Wnt16 Elicits a Protective Effect Against Fractures and Supports Bone Repair in Zebrafish

Bone homeostasis is a dynamic, multicellular process which is required throughout life to maintain bone integrity, prevent fracture and respond to skeletal damage. WNT16 has been linked to bone fragility and osteoporosis in human genome wide association studies, as well as the functional haematopoiesis of leukocytes in vivo. However, the mechanisms by which WNT16 promotes bone health and repair are not fully understood. We used CRISPR-Cas9 to generate mutant zebrafish lacking Wnt16 (wnt16-/-) to study its effect on bone dynamically. wnt16 mutants displayed variable tissue mineral density and were susceptible to spontaneous fractures and the accumulation of bone calluses at an early age. Fractures were induced in the lepidotrichia of the caudal fins of wnt16-/- and wild type (WT) zebrafish; this model was used to probe the mechanisms by which Wnt16 regulates skeletal and immune cell-dynamics in vivo. In WT fins, wnt16 expression increased significantly during the early stages for bone repair. Mineralization of bone during fracture repair was significantly delayed in wnt16 mutants compared to WT zebrafish. Surprisingly, we found no evidence that the recruitment of innate immune cells to fractures or soft callus formation was altered in wnt16 mutants. However, osteoblast recruitment was significantly delayed in wnt16 mutants post-fracture, coinciding with precocious activation of the canonical Wnt signalling pathway. In situ hybridization suggests that canonical Wnt-responsive cells within fractures are osteoblast progenitors, and that osteoblast differentiation during bone repair is coordinated by the dynamic expression of runx2a and wnt16. This study highlights zebrafish as an emerging model for functionally validating osteoporosis-associated genes and investigating fracture repair dynamically in vivo. Using this model, we demonstrate that Wnt16 protects against fracture and supports bone repair, likely by modulating canonical Wnt activity, via runx2a, to facilitate osteoblast differentiation and bone matrix deposition

A robust, semi-automated approach for counting cementum increments imaged with synchrotron X-ray computed tomography

Cementum, the tissue attaching mammal tooth roots to the periodontal ligament, grows appositionally throughout life, displaying a series of circum-annual incremental features. These have been studied for decades as a direct record of chronological lifespan. The majority of previous studies on cementum have used traditional thin-section histological methods to image and analyse increments. However, several caveats have been raised in terms of studying cementum increments in thin-sections. Firstly, the limited number of thin-sections and the two-dimensional perspective they impart provide an incomplete interpretation of cementum structure, and studies often struggle or fail to overcome complications in increment patterns that complicate or inhibit increment counting. Increments have been repeatedly shown to both split and coalesce, creating accessory increments that can bias increment counts. Secondly, identification and counting of cementum increments using human vision is subjective, and it has led to inaccurate readings in several experiments studying individuals of known age. Here, we have attempted to optimise a recently introduced imaging modality for cementum imaging; X-ray propagation-based phase-contrast imaging (PPCI). X-ray PPCI was performed for a sample of rhesus macaque (Macaca mulatta) lower first molars (n = 10) from a laboratory population of known age. PPCI allowed the qualitative identification of primary/annual versus intermittent secondary increments formed by splitting/coalescence. A new method for semi-automatic increment counting was then integrated into a purpose-built software package for studying cementum increments, to count increments in regions with minimal complications. Qualitative comparison with data from conventional cementochronology, based on histological examination of tissue thin-sections, confirmed that X-ray PPCI reliably and non-destructively records cementum increments (given the appropriate preparation of specimens prior to X-ray imaging). Validation of the increment counting algorithm suggests that it is robust and provides accurate estimates of increment counts. In summary, we show that our new increment counting method has the potential to overcome caveats of conventional cementochronology approaches, when used to analyse three-dimensional images provided by X-ray PPCI.Peer reviewe

Regenerating zebrafish scales express a subset of evolutionary conserved genes involved in human skeletal disease

BACKGROUND: Scales are mineralised exoskeletal structures that are part of the dermal skeleton. Scales have been mostly lost during evolution of terrestrial vertebrates whilst bony fish have retained a mineralised dermal skeleton in the form of fin rays and scales. Each scale is a mineralised collagen plate that is decorated with both matrix-building and resorbing cells. When removed, an ontogenetic scale is quickly replaced following differentiation of the scale pocket-lining cells that regenerate a scale. Processes promoting de novo matrix formation and mineralisation initiated during scale regeneration are poorly understood. Therefore, we performed transcriptomic analysis to determine gene networks and their pathways involved in dermal scale regeneration. RESULTS: We defined the transcriptomic profiles of ontogenetic and regenerating scales of zebrafish and identified 604 differentially expressed genes (DEGs). These were enriched for extracellular matrix, ossification, and cell adhesion pathways, but not in enamel or dentin formation processes indicating that scales are reminiscent to bone. Hypergeometric tests involving monogenetic skeletal disorders showed that DEGs were strongly enriched for human orthologues that are mutated in low bone mass and abnormal bone mineralisation diseases (P< 2× 10(−3)). The DEGs were also enriched for human orthologues associated with polygenetic skeletal traits, including height (P< 6× 10(−4)), and estimated bone mineral density (eBMD, P< 2× 10(−5)). Zebrafish mutants of two human orthologues that were robustly associated with height (COL11A2, P=6× 10(−24)) or eBMD (SPP1, P=6× 10(−20)) showed both exo- and endo- skeletal abnormalities as predicted by our genetic association analyses; col11a2(Y228X/Y228X) mutants showed exoskeletal and endoskeletal features consistent with abnormal growth, whereas spp1(P160X/P160X) mutants predominantly showed mineralisation defects. CONCLUSION: We show that scales have a strong osteogenic expression profile comparable to other elements of the dermal skeleton, enriched in genes that favour collagen matrix growth. Despite the many differences between scale and endoskeletal developmental processes, we also show that zebrafish scales express an evolutionarily conserved sub-population of genes that are relevant to human skeletal disease. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12915-021-01209-8

Reply to: Revisiting life history and morphological proxies for early mammaliaform metabolic rates

Non peer reviewe

Collagen pre-strain discontinuity at the bone—Cartilage interface

The bone-cartilage unit (BCU) is a universal feature in diarthrodial joints, which is mechanically-graded and subjected to shear and compressive strains. Changes in the BCU have been linked to osteoarthritis (OA) progression. Here we report existence of a physiological internal strain gradient (pre-strain) across the BCU at the ultrastructural scale of the extracellular matrix (ECM) constituents, specifically the collagen fibril. We use X-ray scattering that probes changes in the axial periodicity of fibril-level D-stagger of tropocollagen molecules in the matrix fibrils, as a measure of microscopic pre-strain. We find that mineralized collagen nanofibrils in the calcified plate are in tensile pre-strain relative to the underlying trabecular bone. This behaviour contrasts with the previously accepted notion that fibrillar pre-strain (or D-stagger) in collagenous tissues always reduces with mineralization, via reduced hydration and associated swelling pressure. Within the calcified part of the BCU, a finer-scale gradient in pre-strain (0.6% increase over ~50μm) is observed. The increased fibrillar pre-strain is linked to prior research reporting large tissue-level residual strains under compression. The findings may have biomechanical adaptative significance: higher in-built molecular level resilience/damage resistance to physiological compression, and disruption of the molecular-level pre-strains during remodelling of the bone-cartilage interface may be potential factors in osteoarthritis-based degeneration

Reptile-like physiology in Early Jurassic stem-mammals

Despite considerable advances in knowledge of the anatomy, ecology and evolution of early mammals, far less is known about their physiology. Evidence is contradictory concerning the timing and fossil groups in which mammalian endothermy arose. To determine the state of metabolic evolution in two of the earliest stem-mammals, the Early Jurassic Morganucodon and Kuehneotherium, we use separate proxies for basal and maximum metabolic rate. Here we report, using synchrotron X-ray tomographic imaging of incremental tooth cementum, that they had maximum lifespans considerably longer than comparably sized living mammals, but similar to those of reptiles, and so they likely had reptilian-level basal metabolic rates. Measurements of femoral nutrient foramina show Morganucodon had blood flow rates intermediate between living mammals and reptiles, suggesting maximum metabolic rates increased evolutionarily before basal metabolic rates. Stem mammals lacked the elevated endothermic metabolism of living mammals, highlighting the mosaic nature of mammalian physiological evolution. Modern mammals are endothermic, but it has not been clear when this type of metabolism evolved. Here, Newham et al. analyse tooth and bone structure in Early Jurassic stem-mammal fossils to estimate lifespan and blood flow rates, which inform about basal and maximum metabolic rates, respectively, and show these stem-mammals had metabolic rates closer to modern ectothermic reptiles than to endothermic mammals.Peer reviewe

Exploring the use of tomography for the quantification of cementum growth patterns across the mammal phylogeny

This project is focused on the application of state-of-the-art imaging technologies and novel analytical techniques in order to improve understanding of the growth and structure of cementum; the mineralised tissue that connects teeth to the peridontium in mammals. Like all vertebrate hard tissues, the growth of cementum is controlled by a series of natural rhythms that control its pattern and pace. This is recorded in circum-annual increments (similar to tree rings) hypothetically created by dietary differences in the production rate of its two key components; hydroxyapatite matrix and collagen fibres. As cementum is only rarely resorbed, these increments can be counted to provide a direct estimate of chronological age, a technique known as cementochronology. Further, the circum-annual rhythm followed by cementum increments may also allow them to record discrete life history events that create severe strain on body metabolism over the course of several months, such as pregnancy. However, the exact causation and rhythm of cementum increments is still poorly understood due to a paucity of direct experimental study. The majority of previous studies of cementochronology have been based on thin-section histology, and several caveats of this approach have undermined confidence in the ability to quantitatively analyse cementum increments. Overall, current study of cementum lacks the application of modern technology and analytical methods that have revolutionised interpretation of microstructures and ultrastructures of other hard tissues such as bone, and increased their use as hallmarks of disease or as records of life history. The application of synchrotron X-ray tomography (SR CT) has here been used to improve understanding of the structure and count of cementum increments in mammals ranging from our oldest ancestors, to some of our closest relatives. The non-destructive nature and high throughput of SR CT has allowed study of large samples of Morganucodon and Kuehneotherium, two of the oldest known fossil mammals from the early Jurassic (~200 million years ago). The three-dimensional perspective and ultra-high resolutions (&lt;500nm voxel size) of SR CT data provided an unprecedented level of detail to study and more precise counts of increments than previous thin-section-based techniques, which in-turn provided minimum estimates of maximum lifespan for both animals. Further, as the lifespans of modern mammals are inversely related to their basal metabolic rate and post-natal growth rate, these estimates have been used to improve understanding of the physiology of early mammals and the evolution of the sophisticated endothermic (warm-blooded) physiology of modern mammals. From this study the potential of SR CT for analyzing cementum growth, and the relationship between cementum growth and life history variables, was made clear. Subsequent SR CT imaging was conducted on a sample of rhesus macaque monkeys (Macaca mulatta), raised in laboratory conditions. One sub-sample consisted of breeding females, another of non-breeding females, as well as a juvenile female and a male. This sample was used to study the optimal experimental settings for SR CT imaging of cementum, and generate an image processing and analysis workflow to automatically count cementum increments and study their shape and texture using computer vision. This workflow was then used to study sexual dimorphism in increment structure, and to investigate the potential for pregnancy events to be recorded in SR CT cementum data in both the M. mulatta sample and samples of C12th and C19th century archaeological humans of known sex (including three C19th individuals of known age and reproductive history). Significant dimorphism in increment shape and texture was found in both taxa. Female increments were found to be significantly more tortuous than males, with lower contrast and less well defined boundaries. Further, increments formed during pregnancy were found to be more chaotic in shape and texture than surrounding increments. Finally, SR CT was used to image the cementum increments of a diverse fauna of fossil mammals from the Middle Jurassic of the UK (Bathonian: ~168-166 million years ago). This fauna can be split into stem mammals (mammaliaforms alongside Morganucodon and Kuehneotherium), and crown mammals (bracketed evolutionarily by living mammals). Using the automative counting technique developed for counting primate cementum increments, it was found that mammaliaforms lived significantly longer maximum lifespans than contemporary crown mammals. This in-turn suggests a proportional disparity in metabolic potential, and that Mid Jurassic crown mammals had developed a similar basal metabolic rate to living mammals of comparable body mass. In summary, the work presented in this thesis has shown that the application of state-of-the-art analysis techniques has the ability to maximise the potential of cementum as a recording structure of disparate elements of life history among mammals. The workflow for imaging, processing and study developed here can be applied to a wide range of life history variables, for both fossil and extant taxa. The automated nature of the image analysis techniques presented overcomes many of the major caveats highlighted in previous thin-section based studies of cementum and their applicability, robusticity and accuracy can only be improved by their continued development in the wider cementochronology community outside of this project

Therian mammals experience an ecomorphological radiation during the Late Cretaceous and selective extinction at the K–Pg boundary

It is often postulated that mammalian diversity was suppressed during the Mesozoic Era and increased rapidly after the Cretaceous–Palaeogene (K– Pg) extinction event.We test this hypothesis by examining macroevolutionary patterns in early therian mammals, the group that gave rise to modern placentals and marsupials. We assess morphological disparity and dietary trends using morphometric analyses of lower molars, and we evaluate generic level taxonomic diversity patterns using techniques that account for sampling biases. In contrast with the suppression hypothesis, our results suggest that an ecomorphological diversification of therians began 10-20 Myr prior to the K–Pg extinction event, led by disparate metatherians and Eurasian faunas. This diversification is concurrent with ecomorphological radiations of multituberculate mammals and flowering plants, suggesting that mammals as a whole benefitted from the ecological rise of angiosperms. In further contrast with the suppression hypothesis, therian disparity decreased immediately after the K–Pg boundary, probably due to selective extinction against ecological specialists and metatherians. However, taxonomic diversity trends appear to have been decoupled from disparity patterns, remaining low in the Cretaceous and substantially increasing immediately after the K–Pg extinction event. The conflicting diversity and disparity patterns suggest that earliest Palaeocene extinction survivors, especially eutherian dietary generalists, underwent rapid taxonomic diversification without considerable morphological diversification.</p

Therian mammals experience an ecomorphological radiation during the Late Cretaceous and selective extinction at the K–Pg boundary

It is often postulated that mammalian diversity was suppressed during the Mesozoic Era and increased rapidly after the Cretaceous–Palaeogene (K– Pg) extinction event.We test this hypothesis by examining macroevolutionary patterns in early therian mammals, the group that gave rise to modern placentals and marsupials. We assess morphological disparity and dietary trends using morphometric analyses of lower molars, and we evaluate generic level taxonomic diversity patterns using techniques that account for sampling biases. In contrast with the suppression hypothesis, our results suggest that an ecomorphological diversification of therians began 10-20 Myr prior to the K–Pg extinction event, led by disparate metatherians and Eurasian faunas. This diversification is concurrent with ecomorphological radiations of multituberculate mammals and flowering plants, suggesting that mammals as a whole benefitted from the ecological rise of angiosperms. In further contrast with the suppression hypothesis, therian disparity decreased immediately after the K–Pg boundary, probably due to selective extinction against ecological specialists and metatherians. However, taxonomic diversity trends appear to have been decoupled from disparity patterns, remaining low in the Cretaceous and substantially increasing immediately after the K–Pg extinction event. The conflicting diversity and disparity patterns suggest that earliest Palaeocene extinction survivors, especially eutherian dietary generalists, underwent rapid taxonomic diversification without considerable morphological diversification.</p