoclast activity compared to WT. Finally, the ultrastructure analysis of WT, Chi/+ and Chi/+LP shows Chi/+ and Chi/+LP osteoblasts with enlarged endoplasmic reticulum cisternae and a high protein content, consistent with intracellular retention of mutated collagen. Nevertheless, the secreted collagen in Chi/+LP appears better organised concerning fibre periodicity compared to Chi/+. Our findings suggest that a reduced mineral content of Chi/+ bone could explain the lower frequency of vertebral column deformities and the restored shape of the vertebral bodies in Chi/+LP animals. This, together with the improved quality of the bone extracellular matrix, suggests that two months of reduced dietary P intake can alleviate the severe bone phenotype in Chi/+ zebrafish.

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

There are several competing hypotheses on tooth origins, with discussions eventually settling in favour of an 'outside-in' scenario, in which internal odontodes (teeth) derived from external odontodes (skin denticles) in jawless vertebrates. The evolution of oral teeth from skin denticles can be intuitively understood from their location at the mouth entrance. However, the basal condition for jawed vertebrates is arguably to possess teeth distributed throughout the oropharynx (i.e. oral and pharyngeal teeth). As skin denticle development requires the presence of ectoderm-derived epithelium and of mesenchyme, it remains to be answered how odontode-forming skin epithelium, or its competence, were 'transferred' deep into the endoderm-covered oropharynx. The 'modified outside-in' hypothesis for tooth origins proposed that this transfer was accomplished through displacement of odontogenic epithelium, that is ectoderm, not only through the mouth, but also via any opening (e.g. gill slits) that connects the ectoderm to the epithelial lining of the pharynx (endoderm). This review explores from an evolutionary and from a developmental perspective whether ectoderm plays a role in (pharyngeal) tooth and denticle formation. Historic and recent studies on tooth development show that the odontogenic epithelium (enamel organ) of oral or pharyngeal teeth can be of ectodermal, endodermal, or of mixed ecto-endodermal origin. Comprehensive data are, however, only available for a few taxa. Interestingly, in these taxa, the enamel organ always develops from the basal layer of a stratified epithelium that is at least bilayered. In zebrafish, a miniaturised teleost that only retains pharyngeal teeth, an epithelial surface layer with ectoderm-like characters is required to initiate the formation of an enamel organ from the basal, endodermal epithelium. In urodele amphibians, the bilayered epithelium is endodermal, but the surface layer acquires ectodermal characters, here termed 'epidermalised endoderm'. Furthermore, ectoderm-endoderm contacts at pouch-cleft boundaries (i.e. the prospective gill slits) are important for pharyngeal tooth initiation, even if the influx of ectoderm via these routes is limited. A balance between sonic hedgehog and retinoic acid signalling could operate to assign tooth-initiating competence to the endoderm at the level of any particular pouch. In summary, three characters are identified as being required for pharyngeal tooth formation: (i) pouch-cleft contact, (ii) a stratified epithelium, of which (iii) the apical layer adopts ectodermal features. These characters delimit the area in which teeth can form, yet cannot alone explain the distribution of teeth over the different pharyngeal arches. The review concludes with a hypothetical evolutionary scenario regarding the persisting influence of ectoderm on pharyngeal tooth formation. Studies on basal osteichthyans with less-specialised types of early embryonic development will provide a crucial test for the potential role of ectoderm in pharyngeal tooth formation and for the 'modified outside-in' hypothesis of tooth origins.

Cells at the Edge: The Dentin-Bone Interface in Zebrafish Teeth.

Bone-producing osteoblasts and dentin-producing odontoblasts are closely related cell types, a result from their shared evolutionary history in the ancient dermal skeleton. In mammals, the two cell types can be distinguished based on histological characters and the cells' position in the pulp cavity or in the tripartite periodontal complex. Different from mammals, teleost fish feature a broad diversity in tooth attachment modes, ranging from fibrous attachment to firm ankylosis to the underlying bone. The connection between dentin and jaw bone is often mediated by a collar of mineralized tissue, a part of the dental unit that has been termed "bone of attachment". Its nature (bone, dentin, or an intermediate tissue type) is still debated. Likewise, there is a debate about the nature of the cells secreting this tissue: osteoblasts, odontoblasts, or yet another (intermediate) type of scleroblast. Here, we use expression of the P/Q rich secretory calcium-binding phosphoprotein 5 (scpp5) to characterize the cells lining the so-called bone of attachment in the zebrafish dentition. scpp5 is expressed in late cytodifferentiation stage odontoblasts but not in the cells depositing the "bone of attachment". nor in bona fide osteoblasts lining the supporting pharyngeal jaw bone. Together with the presence of the osteoblast marker Zns-5, and the absence of covering epithelium, this links the cells depositing the "bone of attachment" to osteoblasts rather than to odontoblasts. The presence of dentinal tubule-like cell extensions and the near absence of osteocytes, nevertheless distinguishes the "bone of attachment" from true bone. These results suggest that the "bone of attachment" in zebrafish has characters intermediate between bone and dentin, and, as a tissue, is better termed "dentinous bone". In other teleosts, the tissue may adopt different properties. The data furthermore support the view that these two tissues are part of a continuum of mineralized tissues. Expression of scpp5 can be a valuable tool to investigate how differentiation pathways diverge between osteoblasts and odontoblasts in teleost models and help resolving the evolutionary history of tooth attachment structures in actinopterygians.

Seadragon genome analysis provides insights into its phenotype and sex determination locus.

The iconic phenotype of seadragons includes leaf-like appendages, a toothless tubular mouth, and male pregnancy involving incubation of fertilized eggs on an open "brood patch." We de novo-sequenced male and female genomes of the common seadragon (Phyllopteryx taeniolatus) and its closely related species, the alligator pipefish (Syngnathoides biaculeatus). Transcription profiles from an evolutionary novelty, the leaf-like appendages, show that a set of genes typically involved in fin development have been co-opted as well as an enrichment of transcripts for potential tissue repair and immune defense genes. The zebrafish mutants for scpp5, which is lost in all syngnathids, were found to lack or have deformed pharyngeal teeth, supporting the hypothesis that the loss of scpp5 has contributed to the loss of teeth in syngnathids. A putative sex-determining locus encoding a male-specific amhr2y gene shared by common seadragon and alligator pipefish was identified.

Miniaturization: How many cells are needed to build a tooth?

BACKGROUND: Organs that develop early in life, and are replaced by a larger version as the animal grows, often represent a miniature version of the adult organ. Teeth constituting the first functional dentition in small-sized teleost fish, such as medaka (Oryzias latipes), are examples of such miniature organs. With a dentin cone as small as the size of one human cell, or even smaller, these teeth raise the question how many dentin-producing cells (odontoblasts) are required to build such a tooth, and whether this number can be as little as one. RESULTS: Based on detailed observations with transmission electron microscopy (TEM) and TEM-based 3D-reconstructions, we show that only one mesenchymal cell qualifies as a true odontoblast. A second mesenchymal cell potentially participates in dentin formation, but only at a late stage of tooth development. Moreover, the fate of these cells appears to be specified very early during tooth development. CONCLUSIONS: Our observations indicate that in this system, one single odontoblast fulfills roles normally exerted by a large and communicating cell population. First-generation teeth in medaka thus provide an exciting model to study integration of multiple functions into a single cell.

Plasticity of the skeleton and skeletal deformities in zebrafish (Danio rerio) linked to rearing density.

The teleost zebrafish (Danio rerio), an established model for human skeletal diseases, is reared under controlled conditions with defined parameters for temperature and photoperiod. Studies aimed at defining the proper rearing density have been performed with regard to behavioural and physiological stress response, sex ratio and reproduction. Studies concerning the effect of rearing density on the skeletal phenotype are lacking. This study analyses the response of the skeleton to different rearing densities and describes the skeletal deformities. Wild-type zebrafish were reared up to 30 dpf (days post-fertilization) in a common environment. From 30 to 90 dpf, animals were reared at three different densities: high density (HD), 32 fish l-1 ; medium density (MD), 8 fish l-1 and low density (LD), 2 fish l-1 . Animals at 30 and 90 dpf were collected and whole-mount stained with Alizarin red S to visualize mineralized tissues. The entire skeleton was analysed for meristic counts and 172 types of deformities. The results showed that the rearing density significantly influenced the specimens' average standard length, which decreased with the increase in the rearing density. Differences in meristic counts among the three groups were not observed. Rearing density-independent malformations affected the ribs, neural arches and the spines of the abdominal region, as well as vertebrae of the caudal complex. The HD group showed the highest number of deformities per specimen, the highest number of observed types of deformities and, together with the MD group, the highest frequency of specimens affected by severe deformities. In particular, the HD group showed deformities affecting arches, spines and vertebral centra in the caudal region of the vertebral column. This study provides evidence of an effect of the rearing density on the development of different skeletal phenotypes.

Photoconvertible fluorescent proteins: a versatile tool in zebrafish skeletal imaging.

One of the most frequently applied techniques in zebrafish (Danio rerio) research is the visualisation or manipulation of specific cell populations using transgenic reporter lines. The generation of these transgenic zebrafish, displaying cell- or tissue-specific expression of frequently used fluorophores such as Green Fluorescent Protein (GFP) or mCherry, is relatively easy using modern techniques. Fluorophores with different emission wavelengths and driven by different promoters can be monitored simultaneously in the same animal. Photoconvertible fluorescent proteins (pcFPs) are different from these standard fluorophores because their emission spectrum is changed when exposed to UV light, a process called photoconversion. Here, the benefits and versatility of using pcFPs for both single and dual fluorochrome imaging in zebrafish skeletal research in a previously generated osx:Kaede transgenic line are illustrated. In this line, Kaede, which is expressed under control of the osterix, otherwise known as sp7, promoter thereby labelling immature osteoblasts, can switch from green to red fluorescence upon irradiation with UV light. First, this study demonstrates that osx:Kaede exhibits an expression pattern similar to a previously described osx:nuGFP transgenic line in both larval and adult stages, hereby validating the use of this line for the imaging of immature osteoblasts. More in-depth experiments highlight different applications for osx:Kaede, such as lineage tracing and its combined use with in vivo skeletal staining and other transgenic backgrounds. Mineral staining in combination with osx:Kaede confirms osteoblast-independent mineralisation of the notochord. Osteoblast lineage tracing reveals migration and dedifferentiation of scleroblasts during fin regeneration. Finally, this study shows that combining two transgenics, osx:Kaede and osc:GFP, with similar emission wavelengths is possible when using a pcFP such as Kaede.

Mucin histochemistry as a tool to assess rostral digestive tract health in a teleost model (Danio rerio).

The zebrafish (Danio rerio), a small agastric teleost fish, has become a popular species to model diseases of the gastrointestinal tract. Remarkably, its rostral digestive tract has been largely understudied. To allow for accurate reporting and to improve the translational potential of this research model, the present study first proposes terminology for the different regions of the rostral digestive tract in zebrafish. Based on markers (in particular mucin histochemistry) which are easy to apply in routine histopathology laboratories, five regions are identified, in line with descriptions for carp, a close relative of zebrafish. The mucin histochemical staining is specific and distinct in each region identified, and can be used as a tool to assess health of the rostral digestive tract. In addition, we identify a transitional zone between the oesophagus and intestinal bulb which may represent a remnant of a rudimentary stomach.

More Bone with Less Minerals? The Effects of Dietary Phosphorus on the Post-Cranial Skeleton in Zebrafish.

Dietary phosphorus (P) is essential for bone mineralisation in vertebrates. P deficiency can cause growth retardation, osteomalacia and bone deformities, both in teleosts and in mammals. Conversely, excess P supply can trigger soft tissue calcification and bone hypermineralisation. This study uses a wide range of complementary techniques (X-rays, histology, TEM, synchrotron X-ray tomographic microscopy, nanoindentation) to describe in detail the effects of dietary P on the zebrafish skeleton, after two months of administering three different diets: 0.5% (low P, LP), 1.0% (regular P, RP), and 1.5% (high P, HP) total P content. LP zebrafish display growth retardation and hypomineralised bones, albeit without deformities. LP zebrafish increase production of non-mineralised bone matrix, and osteoblasts have enlarged endoplasmic reticulum cisternae, indicative for increased collagen synthesis. The HP diet promotes growth, high mineralisation, and stiffness but causes vertebral centra fusions. Structure and arrangement of bone matrix collagen fibres are not influenced by dietary P in all three groups. In conclusion, low dietary P content stimulates the formation of non-mineralised bone without inducing malformations. This indicates that bone formation and mineralisation are uncoupled. In contrast, high dietary P content promotes mineralisation and vertebral body fusions. This new zebrafish model is a useful tool to understand the mechanisms underlying osteomalacia and abnormal mineralisation, due to underlying variations in dietary P levels.

Multiple epithelia are required to develop teeth deep inside the pharynx.

To explain the evolutionary origin of vertebrate teeth from odontodes, it has been proposed that competent epithelium spread into the oropharyngeal cavity via the mouth and other possible channels such as the gill slits [Huysseune et al., 2009, J. Anat. 214, 465-476]. Whether tooth formation deep inside the pharynx in extant vertebrates continues to require external epithelia has not been addressed so far. Using zebrafish we have previously demonstrated that cells derived from the periderm penetrate the oropharyngeal cavity via the mouth and via the endodermal pouches and connect to periderm-like cells that subsequently cover the entire endoderm-derived pharyngeal epithelium [Rosa et al., 2019, Sci. Rep. 9, 10082]. We now provide conclusive evidence that the epithelial component of pharyngeal teeth in zebrafish (the enamel organ) is derived from medial endoderm, as hitherto assumed based on position deep in the pharynx. Yet, dental morphogenesis starts only after the corresponding endodermal pouch (pouch 6) has made contact with the skin ectoderm, and only after periderm-like cells have covered the prospective tooth-forming endodermal epithelium. Manipulation of signaling pathways shown to adversely affect tooth development indicates they act downstream of these events. We demonstrate that pouch-ectoderm contact and the presence of a periderm-like layer are both required, but not sufficient, for tooth initiation in the pharynx. We conclude that the earliest interactions to generate pharyngeal teeth encompass those between different epithelial populations (skin ectoderm, endoderm, and periderm-like cells in zebrafish), in addition to the epithelial-mesenchymal interactions that govern the formation of all vertebrate teeth.

Extensive chondroid bone in juvenile duck limbs hints at accelerated growth mechanism in avian skeletogenesis.

Modern altricial birds are the fastest growing vertebrates, whereas various degrees of precocity (functional maturity) result in slower growth. Diaphyseal osteohistology, the best proxy for inferring relative growth rates in fossils, suggests that in the earliest birds, posthatching growth rates were more variable than in modern representatives, with some showing considerably slow growth that was attributed to their assumed precocial flight abilities. For finding clues how precocial or altricial skeletogenesis and related growth acceleration could be traced in avian evolution, as a case study we investigated the growing limb diaphyseal histology in an ontogenetic series of ducks which, among several other avian taxa, show a combination of altricial wing and precocial leg development. Here we report the unexpected discovery that chondroid bone, a skeletal tissue family intermediate between cartilage and bone, extensively contributes to the development of limb bone shaft in ducks up to at least 30 days posthatching age. To our knowledge, chondroid bone has never been reported in such quantities and with an ontogenetically extended deposition period in post-embryonic, non-pathological periosteal bone formation of any tetrapod limb. It shows transitional cellular/lacunar morphologies and matrix staining properties between cartilage and woven bone and takes a significant part in the diametric growth of the limb bone shaft. Its amount and distribution through duckling ontogeny seems to be associated with the disparate functional and growth trajectories of the altricial wings vs. precocial legs characteristic of duck limb development. The presence of isogenous cell groups in the periosteal chondroid bone implies that cartilage-like interstitial growth took place before matrix mineralization complementing appositional bone growth. Based on these characteristics and on its fast formation rate in all previously reported normal as well as pathological cases, we suggest that chondroid bone in ducks significantly accelerates diametric limb bone growth. Related to this growth acceleration, we hypothesize that chondroid bone may be generally present in the growing limb bones of modern birds and hence may have key skeletogenic importance in achieving extreme avian growth rates and placing birds among the fastest growing vertebrates. Thus, we encourage future studies to test this hypothesis by investigating the occurrence of chondroid bone in a variety of precocial and altricial bird species, and to explore the presence of similar tissues in the growing limbs of other extant and extinct tetrapods in order to understand the evolutionary significance of chondroid bone in accelerated appendicular skeletogenesis.

Periderm invasion contributes to epithelial formation in the teleost pharynx.

The gnathostome pharyngeal cavity functions in food transport and respiration. In amniotes the mouth and nares are the only channels allowing direct contact between internal and external epithelia. In teleost fish, gill slits arise through opening of endodermal pouches and connect the pharynx to the exterior. Using transgenic zebrafish lines, cell tracing, live imaging and different markers, we investigated if pharyngeal openings enable epithelial invasion and how this modifies the pharyngeal epithelium. We conclude that in zebrafish the pharyngeal endoderm becomes overlain by cells with a peridermal phenotype. In a wave starting from pouch 2, peridermal cells from the outer skin layer invade the successive pouches until halfway their depth. Here the peridermal cells connect to a population of cells inside the pharyngeal cavity that express periderm markers, yet do not invade from outside. The latter population expands along the midline from anterior to posterior until the esophagus-gut boundary. Together, our results show a novel role for the periderm as an internal epithelium becomes adapted to function as an external surface.

Beyond the whole-mount phenotype: high-resolution imaging in fluorescence-based applications on zebrafish.

Zebrafish is now widely used in biomedical research as a model for human diseases, but the relevance of the model depends on a rigorous analysis of the phenotypes obtained. Many zebrafish disease models, experimental techniques and manipulations take advantage of fluorescent reporter molecules. However, phenotypic analysis often does not go beyond establishing overall distribution patterns of the fluorophore in whole-mount embryos or using vibratome or paraffin sections with poor preservation of tissue architecture and limited resolution. Obtaining high-resolution data of fluorescent signals at the cellular level from internal structures mostly depends on the availability of expensive imaging technology. Here, we propose a new and easily applicable protocol for embedding and sectioning of zebrafish embryos using in-house prepared glycol methacrylate (GMA) plastic that is suited for preservation of fluorescent signals (including photoactivatable fluorophores) without the need for antibodies. Four main approaches are described, all involving imaging fluorescent signals on semithin (3 µm or less) sections. These include sectioning transgenic animals, whole-mount immunostained embryos, cell tracking, as well as on-section enzyme histochemistry.

Bone without minerals and its secondary mineralization in Atlantic salmon (Salmo salar): the recovery from phosphorus deficiency.

Calcium and phosphorus (P) are the main bone minerals, and P deficiency can cause hypomineralized bones (osteomalacia) and malformations. This study used a P-deficient salmon model to falsify three hypotheses. First, an extended period of dietary P deficiency does not cause pathologies other than osteomalacia. Second, secondary mineralization of non-mineralized bone is possible. Third, secondary mineralization can restore the bones' mineral composition and mechanical properties. For 7 weeks, post-smolt Atlantic salmon (Salmo salar) received diets with regular P content (RP) or with a 50% lowered P content (LP). For additional 9 weeks, RP animals continued on the regular diet (RP-RP). LP animals continued on the LP diet (LP-LP), on a regular P diet (LP-RP) or on a high P diet (LP-HP). After 16 weeks, animals in all groups maintained a non-deformed vertebral column. LP-LP animals continued bone formation albeit without mineralization. Nine weeks of RP diet largely restored the mineral content and mechanical properties of vertebral bodies. Mineralization resumed deep inside the bone and away from osteoblasts. The history of P deficiency was traceable in LP-RP and LP-HP animals as a ring of low-mineralized bone in the vertebral body endplates, but no tissue alterations occurred that foreshadow vertebral body compression or fusion. Large quantities of non-mineralized salmon bone have the capacity to re-mineralize. If 16 weeks of P deficiency as a single factor is not causal for typical vertebral body malformations, other factors remain to be identified. This example of functional bone without minerals may explain why some teleost species can afford to have an extremely low mineralized skeleton.