Ancient vertebrate dermal armor evolved from trunk neural crest.

Bone is an evolutionary novelty of vertebrates, likely to have first emerged as part of ancestral dermal armor that consisted of osteogenic and odontogenic components. Whether these early vertebrate structures arose from mesoderm or neural crest cells has been a matter of considerable debate. To examine the developmental origin of the bony part of the dermal armor, we have performed in vivo lineage tracing in the sterlet sturgeon, a representative of nonteleost ray-finned fish that has retained an extensive postcranial dermal skeleton. The results definitively show that sterlet trunk neural crest cells give rise to osteoblasts of the scutes. Transcriptional profiling further reveals neural crest gene signature in sterlet scutes as well as bichir scales. Finally, histological and microCT analyses of ray-finned fish dermal armor show that their scales and scutes are formed by bone, dentin, and hypermineralized covering tissues, in various combinations, that resemble those of the first armored vertebrates. Taken together, our results support a primitive skeletogenic role for the neural crest along the entire body axis, that was later progressively restricted to the cranial region during vertebrate evolution. Thus, the neural crest was a crucial evolutionary innovation driving the origin and diversification of dermal armor along the entire body axis.

Sox9 is associated with two distinct patterning events during snake lung morphogenesis.

The evolutionary forces that allowed species adaptation to different terrestrial environments and led to great diversity in body shape and size required acquisition of innovative strategies of pattern formation during organogenesis. An extreme example is the formation of highly elongated viscera in snakes. What developmental patterning strategies allowed to overcome the space constraints of the snake's body to meet physiological demands? Here we show that the corn snake uses a Sox2-Sox9 developmental tool kit common to other species to generate and shape the lung in two phases. Initially Sox9 was found at low levels at the tip of the primary lung bud during outgrowth and elongation of the bronchial bud, without driving branching programs characteristic of mammalian lungs. Later, Sox9 induction is recapitulated in the formation of an extensive network of radial septae emerging along the elongated bronchial bud that generates the respiratory region. We propose that altogether these represent key patterning events for formation of both the respiratory faveolar and non-respiratory posterior compartments of the snake's lung.

Pre-oral gut contributes to facial structures in non-teleost fishes.

Despite the wide variety of adaptive modifications in the oral and facial regions of vertebrates, their early oropharyngeal development is considered strictly uniform. It involves sequential formation of the mouth and pharyngeal pouches, with ectoderm outlining the outer surface and endoderm the inner surface, as a rule. At the extreme anterior domain of vertebrate embryos, the ectoderm and endoderm directly juxtapose and initial development of this earliest ecto-endoderm interface, the primary mouth, typically involves ectodermal stomodeal invagination that limits the anterior expansion of the foregut endoderm. Here we present evidence that in embryos of extant non-teleost fishes, oral (stomodeal) formation is preceded by the development of prominent pre-oral gut diverticula (POGD) between the forebrain and roof of the forming mouth. Micro-computed tomography (micro-CT) imaging of bichir, sturgeon and gar embryos revealed that foregut outpocketing at the pre-oral domain begins even before the sequential formation of pharyngeal pouches. The presence of foregut-derived cells in the front of the mouth was further confirmed by in vivo experiments that allowed specific tracing of the early endodermal lining. We show that POGD in sturgeons contribute to the orofacial surface of their larvae, comprising oral teeth, lips, and sensory barbels. To our knowledge, this is the first thorough evidence for endodermal origin of external craniofacial structures in any vertebrate. In bichir and gar embryos, POGD form prominent cranial adhesive organs that are characteristic of the ancient bauplan of free-living chordate larvae. POGD hence seem arguably to be ancestral for all ray-finned fishes, and their topology, pharyngeal-like morphogenesis and gene expression suggest that they are evolutionarily related to the foregut-derived diverticula of early chordate and hemichordate embryos. The formation of POGD might thus represent an ancestral developmental module with deep deuterostome origins.

The remarkable dynamics in the establishment, rearrangement, and loss of dentition during the ontogeny of the sterlet sturgeon.

BACKGROUND: Sturgeons belong to an early-branching lineage often used as a proxy of ancestor-like traits of ray-finned fishes. However, many features of this lineage, such as the transitory presence and the eventual loss of dentition, exemplify specializations that, in fact, provide important information on lineage-specific evolutionary dynamics. RESULTS: Here, we introduce a detailed overview of the dentition during the development of the sterlet sturgeon. The dentition is composed of tooth fields at oral, palatal, and anterior pharyngeal regions. Oral fields are single-rowed, non-renewed and are shed early. Palatal and pharyngeal fields are multi-rowed and renewed from the adjacent superficial epithelium without the presence of the successional dental lamina. The early loss of oral fields and subsequent establishment of palatal and pharyngeal fields leads to a translocation of the functional dentition from the front to the rear of the oropharyngeal cavity until the eventual loss of all teeth. CONCLUSIONS: Our survey shows the sterlet dentition as a dynamic organ system displaying differential composition at different time points in the lifetime of this fish. These dynamics represent a conspicuous feature of sturgeons, unparalleled among extant vertebrates, and appropriate to scrutinize developmental and evolutionary underpinnings of vertebrate odontogenesis.

A simple nuclear contrast staining method for microCT-based 3D histology using lead(II) acetate.

X-ray microtomography (microCT) enables histological-scale 3D imaging of many types of biological samples, but it has yet to rival traditional histology for differentiation of tissue types and cell components. This report presents prima facie results indicating that a simple lead(II) acetate staining solution can impart preferential X-ray contrast to cell nuclei. While not strictly selective for nuclei, the staining reflects local cell-density differences. It can be applied in a single overnight treatment and does not require hematoxylin staining or drying of the sample. The stain is removable with EDTA, and it may enhance early calcifications. A basic protocol is given as a guide for further testing and optimization.

Embryology of the naso-palatal complex in Gekkota based on detailed 3D analysis in Lepidodactylus lugubris and Eublepharis macularius.

The vomeronasal organ (VNO), nasal cavity, lacrimal duct, choanal groove, and associated parts of the superficial (soft tissue) palate are called the naso-palatal complex. Despite the morphological diversity of the squamate noses, little is known about the embryological basis of this variation. Moreover, developmental data might be especially interesting in light of the morpho-molecular discordance of squamate phylogeny, since a 'molecular scenario' implies an occurrence of unexpected scale of homoplasy also in olfactory systems. In this study, we used X-ray microtomography and light microscopy to describe morphogenesis of the naso-palatal complex in two gekkotans: Lepidodactylus lugubris (Gekkonidae) and Eublepharis macularius (Eublepharidae). Our embryological data confirmed recent findings about the nature of some developmental processes in squamates, for example, involvement of the lateral nasal prominence in the formation of the choanal groove. Moreover, our study revealed previously unknown differences between the studied gekkotans and allows us to propose redefinition of the anterior concha of Sphenodon. Interpretation of some described conditions might be problematic in the phylogenetic context, since they represent unknown: squamate, nonophidian squamate, or gekkotan features.

Evolutionary trajectories of tooth histology patterns in modern sharks (Chondrichthyes, Elasmobranchii).

During their evolutionary history, modern sharks developed different tooth mineralization patterns that resulted in very distinct histological patterns of the tooth crown (histotypes). To date, three different tooth histotypes have been distinguished: (i) orthodont teeth, which have a central hollow pulp cavity in the crown, encapsulated by a prominent layer of dentine (orthodentine); (ii) pseudoosteodont teeth, which have their pulp cavities secondarily replaced by a dentinal core of porous dentine (osteodentine), encased by orthodentine; and (iii) osteodont teeth, which lack orthodentine and the whole tooth crown of which consists of osteodentine. The aim of the present study was to trace evolutionary trends of tooth mineralization patterns in modern sharks and to find evidence for the presence of phylogenetic or functional signals. High resolution micro-computed tomography images were generated for the teeth of members of all nine extant shark orders and the putative stem group †Synechodontiformes, represented here by three taxa, to examine the tooth histology non-destructively. Pseudoosteodonty is the predominant state among modern sharks and represents unambiguously the plesiomorphic condition. Orthodonty evolved several times independently in modern sharks, while the osteodont tooth histotype is only developed in lamniform sharks. The two shark orders Heterodontiformes and Pristiophoriformes showed highly modified tooth histologies, with Pristiophorus exhibiting a histology only known from batomorphs (i.e. rays and skates), and Heterodontus showing a histological difference between anterior and posterior teeth, indicating a link between its tooth morphology, histology and durophagous lifestyle. The tooth histotype concept has proven to be a useful tool to reflect links between histology, function and its taxonomic value for distinct taxa; however, a high degree of variation, especially in the pseudoosteodont tooth histotype, demonstrates that the current histotype concept is too simplistic to fully resolve these relationships. The vascularization pattern of the dentine might offer new future research pathways for better understanding functional and phylogenetic signals in the tooth histology of modern sharks.

Development of the squamate naso-palatal complex: detailed 3D analysis of the vomeronasal organ and nasal cavity in the brown anole Anolis sagrei (Squamata: Iguania).

BACKGROUND: Despite the diverse morphology of the adult squamate naso-palatal complex - consisting of the nasal cavity, vomeronasal organ (VNO), choanal groove, lacrimal duct and superficial palate - little is known about the embryology of these structures. Moreover, there are no comprehensive studies concerning development of the nasal cavity and VNO in relation to the superficial palate. In this investigation, we used X-ray microtomography and histological sections to describe embryonic development of the naso-palatal complex of iguanian lizard, the brown anole (Anolis sagrei). The purpose of the study was to describe the mechanism of formation of adult morphology in this species, which combines the peculiar anole features with typical iguanian conditions. Considering the uncertain phylogenetic position of the Iguania within Squamata, embryological data and future comparative studies may shed new light on the evolution of this large squamate clade. RESULTS: Development of the naso-palatal complex was divided into three phases: early, middle and late. In the early developmental phase, the vomeronasal pit originates from medial outpocketing of the nasal pit, when the facial prominences are weakly developed. In the middle developmental phase, the following events can be noted: the formation of the frontonasal mass, separation of the vestibulum, appearance of the lacrimal duct, and formation of the choanal groove, which leads to separation of the VNO from the nasal cavity. In late development, the nasal cavity and the VNO attain their adult morphology. The lacrimal duct establishes an extensive connection with the choanal groove, which eventually becomes largely separated from the oral cavity. CONCLUSIONS: Unlike in other tetrapods, the primordium of the lacrimal duct in the brown anole develops largely beyond the nasolacrimal groove. In contrast to previous studies on squamates, the maxillary prominence is found to participate in the initial fusion with the frontonasal mass. Moreover, formation of the choanal groove occurs due to the fusion of the vomerine cushion to the subconchal fold, rather than to the choanal fold. The loss or significant reduction of the lateral nasal concha is secondary. Some features of anole adult morphology, such as the closure of the choanal groove, may constitute adaptations to vomeronasal chemoreception.

Hard and soft X-ray imaging to resolve human ovarian cortical structures.

Laboratory and synchrotron X-ray tomography are powerful tools for non-invasive studies of biological samples at micrometric resolution. In particular, the development of phase contrast imaging is enabling the visualization of sample details with a small range of attenuation coefficients, thus allowing in-depth analyses of anatomical and histological structures. Reproductive medicine is starting to profit from these techniques, mainly applied to animal models. This study reports the first imaging of human ovarian tissue where the samples consisted of surgically obtained millimetre fragments, properly fixed, stained with osmium tetroxide and included in epoxydic resin. Samples were imaged by the use of propagation phase contrast synchrotron radiation micro-computed tomography (microCT), obtained at the SYRMEP beamline of Elettra light source (Trieste, Italy), and X-ray absorption microCT at the Theoretical Biology MicroCT Imaging Laboratory in Vienna, Austria. The reconstructed microCT images were compared with the soft X-ray absorption and phase contrast images acquired at the TwinMic beamline of Elettra in order to help with the identification of structures. The resulting images allow the regions of the cortex and medulla of the ovary to be distinguished, identifying early-stage follicles and visualizing the distribution of blood vessels. The study opens to further application of micro-resolved 3D imaging to improve the understanding of human ovary's structure and support diagnostics as well as advances in reproductive technologies.

Sox2+ progenitors in sharks link taste development with the evolution of regenerative teeth from denticles.

Teeth and denticles belong to a specialized class of mineralizing epithelial appendages called odontodes. Although homology of oral teeth in jawed vertebrates is well supported, the evolutionary origin of teeth and their relationship with other odontode types is less clear. We compared the cellular and molecular mechanisms directing development of teeth and skin denticles in sharks, where both odontode types are retained. We show that teeth and denticles are deeply homologous developmental modules with equivalent underlying odontode gene regulatory networks (GRNs). Notably, the expression of the epithelial progenitor and stem cell marker sex-determining region Y-related box 2 (sox2) was tooth-specific and this correlates with notable differences in odontode regenerative ability. Whereas shark teeth retain the ancestral gnathostome character of continuous successional regeneration, new denticles arise only asynchronously with growth or after wounding. Sox2+ putative stem cells associated with the shark dental lamina (DL) emerge from a field of epithelial progenitors shared with anteriormost taste buds, before establishing within slow-cycling cell niches at the (i) superficial taste/tooth junction (T/TJ), and (ii) deep successional lamina (SL) where tooth regeneration initiates. Furthermore, during regeneration, cells from the superficial T/TJ migrate into the SL and contribute to new teeth, demonstrating persistent contribution of taste-associated progenitors to tooth regeneration in vivo. This data suggests a trajectory for tooth evolution involving cooption of the odontode GRN from nonregenerating denticles by sox2+ progenitors native to the oral taste epithelium, facilitating the evolution of a novel regenerative module of odontodes in the mouth of early jawed vertebrates: the teeth.

An ancient dental gene set governs development and continuous regeneration of teeth in sharks.

The evolution of oral teeth is considered a major contributor to the overall success of jawed vertebrates. This is especially apparent in cartilaginous fishes including sharks and rays, which develop elaborate arrays of highly specialized teeth, organized in rows and retain the capacity for life-long regeneration. Perpetual regeneration of oral teeth has been either lost or highly reduced in many other lineages including important developmental model species, so cartilaginous fishes are uniquely suited for deep comparative analyses of tooth development and regeneration. Additionally, sharks and rays can offer crucial insights into the characters of the dentition in the ancestor of all jawed vertebrates. Despite this, tooth development and regeneration in chondrichthyans is poorly understood and remains virtually uncharacterized from a developmental genetic standpoint. Using the emerging chondrichthyan model, the catshark (Scyliorhinus spp.), we characterized the expression of genes homologous to those known to be expressed during stages of early dental competence, tooth initiation, morphogenesis, and regeneration in bony vertebrates. We have found that expression patterns of several genes from Hh, Wnt/ß-catenin, Bmp and Fgf signalling pathways indicate deep conservation over ~450 million years of tooth development and regeneration. We describe how these genes participate in the initial emergence of the shark dentition and how they are redeployed during regeneration of successive tooth generations. We suggest that at the dawn of the vertebrate lineage, teeth (i) were most likely continuously regenerative structures, and (ii) utilised a core set of genes from members of key developmental signalling pathways that were instrumental in creating a dental legacy redeployed throughout vertebrate evolution. These data lay the foundation for further experimental investigations utilizing the unique regenerative capacity of chondrichthyan models to answer evolutionary, developmental, and regenerative biological questions that are impossible to explore in classical models.

Open data and digital morphology.

Over the past two decades, the development of methods for visualizing and analysing specimens digitally, in three and even four dimensions, has transformed the study of living and fossil organisms. However, the initial promise that the widespread application of such methods would facilitate access to the underlying digital data has not been fully achieved. The underlying datasets for many published studies are not readily or freely available, introducing a barrier to verification and reproducibility, and the reuse of data. There is no current agreement or policy on the amount and type of data that should be made available alongside studies that use, and in some cases are wholly reliant on, digital morphology. Here, we propose a set of recommendations for minimum standards and additional best practice for three-dimensional digital data publication, and review the issues around data storage, management and accessibility.

Past climate change on Sky Islands drives novelty in a core developmental gene network and its phenotype.

BACKGROUND: A fundamental and enduring problem in evolutionary biology is to understand how populations differentiate in the wild, yet little is known about what role organismal development plays in this process. Organismal development integrates environmental inputs with the action of gene regulatory networks to generate the phenotype. Core developmental gene networks have been highly conserved for millions of years across all animals, and therefore, organismal development may bias variation available for selection to work on. Biased variation may facilitate repeatable phenotypic responses when exposed to similar environmental inputs and ecological changes. To gain a more complete understanding of population differentiation in the wild, we integrated evolutionary developmental biology with population genetics, morphology, paleoecology and ecology. This integration was made possible by studying how populations of the ant species Monomorium emersoni respond to climatic and ecological changes across five 'Sky Islands' in Arizona, which are mountain ranges separated by vast 'seas' of desert. Sky Islands represent a replicated natural experiment allowing us to determine how repeatable is the response of M. emersoni populations to climate and ecological changes at the phenotypic, developmental, and gene network levels. RESULTS: We show that a core developmental gene network and its phenotype has kept pace with ecological and climate change on each Sky Island over the last ~90,000 years before present (BP). This response has produced two types of evolutionary change within an ant species: one type is unpredictable and contingent on the pattern of isolation of Sky lsland populations by climate warming, resulting in slight changes in gene expression, organ growth, and morphology. The other type is predictable and deterministic, resulting in the repeated evolution of a novel wingless queen phenotype and its underlying gene network in response to habitat changes induced by climate warming. CONCLUSION: Our findings reveal dynamics of developmental gene network evolution in wild populations. This holds important implications: (1) for understanding how phenotypic novelty is generated in the wild; (2) for providing a possible bridge between micro- and macroevolution; and (3) for understanding how development mediates the response of organisms to past, and potentially, future climate change.

Testing hypotheses of bat baculum function with 3D models derived from microCT.

The baculum (os penis) has been extensively studied as a taxon-specific character in bats and other mammals but its mechanical function is still unclear. There is a wide consensus in the literature that the baculum is probably a sexually selected character. Using a novel approach combining postmortem manipulation and three-dimensional (3D) imaging, we tested two functional hypotheses in the common noctule bat Nyctalus noctula, the common pipistrelle Pipistrellus pipistrellus, and Nathusius' pipistrelle Pipistrellus nathusii: (i) whether the baculum can protect the distal urethra and urethral opening from compression during erection and copulation; and (ii) whether the baculum and corpora cavernosa form a functional unit to support both the penile shaft and the more distal glans tip. In freshly dead or frozen and thawed bats, we compared flaccid penises with artificially 'erect' penises that were inflated with 10% formalin. Penises were stained with alcoholic iodine and imaged with a lab-based high-resolution x-ray microtomography system. Analysis of the 3D images enabled us to compare the changes in relative positions of the baculum, corpora cavernosa, urethra, and corpus spongiosum with one another between flaccid and 'erect' penises. Our results support both functional hypotheses, indicating that the baculum probably performs two different roles during erection. Our approach should prove valuable for comparing and testing the functions of different baculum morphologies in bats and other mammals. Moreover, we have validated an essential component of the groundwork necessary to extend this approach with finite element analysis for quantitative 3D biomechanical modeling of penis function.

The lateral mesodermal divide: an epigenetic model of the origin of paired fins.

By examining development at the level of tissues and processes, rather than focusing on gene expression, we have formulated a general hypothesis to explain the dorso-ventral and anterior-posterior placement of paired appendage initiation sites in vertebrates. According to our model, the number and position of paired appendages are due to a commonality of embryonic tissue environments determined by the global interactions involving the two separated layers (somatic and visceral) of lateral plate mesoderm along the dorso-ventral and anterior-posterior axes of the embryo. We identify this distribution of developmental conditions, as modulated by the separation/contact of the two LPM layers and their interactions with somitic mesoderm, ectoderm, and endoderm as a dynamic developmental entity which we have termed the lateral mesodermal divide (LMD). Where the divide results in a certain tissue environment, fin bud initiation can occur. According to our hypothesis, the influence of the developing gut suppresses limb initiation along the midgut region and the ventral body wall owing to an "endodermal predominance." From an evolutionary perspective, the lack of gut regionalization in agnathans reflects the ancestral absence of these conditions, and the elaboration of the gut together with the concomitant changes to the LMD in the gnathostomes could have led to the origin of paired fins.

Thumbs down: a molecular-morphogenetic approach to avian digit homology.

Avian forelimb digit homology remains one of the standard themes in comparative biology and EvoDevo research. In order to resolve the apparent contradictions between embryological and paleontological evidence a variety of hypotheses have been presented in recent years. The proposals range from excluding birds from the dinosaur clade, to assignments of homology by different criteria, or even assuming a hexadactyl tetrapod limb ground state. At present two approaches prevail: the frame shift hypothesis and the pyramid reduction hypothesis. While the former postulates a homeotic shift of digit identities, the latter argues for a gradual bilateral reduction of phalanges and digits. Here we present a new model that integrates elements from both hypotheses with the existing experimental and fossil evidence. We start from the main feature common to both earlier concepts, the initiating ontogenetic event: reduction and loss of the anterior-most digit. It is proposed that a concerted mechanism of molecular regulation and developmental mechanics is capable of shifting the boundaries of hoxD expression in embryonic forelimb buds as well as changing the digit phenotypes. Based on a distinction between positional (topological) and compositional (phenotypic) homology criteria, we argue that the identity of the avian digits is II, III, IV, despite a partially altered phenotype. Finally, we introduce an alternative digit reduction scheme that reconciles the current fossil evidence with the presented molecular-morphogenetic model. Our approach identifies specific experiments that allow to test whether gene expression can be shifted and digit phenotypes can be altered by induced digit loss or digit gain.

Are accessory hearing structures linked to inner ear morphology? Insights from 3D orientation patterns of ciliary bundles in three cichlid species.

BACKGROUND: Cichlid fishes show considerable diversity in swim bladder morphology. In members of the subfamily Etroplinae, the connection between anterior swim bladder extensions and the inner ears enhances sound transmission and translates into an improved hearing ability. We tested the hypothesis that those swim bladder modifications coincide with differences in inner ear morphology and thus compared Steatocranus tinanti (vestigial swim bladder), Hemichromis guttatus (large swim bladder without extensions), and Etroplus maculatus (intimate connection between swim bladder and inner ears). METHODOLOGY AND RESULTS: We applied immunostaining together with confocal imaging and scanning electron microscopy for the investigation of sensory epithelia, and high-resolution, contrast-enhanced microCT imaging for characterizing inner ears in 3D, and evaluated otolith dimensions. Compared to S. tinanti and H. guttatus, inner ears of E. maculatus showed an enlargement of all three maculae, and a particularly large lacinia of the macula utriculi. While our analysis of orientation patterns of ciliary bundles on the three macula types using artificially flattened maculae uncovered rather similar orientation patterns of ciliary bundles, interspecific differences became apparent when illustrating the orientation patterns on the 3D models of the maculae: differences in the shape and curvature of the lacinia of the macula utriculi, and the anterior arm of the macula lagenae resulted in an altered arrangement of ciliary bundles. CONCLUSIONS: Our results imply that improved audition in E. maculatus is associated not only with swim bladder modifications but also with altered inner ear morphology. However, not all modifications in E. maculatus could be connected to enhanced auditory abilities, and so a potential improvement of the vestibular sense, among others, also needs to be considered. Our study highlights the value of analyzing orientation patterns of ciliary bundles in their intact 3D context in studies of inner ear morphology and physiology.

A unique swim bladder-inner ear connection in a teleost fish revealed by a combined high-resolution microtomographic and three-dimensional histological study.

BACKGROUND: In most modern bony fishes (teleosts) hearing improvement is often correlated with a close morphological relationship between the swim bladder or other gas-filled cavities and the saccule or more rarely with the utricle. A connection of an accessory hearing structure to the third end organ, the lagena, has not yet been reported. A recent study in the Asian cichlid Etroplus maculatus provided the first evidence that a swim bladder may come close to the lagena. Our study was designed to uncover the swim bladder-inner ear relationship in this species. We used a new approach by applying a combination of two high-resolution techniques, namely microtomographic (microCT) imaging and histological serial semithin sectioning, providing the basis for subsequent three-dimensional reconstructions. Prior to the morphological study, we additionally measured auditory evoked potentials at four frequencies (0.5, 1, 2, 3 kHz) to test the hearing abilities of the fish. RESULTS: E. maculatus revealed a complex swim bladder-inner ear connection in which a bipartite swim bladder extension contacts the upper as well as the lower parts of each inner ear, a condition not observed in any other teleost species studied so far. The gas-filled part of the extension is connected to the lagena via a thin bony lamella and is firmly attached to this bony lamella with connective material. The second part of the extension, a pad-like structure, approaches the posterior and horizontal semicircular canals and a recessus located posterior to the utricle. CONCLUSIONS: Our study is the first detailed report of a link between the swim bladder and the lagena in a teleost species. We suggest that the lagena has an auditory function in this species because the most intimate contact exists between the swim bladder and this end organ. The specialized attachment of the saccule to the cranial bone and the close proximity of the swim bladder extension to the recessus located posterior to the utricle indicate that the saccule and the utricle also receive parallel inputs from the swim bladder extension. We further showed that a combination of non-destructive microCT imaging with histological analyses on the same specimen provides a powerful tool to decipher and interpret fine structures and to compensate for methodological artifacts.

3D atlas of the human fetal chondrocranium in the middle trimester.

The chondrocranium provides the key initial support for the fetal brain, jaws and cranial sensory organs in all vertebrates. The patterns of shaping and growth of the chondrocranium set up species-specific development of the entire craniofacial complex. The 3D development of chondrocranium have been studied primarily in animal model organisms, such as mice or zebrafish. In comparison, very little is known about the full 3D human chondrocranium, except from drawings made by anatomists many decades ago. The knowledge of human-specific aspects of chondrocranial development are essential for understanding congenital craniofacial defects and human evolution. Here advanced microCT scanning was used that includes contrast enhancement to generate the first 3D atlas of the human fetal chondrocranium during the middle trimester (13 to 19 weeks). In addition, since cartilage and bone are both visible with the techniques used, the  endochondral ossification of cranial base was mapped since this region is so critical for brain and jaw growth. The human 3D models are published as a scientific resource for human development.

Sensory epithelia of the fish inner ear in 3D: studied with high-resolution contrast enhanced microCT.

INTRODUCTION: While a number of studies have illustrated and analyzed 3D models of inner ears in higher vertebrates, inner ears in fishes have rarely been investigated in 3D, especially with regard to the sensory epithelia of the end organs, the maculae. It has been suggested that the 3D curvature of these maculae may also play an important role in hearing abilities in fishes. We therefore set out to develop a fast and reliable approach for detailed 3D visualization of whole inner ears as well as maculae. RESULTS: High-resolution microCT imaging of black mollies Poecilia sp. (Poeciliidae, Teleostei) and Steatocranus tinanti (Cichlidae, Teleostei) stained with phosphotungstic acid (PTA) resulted in good tissue contrast, enabling us to perform a reliable 3D reconstruction of all three sensory maculae of the inner ears. Comparison with maculae that have been 3D reconstructed based on histological serial sections and phalloidin-stained maculae showed high congruence in overall shape of the maculae studied here. CONCLUSIONS: PTA staining and subsequent high-resolution contrast enhanced microCT imaging is a powerful method to obtain 3D models of fish inner ears and maculae in a fast and more reliable manner. Future studies investigating functional morphology, phylogenetic potential of inner ear features, or evolution of hearing and inner ear specialization in fishes may benefit from the use of 3D models of inner ears and maculae.