The evolution of habitual bipedalism was a defining milestone in human history, yet fossil evidence indicates substantial variation in how bipedality was practiced. Trabecular bone, which responds throughout life to joint loading and orientation, offers a powerful way to investigate these differences. Interpreting variation in fossil hominins, however, requires a reliable baseline of intraspecific variation in modern humans, accounting for differences in lifestyle, mobility, and habitual behaviors. Many modern skeletal collections derive from postindustrial populations whose activity patterns, footwear use, and health profiles differ markedly from past populations, potentially biasing interpretations. Thus, habitually shod, postindustrial humans may not be the best proxy for the loading environments experienced by early hominins, whereas unshod individuals provide a closer approximation of barefoot locomotion. To explore the effects of footwear on internal bone structure, we examined trabecular architecture in the talus of habitually shod (N = 40) modern humans (Homo sapiens) from recent donation-based skeletal collections and habitually unshod (N = 16) individuals from the Andaman Island, Khoisan, and Aka populations, representing diverse ecological and subsistence contexts, using high-resolution micro-computed tomographic scans. Trabecular parameters-including bone volume fraction (BV/TV), relative BV/TV (rBV/TV), trabecular thickness (Tb.Th), separation (Tb.Sp), number (Tb.N), and degree of anisotropy (DA)-were quantified using canonical holistic morphometric analysis (cHMA). Shod individuals exhibited higher mean BV/TV and greater variability in BV/TV, suggesting that the talus remains heavily loaded due to its central role in weight bearing during bipedal gait, and that different types of footwear create variable force distributions across the ankle. The spatial distribution of rBV/TV revealed further differences between the groups: shod individuals showed higher rBV/TV in the medial and posterior talus, consistent with more dorsiflexed and medially loaded ankle postures, whereas unshod individuals had elevated rBV/TV in the talar head and neck, potentially reflecting loading associated to squatting or climbing. Shod individuals also exhibited higher mean DA indicating more strongly organized and directionally constrained trabeculae. Mean BV/TV was likewise higher in the shod group and primarily driven by greater trabecular thickness, as trabecular separation and number were broadly similar between groups, suggesting loading differences associated with footwear primarily influence BV/TV and trabecular anisotropy. By establishing clear patterns of trabecular variation between shod and unshod modern humans, this study provides a robust comparative framework for interpreting locomotor behavior in fossil hominins, while highlighting the need to further disentangle the effects of footwear from differences in body size, stature, subsistence strategy, and other aspects of lifestyle among human populations. Recognizing how habitual behavior influences internal bone architecture is essential for reconstructing past movement patterns and understanding the evolutionary pathways of bipedalism.
Diffusible iodine-based contrast-enhanced computed tomography (diceCT) approaches have improved our ability to non-destructively sample soft tissue anatomy in animals. Key aspects of muscle fiber architecture, like muscle fiber length and pennation, can in theory be reconstructed from diceCT scans; however, manual segmentation is laborious and time-consuming. Several software applications have been developed to automate reconstruction of muscle fibers from diceCT data, including both open-source and proprietary options. This paper expands on the GoodFibes package, an R-language based toolkit for detecting, reconstructing, and visualizing muscle fibers from diceCT image stack datasets, to include the analysis of pennation angle in 3D. Fiber orientation is determined via principal component analysis of fiber path coordinate data, and muscle fibers may be aligned in the R environment to a tendon or line of action, permitting the direct calculation of pennation angle. I demonstrate the effectiveness of this approach using an ant mandibular muscle dataset that has been previously studied using manual digital dissection as well as both open source and proprietary approaches for muscle fiber detection and analysis. I also apply these new utilities to a comparative case study of Percid fish biting muscles. Overall, I find that this approach is effective at estimating muscle pennation angles in the ant dataset, with performance comparable to other software applications. Application of these new utilities to Percid jaw muscles reveals a pattern of divergent pennation between the adductor mandibulae (AM) pars malaris and AM pars rictostegalis in the Darter subfamily, a group of small, benthic fishes with innovative jaw anatomy and kinematics. Large, predatory Percids and small, benthic species living in streams may have different strategies for maximizing bite force proportional to body size. Overall, these additions expand the growing open-source toolkit for the analysis of diceCT data, and in particular the flexible R programming language environment that is widely used by biologists.
Proboscideans exhibit limb morphologies specialised for supporting extreme body masses under predominantly compressive loading. These graviportal adaptations include repositioning of the limb bones closer to the trunk, resulting in a columnar stance. Among proboscideans, two humeral morphotypes have been proposed (slender and robust), yet their functional significance under compressive loading remains poorly understood. Finite element analysis (FEA) is applied to evaluate biomechanical performances of proboscidean humeri subject to axial compression, simulating conditions associated with a columnar stance and deviations from it. Our results reveal clear differences between morphotypes, with slender humeri performing worse than the robust morphotype and functioning optimally under near-axial loading. Robust humeri consistently exhibit lower peak stress under both neutral and off-axis loading and show considerable resistance to mediolateral stresses. Across all taxa, stress magnitudes generally increase when deviating from a neutral orientation, especially during posterior loading orientations, highlighting the mechanical importance of maintaining columnar limb posture. These findings support the hypothesis that robust morphologies are better adapted to resist multidirectional stresses, while slender morphologies favour energetic and locomotor efficiency. This study demonstrates the applicability of FEA in assessing compressive performance in postcranial elements and provides a novel framework for investigating biomechanical adaptations in graviportal taxa.
The morphology and internal structure of tetrapod limb bones reflect complex interactions between functional demands, mechanical loading, and environmental conditions. In most tetrapods, limb bone microanatomy shows a clear ecological signal, with aquatic taxa typically displaying increased bone compactness and terrestrial taxa exhibiting more open medullary cavities. Testudines represent a notable exception to this pattern, as both terrestrial and aquatic turtles possess unusually compact long bones, suggesting that factors beyond habitat alone shape their skeletal design. Here, we present the first comprehensive whole-bone, longitudinal analysis of cortical bone mass, geometry, tissue quality, and mechanostat-related distribution/quality (d/q) relationships in turtle long bones, aiming to evaluate how locomotor ecology modulates bone design within the constraints of the turtle bauplan. We analyzed femora and humeri of four turtle species spanning a terrestrial-aquatic gradient (Chelonoidis carbonarius, Chelonoidis chilensis, Trachemys dorbignii, and Phrynops hilarii) using peripheral quantitative computed tomography (pQCT). Ten serial cross-sections along each bone were assessed to quantify cortical bone mineral content, cortical area, volumetric bone mineral density, cross-sectional moments of inertia, bone strength indices, and d/q relationships between tissue stiffness and cross-sectional design. Statistical comparisons focused on mid-diaphyseal regions, with interspecific differences evaluated using ANOVA, and mechanostat-related patterns examined through correlation analyses and ANCOVA. All species exhibited the characteristic turtle "over-designed" condition, with abundant cortical bone and peak mass and density at midshaft. However, marked interspecific differences emerged in bone geometry and in the organization of cortical tissue. The terrestrial species, particularly Chelonoidis carbonarius, showed higher cortical mass and larger moments of inertia, reflecting enhanced resistance to bending under weight-bearing locomotion. In contrast, the more aquatic Phrynops hilarii displayed lower moments of inertia and flatter or near-zero d/q slopes, indicating a biomechanical strategy favoring compressive stiffness via endosteal infilling rather than structural optimization for bending. Intermediate species occupied transitional positions along this gradient. These results demonstrate that, despite strong phylogenetic constraints associated with the turtle bauplan, the spatial distribution of cortical bone is finely tuned to locomotor ecology. The bone mechanostat in turtles operates within an "over-designed" framework to produce distinct structural solutions for terrestrial versus aquatic loading regimes. Our findings provide a quantitative link between habitat, locomotor mode, and long-bone design, revealing that turtle limbs are not simply overbuilt, but precisely engineered for their mechanical environments.
Recent advances in shape quantification techniques have revolutionised the field of evolutionary morphology by providing a time-efficient alternative to well-established methods such as geometric morphometrics. In particular, landmark-free methods are at the forefront of new developments in shape analysis, but concerns over their reproducibility and sensitivity to non-biological variation have hindered more widespread adoption. A primary source of non-biological bias in shape analysis is misalignment and incorrect scaling of specimens, which can result in capturing anatomically incongruent aspects of shape in otherwise homologous structures. Here, we compare methods for mesh alignment and scaling using a recently described landmark-free approach, Deterministic Atlas Analysis (DAA). We find similarities between morphospaces derived from meshes aligned using most alignment protocols, but with large differences in variance structure. Surprisingly, the largest differences are not found between Procrustes-based and non-Procrustes-based approaches, but between the Procrustes-based method with internal scaling and all other alignment approaches. More broadly, scaling method has the greatest impact on shape variation captured by DAA. Based on these results, we make recommendations to ensure that alignment strategy maintains homology in landmark-free morphometry. As in all morphometric analyses, violating principles of homology can result in spurious or inaccurate biological inferences in macroevolutionary studies of anatomy and must be explicitly considered in designing methodological frameworks.
GABAergic interneurons play a central role in shaping cortical microcircuit function, yet the anatomical organization of major interneuron populations across different subdivisions of the human prefrontal cortex remains poorly characterized. The present study examined the laminar distribution and regional organization of major GABAergic interneuron populations in the dorsal (Brodmann area 9) and ventral (Brodmann area 14r) portions of the human prefrontal cortex. Postmortem human prefrontal cortex tissue from five normotypical adult male brains was analysed using the following molecular markers: calretinin (CR), parvalbumin (PV), calbindin (CB), and somatostatin (SOM). Double-labelling immunofluorescence was used to assess overlaps between different GABAergic interneuron populations, and RNAscope in situ hybridization was used to confirm their neurotransmitter phenotype. All analysed populations demonstrated a GABAergic molecular phenotype. CR+ neurons constituted the largest interneuron population in both analysed cortical regions and showed marked supragranular enrichment. CB+ neurons were also concentrated in upper cortical layers and frequently co-expressed SOM. By contrast, PV+ neurons were concentrated in layers III-IV, whereas SOM+ neurons were distributed more uniformly across layers II-VI. The principal regional difference was a significantly higher proportion of CR+ neurons in BA14r compared with BA9 (10.24% vs. 8.21% of all neurons; p = 0.0248), while exploratory analyses additionally indicated a higher CR/PV ratio and a lower PV/SOM ratio. These differences were driven predominantly by variation in supragranular interneuron composition, while infragranular organization remained comparatively similar between the two cortical regions. These findings support region-specific specialization of inhibitory interneuron organization in the human prefrontal cortex and suggest that differences in supragranular inhibitory circuitry may contribute to the distinct functional architecture of dorsal and ventral prefrontal cortico-cortical networks.
Studies in anatomy are frequently performed through dissection and histology as investigative methods. These techniques are, however, destructive and struggle to capture the peripheral nerves over larger areas, such as the pelvis, and their trajectories thus remain elusive. This study aimed to explore the capabilities of non-destructive micro-CT in identifying small nerves in the periphery of the pelvic area and map them in human fetal development. Six human fetal pelvic samples were derived from the Dutch Fetal Biobank; samples were contrast enhanced with B-Lugol and scanned through micro-CT imaging. Scans were segmented using Avizo Amira software to quantify nerves entering the rectum by crossing the longitudinal muscle layer and visualize their trajectory in relationship to pelvic organs across development age range and sex. Results were verified with haematoxylin and eosin stained histological slices. The following conclusions were found: (1) Micro-CT is able to resolve nerves with a diameter larger than 60 μm in the periphery of the pelvic area in full fetal pelvises of 4 cm in size. (2) Both above and below the level of the levator ani, small lateral nerves enter the anal canal by penetrating the longitudinal muscle layer. (3) Males appear to have more crossing nerves than females. (4) A developmental shift was observed with more nerve crossings below the levator ani muscle in older fetuses. Micro-CT was successfully applied to scan human fetal pelvic samples to study nerves and provides a new way to analyse topographical nerve development. Microscale nerves cross to the anal canal at all levels, which should be taken into account when operating in this area.
The pectoral girdle of terrestrial tetrapods functions as a critical interface for transmitting loads between the trunk and forelimbs. While mammals with a parasagittal forelimb posture mainly rely on the scapula as the principal element for load bearing, talpids (Talpidae) exhibit a markedly specialised pectoral girdle related to a hyper-abducted forelimb posture. In talpids, the humero-clavicular articulation plays an important functional role, suggesting that load-transmission mechanisms within the pectoral girdle may differ from those of typical terrestrial mammals. However, because the pectoral girdle of talpids is highly developed as a fossorial adaptation, it is difficult to infer the mechanical environments experienced by each skeletal element from external morphology alone, and the mechanical roles of the pectoral girdle elements are poorly understood. To examine whether posture-related reorganisation of load-transmission pathways is reflected in bone microstructure, collagen fibre orientation weighted mean greyscale level (CFO-WMGL) and cortical bone robusticity (relative robusticity indicator: RRI) in the clavicle and scapula were evaluated in fossorial talpids (Mogera imaizumii, Mogera wogura and Urotrichus talpoides) and compared with those of the parasagittal outgroup Suncus murinus. Talpids exhibited high RRI values in both the clavicle and scapula, indicating overall mechanical reinforcement of the pectoral girdle. In contrast, CFO-WMGL showed element-specific differences among taxa. In Mogera, CFO-WMGL values were consistently higher in the clavicle than in the scapula, suggesting that the clavicle played a relatively greater load-bearing role within the pectoral girdle. Conversely, S. murinus exhibited histological characters consistent with the scapula functioning as the principal element for load bearing. These distinct histological patterns indicate that the pectoral girdle is not a mechanically uniform structure, but rather an integrated functional system in which each skeletal element performs distinct mechanical roles. These findings suggest that the evolution of the forelimb posture in talpids was accompanied by a reorganisation of load-transmission pathways within the pectoral girdle, involving a relative increase in the load-bearing role of the clavicle compared with the scapula. This study demonstrates that bone histological characters can provide insights into skeletal function that are not apparent from external morphology alone. This approach may offer a useful framework for reconstructing load-transmission mechanisms in both extant and extinct tetrapods and for investigating how the mechanical demands imposed on each skeletal element have changed during evolution. Future comparative studies across a broader range of taxa will be essential for evaluating the generality of these relationships and for advancing our understanding of the mechanical reorganisation of the vertebrate musculoskeletal system throughout evolutionary history.
The history of anatomical dissection in nineteenth-century Britain has largely been reconstructed from archival sources, including legislation, institutional records and medical writings. Archaeological evidence for the material practices of anatomy schools, however, remains comparatively limited. In 2010, redevelopment within the main quadrangle of University College London (UCL) revealed a substantial assemblage of human and animal skeletal remains associated with the institution's early anatomy school. Excavation recovered more than 8700 human skeletal fragments and over 800 animal bones, together with artefacts dating to the late nineteenth century. Osteological analysis indicates a minimum of 38 individuals, predominantly adults aged 30-50 years, with an overall male:female ratio of approximately 2:1. Only a small proportion of bones exhibited cut marks consistent with dissection, suggesting that the assemblage partly represents the discard of a curated teaching collection rather than solely the residue of routine student dissection. The associated animal remains, dominated by domesticated taxa including cattle, sheep/goat and horse, indicate that comparative anatomy formed an integral component of the same pedagogical environment. Considered in the context of the Anatomy Act of 1832 and the expansion of metropolitan medical education, the assemblage provides rare material evidence for the procurement, pedagogical use, preparation, curation and eventual disposal of both human and animal anatomical material at one of Britain's earliest secular medical schools. The quadrangle assemblage therefore offers a rare opportunity to reflect on the everyday practice of anatomy and the complex relationship between medical education, comparative anatomy, society and the bodies of the nineteenth-century poor in London.
The superior hypogastric plexus (SHP) and hypogastric nerves (HN) are components of the autonomic nervous system required for sympathetic regulation of the pelvic viscera. Despite their functional importance, the three-dimensional (3D) distribution and immunohistochemical composition of neurons in these structures remain poorly characterized. In this study, we used a multiscale imaging approach to generate a 3D anatomical and immunohistochemical characterization of the SHP and HN in adult humans. Both embalmed (body donor program) and unembalmed paraformaldehyde (PFA)-fixed specimens (organ donor program) were cleared using a modified Adipo-Clear/iDISCO protocol and imaged by light sheet fluorescence microscopy. Thousands of neuronal cell bodies were identified within the HN, demonstrating that this nerve does not function solely as a conduit for axons. In one HN sample analyzed along its whole length by immunohistochemistry, more than 90% of the neurons were tyrosine hydroxylase-immunoreactive (TH-IR), so presumed to be noradrenergic. Neuronal cell bodies in both the SHP and HN were arranged in clusters of diverse size, embedded within nerve tracts rather than in discrete ganglia. High-resolution confocal microscopy of cryosections confirmed the presence of numerous TH-IR (presumed noradrenergic) neuronal cell bodies and axons in the SHP and HN. Non-noradrenergic axonal populations were also abundant. Putative afferent axons (calcitonin gene-related peptide- and substance P-immunoreactive) traversed the SHP and HN and occasionally encircled individual ganglion neurons, raising the possibility of direct sensory-motor communication at these sites. Collectively, these findings provide a new 3D anatomical and immunohistochemical characterization of the SHP and HN in the adult human. This has important implications for understanding normal pelvic autonomic function and pathophysiology of genitourinary disorders. These data and further application of our imaging approach will inform the improvement of nerve-sparing surgical techniques in the pelvic region and the development of targeted neuromodulation approaches.
Although our understanding of ovary structure in mites is limited, it has been shown that the female gonads in these arachnids are highly diverse compared with other chelicerates. Unlike other chelicerates, which typically have a panoistic ovary with a relatively consistent architecture, mites also possess a meroistic ovary. In the panoistic ovary, germline cells differentiate into oocytes, whereas in the meroistic ovary, the germline cells differentiate into oocytes and their accompanying nurse cells. Both types of ovaries have been observed in the two mite lineages, Acariformes and Parasitiformes. Hydrachnidia (Acariformes), also known as water mites, are one of the largest groups of freshwater arthropods. However, despite their common occurrence, they are less explored compared with freshwater insects and crustaceans. Data on the structure of the ovary in water mites are scarce and originate from the 20 c. The aim of the study is to analyze the ovary structure of adult females representing five families of water mites: Arrenuridae, Hydrachnidae, Hydrodromidae, Pionidae, and Limnesiidae, using light, fluorescence, and transmission electron microscopy. Serial block-face scanning electron microscopy technique was applied for 3D reconstruction of the germline cysts. Our results provide new data on the structure of the ovary in water mites, challenging earlier literature data on the panoistic ovary in this group. In all species examined, the presence of a meroistic ovary has been confirmed. The ovary contains a number of germline cysts composed of several germline cells diversified into one oocyte and a few nurse cells. Within the cysts, each cell (oocyte and nurse cells) is connected by a cytoplasmic bridge to a central cytoplasm. The oocytes start previtellogenesis and protrude on the ovary surface connected to the latter by long oocyte stalks. The nurse cells remain in the ovary wall; their nuclei are highly branched and polyploid. The nurse cells provide macromolecules and organelles to oocytes transferred by trophic cords in a microtubular-dependent manner. The results of our study indicate that the overall architecture of the ovary, the structure of the germline cysts, the pattern of germline cell differentiation, and the trophic support remain consistent across Hydrachnidia.
Cetaceans primarily generate thrust through dorsoventral oscillations of the caudal flukes, while their flippers are generally associated with lift generation and torque production during manoeuvring. However, the humpback whale, which possesses the largest forelimbs among extant and extinct vertebrates, has been observed performing active flipper strokes that contribute to forward propulsion. This behaviour represents a unique combination of axial swimming and underwater flight among cetaceans. In this study we investigate the anatomical correlates of this rarely observed locomotor behaviour and examine how the distinctive flipper anatomy of the humpback whale compares to that of other cetaceans. We focus in particular on the musculoskeletal and tendinous organisation of the flipper. We compared the flipper musculoskeletal anatomy of the humpback whale with that of seven other cetacean species (two mysticetes and five odontocetes). A novel protocol to acquire images during dissections involving ultraviolet (UV) light was employed to enhance the contrast between skeletal elements, musculature and connective tissues, with a particular emphasis on tendons and entheses. Although all flippers are highly vascularised, antebrachial musculature is strongly reduced across cetaceans, a condition that is especially pronounced in humpback whales and several odontocete lineages that lack antebrachial muscles. These findings suggest that active control of the flipper is primarily achieved proximally at the shoulder joint between the scapula and humerus, where the musculature is markedly developed. This proximal control pattern appears to be shared among cetaceans despite substantial differences in flipper size and external morphology. Beyond its anatomical implications this study provides photographic documentation of key flipper structures across cetacean groups and demonstrates that UV-light imaging is an effective tool for revealing internal soft-tissue anatomy. This approach is likely to be valuable for the study of other anatomical systems.
Subplate neurons (SpNs) are among the earliest-born and maturing neurons in the developing cerebral cortex. They arise from multiple origins and can be classified into several subgroups based on morphology, connectivity, and gene expression. These neurons play essential roles in cortical circuit formation, yet their cellular diversity and transcriptional dynamics remain incompletely understood. Here, we characterized transcriptomic profiles of SpN subpopulations in embryonic mouse cortex using Lpar1-EGFP and NeuroD1/Cre-ERT2 (D1B) reporter lines. We applied complementary approaches of gene expression profiling, including bulk microarray analysis, single-cell RNA sequencing (scRNA-seq), and Visium spatial transcriptomics. At embryonic day 17 (E17), scRNA-seq identified 10 distinct Lpar1-EGFP-positive SpN clusters, which spatial transcriptomics mapped to specific cortical regions. While many markers showed enrichment within the subplate region, others extended into the hippocampus and ventral pallium (including the amygdala, claustrum, and endopiriform nucleus). Integrated analysis of Lpar1-EGFP and D1B lines revealed both overlapping and unique gene expression signatures, highlighting dynamic markers of subplate identity. Comparisons between E15 and E17 datasets showed substantial transcriptional shifts, suggesting rapid developmental changes in SpN subgroups. Validation with in situ hybridization and RNAscope confirmed the selectivity of key markers, including Cryab, Cdh13, Nr4a2, and Lmo3. Together, these findings provide a molecular framework for further classifying SpN subtypes and identifying candidate markers for transient versus persistent populations, thereby advancing our understanding of early cortical development.
Order Lamniformes consists of 15 extant shark species that are ecologically diverse and utilize different swimming modes and speeds. Family Lamnidae includes the shortfin mako, porbeagle, and white shark which are fast, athletic sharks that swim using oscillations confined to the caudal body and fin. Other lamniforms, like the common thresher shark (Alopiidae), sand tiger (Carchariidae), and basking shark (Cetorhinidae), swim via oscillations that begin anteriorly, impacting a greater proportion of the axial body. Swimming oscillations subject the body to repeated bending cycles, including the cartilaginous vertebral column, the main longitudinal axis of the body. Vertebrae are mineralized with the amount and arrangement varying among species. We investigated morphological variation in lamniform shark vertebrae to understand adaptations to locomotive demands among species. We examined vertebral morphology and mineral architecture of lamniform centra across three body regions (anterior, middle, and posterior) and among six species (shortfin mako, porbeagle, sand tiger, white, common thresher, and basking shark) through micro-computed tomography scans. We analyzed morphology and structure of 139 vertebrae from 24 sharks using meristics, principal component analyses, and 3D landmark-based geometric morphometrics. Through 3D quantification, we identified regional patterns in centrum size and mineral amount which also varied across shark families. In the lamnids, centra morphometrics are largest in the mid-body and decrease posteriorly simultaneous with increased counts of lamellae. Together, these trends suggest the middle body region is stabilized while allowing for rapid lateral oscillations at the precaudal pit. Cranio-caudally compressed centra with high quantities of lamellae were characteristic for common thresher shark centra, likely to support loading in multiple planes from extreme axial bending during tail-whipping behaviors. In the sand tiger and basking shark, we quantified opposing trends-large anterior centra decreased in size along the column with reduced mineralization for slow swimming. We calculated scaling relationships of mineral volume with shark size and identified a negative allometric relationship, suggesting adult sharks may adapt internal architecture rather than contributing to overall centrum size. This comprehensive analysis of calcified structure in lamniform shark centra provides a greater understanding of skeletal tissues and the adaptation of mineralized cartilage to support swimming and ecological needs.
Cilia are finger-like organelles protruding from the cell membrane, and dysfunction of cilia can cause human diseases called ciliopathies with a range of phenotypes. Some ciliopathies include pituitary phenotypes, and previous studies have shown a role for non-motile primary cilia in pituitary organogenesis. In this study, we generated a pituitary gland-conditional knockout of the ciliary basal body protein CEP164 to investigate the role of primary cilia in pituitary development and postnatal function. Our mouse model shows a loss of pituitary primary cilia between e14.5 and e18.5, although pituitary organogenesis and morphology appear normal in late embryos and postpubertal mice of both sexes. No physiological defects were noted in postpubertal mice, although there were minor changes in the expression of Sox2 and Prl transcripts. We conclude that primary cilia are dispensable in late pituitary organogenesis, therefore suggesting the role of primary cilia occurs during early pituitary development. Further studies may investigate whether cilia are required for specific responses to physiological or pathological conditions where endocrine needs are altered.
Many developmental and metabolic effects of growth hormone (GH) on vertebrate life history traits have been widely studied in biological and biomedical contexts. The scope of alterations in GH/GH receptor (GHR) interactions has generally focused on molecular, cellular, histological, and physiological framing, leaving a gap in understanding of how the cumulative organism- and lifespan-scale manifestations of morphological changes attributable to GH/GHR perturbations may both be influenced by and also influence smaller-scale study results. The pilot study conducted herein used micro-computed tomography (μCT) to survey and characterize the axial and appendicular skeletons of adult male and female bGH (transgenic overexpression of bovine GH) mice and to compare them with those of age- and sex-matched wild-type (WT) controls. Male and female bGH mice in our sample were larger (by linear measurements of skeletal elements) and leaner but not heavier than their WT counterparts. bGH mice exhibit thoracic kyphosis and radiographically detectable incipient sacralization of the last lumbar vertebra, as well as robust and altered muscle attachment sites in both girdle and long bones, re-orientation of the acetabulum, and dysmorphology of the femur at both hip and knee joints. bGH mice generally lack clear, radiologically determinable differentiation of long bone growth plates and bear larger and differently proportioned sesamoids at the elbow and knee. They are also preferentially subject to the accumulation of inferred heterotopic calcification (IHC) and other radiodense soft tissue (RST) around joints and entheses. Female bGH mice exhibit variable and aberrant morphology in the humerus, innominate, and femur not seen in other groups, and show the most size and shape variation within the four genotype × sex groupings. Our survey illustrates widespread musculoskeletal impacts of excessive GH into adulthood in the model. These data provide an initial whole-skeleton framework for further efforts characterizing molecular-, cellular-, and tissue-scale alterations ultimately influencing the bGH mouse model skeletal phenotype. This pilot work serves to contextualize future preclinical studies as well as broader investigations into how GH and its metabolic cascade may affect vertebrate morphological and histological development, shape and size disparity, and dimorphism.
The accurate alignment of serial histological sections is essential for preserving anatomical continuity in 3D reconstruction. Although automated registration tools can efficiently correct global alignment errors, they often fail to resolve local misalignments caused by sectioning artifacts, tissue deformation, staining variability, or missing slices. Thus, we propose a practical two-step registration workflow that uses MultiStackReg (an ImageJ/Fiji plugin) for automatic alignment and AlignRef (a standalone application for interactive adjustment) for manual refinement. In the first step, MultiStackReg performs global registration by using rigid body transformations. In the second step, AlignRef corrects residual misalignments through semi-transparent overlay visualization, keyboard-based translation and rotation, and batch propagation of recorded transformations across selected slice ranges. We applied our workflow to 135 serial sections of a Carnegie Stage 15 embryo from the Virtual Human Embryo dataset that were stained with hematoxylin and eosin. MultiStackReg resolved most global inconsistencies, whereas AlignRef enabled the precise adjustment of subtle local deviations, particularly in curved structures such as neural tubes and limb buds. After automatic registration with MultiStackReg, the subsequent manual refinement step using AlignRef was completed in approximately 30 min and produced a suitable stack for 3D reconstruction. This two-step workflow balances automation with expert-guided correction and provides an accessible, reproducible, and anatomically precise method for the serial section alignment of morphological and developmental anatomy.
Functional reconstructions of extinct mammals often infer joint mobility from osteological geometry, yet the mobility envelope in life emerged from bones embedded within a layered soft-tissue system. More broadly, passive joint mobility provides an anatomical bridge between osteological form, soft-tissue constraints, and biological movement. Ex vivo range-of-motion (ROM) datasets that explicitly partition soft-tissue contributions across multiple joints and postural types remain scarce for mammals. Here, we quantify layer-by-layer effects on flexion-extension-dominant passive ex vivo limb joint ROM across six major limb joints (shoulder, elbow, wrist [carpal joint], hip, knee [stifle] and ankle [hock]) in four quadrupedal mammals spanning differing postures: rabbit (Oryctolagus cuniculus), chilla fox (Lycalopex griseus), pig (Sus scrofa domestica) and pudu deer (Pudu puda) (one specimen per taxon). ROM was measured sequentially under four anatomical conditions that progressively isolate tissue contributions: intact (S+M+CL+O), myofascial (M+CL+O), capsulo-ligamentous (CL+O) and osteology-only (O). Three trained evaluators passively moved each joint to maximal flexion and extension endpoints. Endpoints were recorded using calibrated photographs, and joint angles were quantified via vector-based analysis by a single experienced assessor. Between-evaluator dispersion was summarised using SD and coefficient of variation (CV%). Passive ROM did not increase monotonically with tissue removal. Instead, trajectories were strongly joint- and taxon-specific, with frequent intermediate-condition maxima and, in several distal joints, marked reductions in the osteology-only condition after capsulo-ligamentous removal. The wrist showed the most pronounced non-monotonicity, commonly peaking at CL+O and decreasing sharply in O, in some cases, to values below the intact state. By contrast, proximal joints more often exhibited large net expansions across the dissection sequence, although the condition producing maximal ROM varied among taxa and joints. Measurement dispersion also varied by joint and condition, tending to be higher in intact states and in joints that were more complex to manipulate consistently, especially distal joints, consistent with less sharply defined passive endpoints when multiple layers contribute distributed resistance. These results indicate that osteology-only ROM is not a reliable upper bound on biologically feasible flexion-extension-dominant passive motion, because periarticular tissues can both constrain excursion and stabilise alignment within multi-element joint complexes. Accordingly, layer-resolved ROM series provide an empirical anatomical line of evidence that can guide sensitivity analyses in musculoskeletal reconstructions and support cautious calibration of morphofunctional spaces for movement interpretation, including future palaeobiological applications.
The forelimbs of mammals are involved in many crucial behaviours for an animal's ecology, including locomotion. It has been shown that forelimb morphology and locomotor mechanisms are greatly impacted by functional constraints induced by the properties of the media across or in which the animal moves. These functional constraints are thought to drive an important part of bone shape, as bone directly remodels in response to both muscle and external forces. Due to its anatomical particularities, the forelimb of fully fossorial moles is of particular interest to better understand fossorial adaptations and has already been studied extensively. Recently, some studies focusing on two European mole species, Talpa europaea and the recently described Talpa aquitania, highlighted inter and intraspecific variations in the inner ear and forelimb bones morphology, which could be linked to locomotor performances. To better understand the specificity of their fossorial adaptations, we focus, in the present study, on these two species' musculature. Performing anatomical dissections, and in accordance with the literature, we provided a redescription of 39 extrinsic and intrinsic muscles inserting on the forelimb bones. We also made a quantitative comparison of muscles features, both at the inter and intraspecific level. Especially, we focus on muscle Physiological Cross-Sectional Area (PCSA), a measure considered as a good estimator of the force-producing capacity of a muscle. Finally, to investigate relationships between bone shape and muscle force, we quantified covariations between shape data and muscle PCSA. Our results highlighted inter and intraspecific (in T. aquitania) variations in muscle force-producing capacity, which is consistent with the studies of the inner ear and forelimb bones. Focusing on the relationship between muscle PCSA and shape of ulna and humerus, we showed that shoulder extensors, carpal/digital extensors, and carpal/digital flexors' PCSA were highly integrated with humerus shape and that elbow extensors' PCSA were highly integrated with the ulna shape. These results are rather consistent with insertion sites of these muscles on bones.
Living crocodylians (alligators, caimans, gavials and 'true' crocodiles) are the remnants of a broader clade, Crocodylomorpha, that displays high ecomorphological diversity, particularly in skull shape. However, there are several instances of ecomorphological convergence between distant lineages, especially pertaining to the independent acquisition of a long, slender, longirostrine snout. In some instances, it is difficult to determine whether extinct species belong to one longirostrine lineage or another, with this problem especially prevalent with regards to the gavialoid crocodylian clade. The recent application of computed tomography (CT) scanning to the skulls of vertebrate species has revealed a plethora of previously hidden information on their endocranial anatomy. This in turn has the potential to shed new light on a group's evolutionary interrelationships, with endocranial data potentially helping to distinguish between shared phylogenetic ancestry of an anatomical feature and ecomorphological convergence. Although CT scans of skulls of several crocodylian species have been evaluated, previous studies have focused on describing the endocranial anatomy of an individual species or assessing intraclade variation of a specific morphological feature. Here, we evaluate the endocranial anatomy of Crocodylia based on a combination of published and newly presented CT-scan data for 43 extant and extinct species, including representatives from the three major subclades, Alligatoroidea (13 species), Crocodyloidea (14 species), and Gavialoidea (16 species), alongside four non-crocodylian eusuchians. Alligatoroids possess a sigmoidal encephalic endocast, an endosseous labyrinth wherein the area of the anterior semicircular canal is more than three times that of its posterior counterpart, as well as snout sinuses lateral and ventral to the nasal cavity. Crocodyloids have dorsoventrally tall pharyngotympanic tubes, antorbital and postvestibular sinuses, and a cerebrum that has its greatest transverse width at its midpoint. Gavialoids have a relatively 'simple' endocranial morphology, with a straight encephalic endocast, and reduced or absent snout sinuses. Several extinct gavialoids are also characterised by depressions on the internal surface of the prefrontal bones, which have been previously hypothesised to be osteological correlates for salt glands; however, similar depressions in some extant species of Crocodylus appear to be associated with airspace instead. We identify several differences between the endocranial anatomy of phylogenetically distant longirostrine crocodylomorphs: by contrast with thalattosuchians, crocodylians lack a confluent dorsal dural venous sinus and orbital arteries, whereas they possess an intertympanic sinus and nasolacrimal ducts. Our study demonstrates the viability of endocranial data for identifying phylogenetically informative morphological features in crocodylomorphs more broadly.
