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.

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.

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.

OCT Meets micro-CT: A Subject-Specific Correlative Multimodal Imaging Workflow for Early Chick Heart Development Modeling.

Structural and Doppler velocity data collected from optical coherence tomography have already provided crucial insights into cardiac morphogenesis. X-ray microtomography and other ex vivo methods have elucidated structural details of developing hearts. However, by itself, no single imaging modality can provide comprehensive information allowing to fully decipher the inner workings of an entire developing organ. Hence, we introduce a specimen-specific correlative multimodal imaging workflow combining OCT and micro-CT imaging which is applicable for modeling of early chick heart development-a valuable model organism in cardiovascular development research. The image acquisition and processing employ common reagents, lab-based micro-CT imaging, and software that is free for academic use. Our goal is to provide a step-by-step guide on how to implement this workflow and to demonstrate why those two modalities together have the potential to provide new insight into normal cardiac development and heart malformations leading to congenital heart disease.

Evolutionary functional morphology of the proboscis and feeding apparatus of hawk moths (Sphingidae: Lepidoptera).

The morphology of the proboscis and associated feeding organs was studied in several nectar-feeding hawk moths, as well as a specialized honey-feeder and two supposedly nonfeeding species. The proboscis lengths ranged from a few millimeters to more than 200 mm. Despite the variation in proboscis length and feeding strategy, the principle external and internal composition of the galeae, the stipes pump, and the suction pump were similar across all species. The morphology of the smooth and slender proboscis is highly conserved among all lineages of nectar-feeding Sphingidae. Remarkably, they share a typical arrangement of the sensilla at the tip. The number and length of sensilla styloconica are independent from proboscis length. A unique proboscis morphology was found in the honey-feeding species Acherontia atropos. Here, the distinctly pointed apex displays a large subterminal opening of the food canal, and thus characterizes a novel type of piercing proboscis in Lepidoptera. In the probably nonfeeding species, the rudimentary galeae are not interlocked and the apex lacks sensilla styloconica; galeal muscles, however, are present. All studied species demonstrate an identical anatomy of the stipes, and suction pump, regardless of proboscis length and diet. Even supposedly nonfeeding Sphingidae possess all organs of the feeding apparatus, suggesting that their proboscis rudiments might still be functional. The morphometric analyses indicate significant positive correlations between galea lumen volume and stipes muscle volume as well as the volume of the food canal and the muscular volume of the suction pump. Size correlations of these functionally connected organs reflect morphological fine-tuning in the evolution of proboscis length and function.

Anuran development: A reinvestigation of the conus arteriosus and gill formation in Bufo bufo throughout metamorphosis using micro-CT.

Using high-resolution X-ray micro-CT imaging of whole Bufo bufo specimens, we acquired detailed 3D descriptions of the changing morphology of the cardiac outflow structures, in particular the conus arteriosus through larval development and the transition. Previous findings regarding anuran conal structures were contradictory, depending on the specifics of the 2D imaging methods used by different authors. Our descriptions of conal morphology at different developmental stages show that early tadpoles initially only have one opening at the ventricular-conal junction and only one cavum within their conus; however, the forming septum coni soon divides the conus into two chambers, the cavum pulmocutaneum and the cavum aorticum. This is accompanied by the development of a second small opening at the ventricular-conal junction. The separated chambers continue into the aortic arches. Following the aortic arches into the area where gills will form, we describe how blood vessels associated with the external gills develop from vessels arising from the truncus arteriosus. The external gills soon undergo partial absorption. During the transition from external to internal gills, the gill filaments retreat asymmetrically into a gill chamber formed by a hyoidal cover contacting the animal's ventral side, leaving only a single opening on the animal's left side, the opercular spout. B. bufo retains its internal gills up to metamorphic climax, with the aortic arches arising from the conus arteriosus still leading into the gills. Our 3D image data are publicly available and will provide a sound morphological basis for future studies.

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.

Ultrastructural studies of developing egg tooth in grass snake Natrix natrix (Squamata, Serpentes) embryos, supported by X-ray microtomography analysis.

The egg tooth development is similar to the development of all the other vertebrate teeth except earliest developmental stages because the egg tooth develops directly from the oral epithelium instead of the dental lamina similarly to null generation teeth. The developing egg tooth of Natrix natrix changes its curvature differently than the egg tooth of the other investigated unidentates due to the presence of the rostral groove. The developing grass snake egg tooth comprises dental pulp and the enamel organ. The fully differentiated enamel organ consists of outer enamel epithelium, stellate reticulum, and ameloblasts in its inner layer. The enamel organ directly in contact with the oral cavity is covered with periderm instead of outer enamel epithelium. Stellate reticulum cells in the grass snake egg tooth share intercellular spaces with the basal part of ameloblasts and are responsible for their nutrition. Ameloblasts during egg tooth differentiation pass through the following stages: presecretory, secretory, and mature. The ameloblasts from the grass snake egg tooth show the same cellular changes as reported during mammalian amelogenesis but are devoid of Tomes' processes. Odontoblasts of the developing grass snake egg tooth pass through the following classes: pre-odontoblasts, secretory odontoblasts, and ageing odontoblasts. They have highly differentiated secretory apparatus and in the course of their activity accumulate lipofuscin. Grass snake odontoblasts possess processes which are poor in organelles. In developing egg tooth cilia have been identified in odontoblasts, ameloblasts and cells of the stellate reticulum. Dental pulp cells remodel collagen matrix during growth of the grass snake egg tooth. They degenerate in a way previously not described in other teeth.

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.

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.

Secondary ossification center induces and protects growth plate structure.

Growth plate and articular cartilage constitute a single anatomical entity early in development but later separate into two distinct structures by the secondary ossification center (SOC). The reason for such separation remains unknown. We found that evolutionarily SOC appears in animals conquering the land - amniotes. Analysis of the ossification pattern in mammals with specialized extremities (whales, bats, jerboa) revealed that SOC development correlates with the extent of mechanical loads. Mathematical modeling revealed that SOC reduces mechanical stress within the growth plate. Functional experiments revealed the high vulnerability of hypertrophic chondrocytes to mechanical stress and showed that SOC protects these cells from apoptosis caused by extensive loading. Atomic force microscopy showed that hypertrophic chondrocytes are the least mechanically stiff cells within the growth plate. Altogether, these findings suggest that SOC has evolved to protect the hypertrophic chondrocytes from the high mechanical stress encountered in the terrestrial environment.

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.

Dental cell type atlas reveals stem and differentiated cell types in mouse and human teeth.

Understanding cell types and mechanisms of dental growth is essential for reconstruction and engineering of teeth. Therefore, we investigated cellular composition of growing and non-growing mouse and human teeth. As a result, we report an unappreciated cellular complexity of the continuously-growing mouse incisor, which suggests a coherent model of cell dynamics enabling unarrested growth. This model relies on spatially-restricted stem, progenitor and differentiated populations in the epithelial and mesenchymal compartments underlying the coordinated expansion of two major branches of pulpal cells and diverse epithelial subtypes. Further comparisons of human and mouse teeth yield both parallelisms and differences in tissue heterogeneity and highlight the specifics behind growing and non-growing modes. Despite being similar at a coarse level, mouse and human teeth reveal molecular differences and species-specific cell subtypes suggesting possible evolutionary divergence. Overall, here we provide an atlas of human and mouse teeth with a focus on growth and differentiation.

Do all geckos hatch in the same way? Histological and 3D studies of egg tooth morphogenesis in the geckos Eublepharis macularius Blyth 1854 and Lepidodactylus lugubris Duméril & Bibron 1836.

The egg tooth of squamates evolved to facilitate hatching from mineralized eggshells. Squamate reptiles can assist their hatching with a single unpaired egg tooth (unidentates) or double egg teeth (geckos and dibamids). Egg tooth ontogeny in two gekkotan species, the leopard gecko Eublepharis macularius and the mourning gecko Lepidodactylus lugubris, was compared using microtomography, scanning electron microscopy, and light microscopy. Investigated species are characterized by different hardnesses of their eggshells. Leopard geckos eggs have a relatively soft and flexible parchment (leathery) shell, while eggshells of mourning geckos are hard and rigid. Embryos of both species, like other Gekkota, have double egg teeth, but the morphology of these structures differs between the investigated species. These differences in shape, localization, and spatial orientation were present from the earliest stages of embryonic development. In mourning gecko, anlagen of differentiating egg teeth change their position on the palate during embryonic development. Initially they are separated by condensed mesenchyme, but later in development, their enamel organs are connected. In leopard geckos, the localization of egg tooth germs does not change, but their spatial orientation does. Egg teeth of this species shift from inward to outward orientation. This is likely related to differences in structure and mechanical properties of eggshells in the studied species. In investigated species, two hatching mechanisms are possible during emergence of young individuals. We speculate that mourning geckos break the eggshell through puncturing action with egg teeth, similar to the pipping phase of chick and turtles embryos. Egg teeth of leopard geckos cut egg membranes similarly to most squamates. Our results also revealed differences in egg tooth implantation between Gekkota and Unidentata: gekkotan egg teeth are subthecodont (in shallow sockets), while those in unidentates are acrodont (attached to the top of the alveolar ridge). © 2020 Wiley Periodicals LLC.

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.

Delivery of Iron Oxide Nanoparticles into Primordial Germ Cells in Sturgeon.

Nanoparticles are finding increasing applications in diagnostics, imaging and therapeutics in medicine. Iron oxide nanoparticles (IONs) have received significant interest of scientific community due to their distinctive properties. For the first time, we have delivered IONs into germ cells in any species. Our results showed that sturgeon primordial germ cells (PGCs) delivered with IONs could be detected until seven days post fertilization (dpf) under fluorescent microscope and at 22 dpf by micro-CT. Delivery of IONs into cells could be helpful for studying germ cell biology and the improvement of germ cell-based bio-technologies as isolation of PGCs using magnetic activated cell sorting or application of hyperthermia for a host sterilization purpose. Intriguingly, in our study, we did not find any toxic effects of IONs on the survival and hatching rates of sturgeon embryos when compared with embryos injected with FITC-dextran only.

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.

Bichir external gills arise via heterochronic shift that accelerates hyoid arch development.

In most vertebrates, pharyngeal arches form in a stereotypic anterior-to-posterior progression. To gain insight into the mechanisms underlying evolutionary changes in pharyngeal arch development, here we investigate embryos and larvae of bichirs. Bichirs represent the earliest diverged living group of ray-finned fishes, and possess intriguing traits otherwise typical for lobe-finned fishes such as ventral paired lungs and larval external gills. In bichir embryos, we find that the anteroposterior way of formation of cranial segments is modified by the unique acceleration of the entire hyoid arch segment, with earlier and orchestrated development of the endodermal, mesodermal, and neural crest tissues. This major heterochronic shift in the anteroposterior developmental sequence enables early appearance of the external gills that represent key breathing organs of bichir free-living embryos and early larvae. Bichirs thus stay as unique models for understanding developmental mechanisms facilitating increased breathing capacity.

New avatars for Myriapods: Complete 3D morphology of type specimens transcends conventional species description (Myriapoda, Chilopoda).

We present high-resolution X-ray microtomography (microCT) to enhance the standard morphological description of a recently described centipede, Eupolybothrus liburnicus Akkari, Komericki, Weigand, Edgecombe and Stoev, 2017. The 3D images of the holotype and paratype specimens are considered here as cybertypes for the species-a universal and virtual representation of the type material. This 'avatar' of the holotype is the first published male centipede cybertype. The microtomographic data of both types revealed further characters of systematic value and allowed us to hypothesise on the function of some of the male secondary structures and the mating behaviour of the species. Additionally, we compared part of the female reproductive system of E. liburnicus to species from the same genus, including E. cavernicolus Stoev & Komericki 2013, its closest congener. The high-resolution 3D image data have been uploaded to an open repository (MorphoSource.org) to serve in any subsequent study on the species and genus, as we believe this would catalyse biosystematic research on this and other arthropod groups.

Dispersal of thermophilic beetles across the intercontinental Arctic forest belt during the early Eocene.

Massive biotic change occurred during the Eocene as the climate shifted from warm and equable to seasonal and latitudinally stratified. Mild winter temperatures across Arctic intercontinental land bridges permitted dispersal of frost-intolerant groups until the Eocene-Oligocene boundary, while trans-Arctic dispersal in thermophilic groups may have been limited to the early Eocene, especially during short-lived hyperthermals. Some of these lineages are now disjunct between continents of the northern hemisphere. Although Eocene climate change may have been one of the most important drivers of these ancient patterns in modern animal and plant distributions, its particular events are rarely implicated or correlated with group-specific climatic requirements. Here we explored the climatic and geological drivers of a particularly striking Neotropical-Oriental disjunct distribution in the rove beetle Bolitogyrus, a suspected Eocene relict. We integrated evidence from Eocene fossils, distributional and climate data, paleoclimate, paleogeography, and phylogenetic divergence dating to show that intercontinental dispersal of Bolitogyrus ceased in the early Eocene, consistent with the termination of conditions required by thermophilic lineages. These results provide new insight into the poorly known and short-lived Arctic forest community of the Early Eocene and its surviving lineages.