A comparative approach for characterizing the relationship among morphology, range-of-motion and locomotor behaviour in the primate shoulder.

Shoulder shape directly impacts forelimb function by contributing to glenohumeral (GH) range-of-motion (ROM). However, identifying traits that contribute most to ROM and visualizing how they do so remains challenging, ultimately limiting our ability to reconstruct function and behaviour in fossil species. To address these limitations, we developed an in silico proximity-driven model to simulate and visualize three-dimensional (3D) GH rotations in living primate species with diverse locomotor profiles, identify those shapes that are most predictive of ROM using geometric morphometrics, and apply subsequent insights to interpret function and behaviour in the fossil hominin Australopithecus sediba. We found that ROM metrics that incorporated 3D rotations best discriminated locomotor groups, and the magnitude of ROM (mobility) was decoupled from the anatomical location of ROM (e.g. high abduction versus low abduction). Morphological traits that enhanced mobility were decoupled from those that enabled overhead positions, and all non-human apes possessed the latter but not necessarily the former. Model simulation in A. sediba predicted high mobility and a ROM centred at lower abduction levels than in living apes but higher than in modern humans. Together these results identify novel form-to-function relationships in the shoulder and enhance visualization tools to reconstruct past function and behaviour.

The developmental-genetics of canalization.

Canalization, or robustness to genetic or environmental perturbations, is fundamental to complex organisms. While there is strong evidence for canalization as an evolved property that varies among genotypes, the developmental and genetic mechanisms that produce this phenomenon are very poorly understood. For evolutionary biology, understanding how canalization arises is important because, by modulating the phenotypic variation that arises in response to genetic differences, canalization is a determinant of evolvability. For genetics of disease in humans and for economically important traits in agriculture, this subject is important because canalization is a potentially significant cause of missing heritability that confounds genomic prediction of phenotypes. We review the major lines of thought on the developmental-genetic basis for canalization. These fall into two groups. One proposes specific evolved molecular mechanisms while the other deals with robustness or canalization as a more general feature of development. These explanations for canalization are not mutually exclusive and they overlap in several ways. General explanations for canalization are more likely to involve emergent features of development than specific molecular mechanisms. Disentangling these explanations is also complicated by differences in perspectives between genetics and developmental biology. Understanding canalization at a mechanistic level will require conceptual and methodological approaches that integrate quantitative genetics and developmental biology.

Nonlinear gene expression-phenotype relationships contribute to variation and clefting in the A/WySn mouse.

BACKGROUND: Cleft lip and palate is one of the most common human birth defects, but the underlying etiology is poorly understood. The A/WySn mouse is a spontaneously occurring model of multigenic clefting in which 20% to 30% of individuals develop an orofacial cleft. Recent work has shown altered methylation at a specific retrotransposon insertion downstream of the Wnt9b locus in clefting animals, which results in decreased Wnt9b expression. RESULTS: Using a newly developed protocol that allows us to measure morphology, gene expression, and DNA methylation in the same embryo, we relate gene expression in an individual embryo directly to its three-dimensional morphology for the first time. We find that methylation at the retrotransposon relates to Wnt9b expression and morphology. IAP methylation relates to shape of the nasal process in a manner consistent with clefting. Embryos with low IAP methylation exhibit increased among-individual variance in facial shape. CONCLUSIONS: Methylation and gene expression relate nonlinearly to nasal process morphology. Individuals at one end of a continuum of phenotypic states display a clinical phenotype and increased phenotypic variation. Variable penetrance and expressivity in this model is likely determined both by among-individual variation in methylation and changes in phenotypic robustness along the underlying liability distribution for orofacial clefting.

Anatomic shoulder parameters and their relationship to the presence of degenerative rotator cuff tears and glenohumeral osteoarthritis: a systematic review and meta-analysis.

BACKGROUND: Scapular anatomy, as measured by the acromial index (AI), critical shoulder angle (CSA), lateral acromial angle (LAA), and glenoid inclination (GI), has emerged as a possible contributor to the development of degenerative shoulder conditions such as rotator cuff tears and glenohumeral osteoarthritis. The purpose of this study was to investigate the published literature on influences of scapular morphology on the development of degenerative shoulder conditions. METHODS: A systematic review of the Embase and PubMed databases was performed to identify published studies on the potential influence of scapular bony morphology on the development of degenerative rotator cuff tears and glenohumeral osteoarthritis. The studies were reviewed by 2 authors. The findings were summarized for various anatomic parameters. A meta-analysis was completed for parameters reported in more than 5 related publications. RESULTS: A total of 660 unique titles and 55 potentially relevant abstracts were reviewed with 30 published articles identified for inclusion. The AI, CSA, LAA, and GI were the most commonly reported bony measurements. Increased CSA and AI correlated with rotator cuff tears, whereas lower CSA appeared to be related to the presence of glenohumeral osteoarthritis. Decreased LAA correlated with degenerative rotator cuff tears. Five articles reported on the GI with mixed results on shoulder pathology. DISCUSSION: Degenerative rotator cuff tears appear to be significantly associated with the AI, CSA, and LAA. There does not appear to be a significant relationship between the included shoulder parameters and the development of osteoarthritis.

A dynamic Shh expression pattern, regulated by SHH and BMP signaling, coordinates fusion of primordia in the amniote face.

The mechanisms of morphogenesis are not well understood, yet shaping structures during development is essential for establishing correct organismal form and function. Here, we examine mechanisms that help to shape the developing face during the crucial period of facial primordia fusion. This period of development is a time when the faces of amniote embryos exhibit the greatest degree of similarity, and it probably results from the necessity for fusion to occur to establish the primary palate. Our results show that hierarchical induction mechanisms, consisting of iterative signaling by Sonic hedgehog (SHH) followed by Bone morphogenetic proteins (BMPs), regulate a dynamic expression pattern of Shh in the ectoderm covering the frontonasal (FNP) and maxillary (MxP) processes. Furthermore, this Shh expression domain contributes to the morphogenetic processes that drive the directional growth of the globular process of the FNP toward the lateral nasal process and MxP, in part by regulating cell proliferation in the facial mesenchyme. The nature of the induction mechanism that we discovered suggests that the process of fusion of the facial primordia is intrinsically buffered against producing maladaptive morphologies, such as clefts of the primary palate, because there appears to be little opportunity for variation to occur during expansion of the Shh expression domain in the ectoderm of the facial primordia. Ultimately, these results might explain why this period of development constitutes a phylotypic stage of facial development among amniotes.

Fossil hominin shoulders support an African ape-like last common ancestor of humans and chimpanzees.

Reconstructing the behavioral shifts that drove hominin evolution requires knowledge of the timing, magnitude, and direction of anatomical changes over the past ∼6-7 million years. These reconstructions depend on assumptions regarding the morphotype of the Homo-Pan last common ancestor (LCA). However, there is little consensus for the LCA, with proposed models ranging from African ape to orangutan or generalized Miocene ape-like. The ancestral state of the shoulder is of particular interest because it is functionally associated with important behavioral shifts in hominins, such as reduced arboreality, high-speed throwing, and tool use. However, previous morphometric analyses of both living and fossil taxa have yielded contradictory results. Here, we generated a 3D morphospace of ape and human scapular shape to plot evolutionary trajectories, predict ancestral morphologies, and directly test alternative evolutionary hypotheses using the hominin fossil evidence. We show that the most parsimonious model for the evolution of hominin shoulder shape starts with an African ape-like ancestral state. We propose that the shoulder evolved gradually along a single morphocline, achieving modern human-like configuration and function within the genus Homo. These data are consistent with a slow, progressive loss of arboreality and increased tool use throughout human evolution.

Embryonic bauplans and the developmental origins of facial diversity and constraint.

A central issue in biology concerns the presence, timing and nature of phylotypic periods of development, but whether, when and why species exhibit conserved morphologies remains unresolved. Here, we construct a developmental morphospace to show that amniote faces share a period of reduced shape variance and convergent growth trajectories from prominence formation through fusion, after which phenotypic diversity sharply increases. We predict in silico the phenotypic outcomes of unoccupied morphospaces and experimentally validate in vivo that observed convergence is not due to developmental limits on variation but instead from selection against novel trajectories that result in maladaptive facial clefts. These results illustrate how epigenetic factors such as organismal geometry and shape impact facial morphogenesis and alter the locus of adaptive selection to variation in later developmental events.

Signals from the brain induce variation in avian facial shape.

BACKGROUND: How developmental mechanisms generate the phenotypic variation that is the raw material for evolution is largely unknown. Here, we explore whether variation in a conserved signaling axis between the brain and face contributes to differences in morphogenesis of the avian upper jaw. In amniotes, including both mice and avians, signals from the brain establish a signaling center in the ectoderm (the Frontonasal ectodermal zone or "FEZ") that directs outgrowth of the facial primordia. RESULTS: Here we show that the spatial organization of this signaling center differs among avians, and these correspond to Sonic hedgehog (Shh) expression in the basal forebrain and embryonic facial shape. In ducks this basal forebrain domain is present almost the entire width, while in chickens it is restricted to the midline. When the duck forebrain is unilaterally transplanted into stage matched chicken embryos the face on the treated side resembles that of the donor. CONCLUSIONS: Combined with previous findings, these results demonstrate that variation in a highly conserved developmental pathway has the potential to contribute to evolutionary differences in avian upper jaw morphology. Developmental Dynamics 244:1133-1143, 2015. © 2015 Wiley Periodicals, Inc.

Divergence of craniofacial developmental trajectories among avian embryos.

BACKGROUND: Morphological divergence among related species involves changes to developmental processes. When such variation arises in development has garnered considerable theoretical interest relating to the broader issue of how development may constrain evolutionary change. The hourglass model holds that while early developmental events may be highly evolvable, there is a phylotypic stage when key developmental events are conserved. Thus, evolutionary divergence among related species should tend to arise after such a stage of reduced evolvability and, consequently, reduced variation among species. We test this prediction by comparing developmental trajectories among three avian species of varying relatedness (chick, quail, and duck) to locate their putative point of divergence. Three-dimensional geometric morphometrics and trajectory analyses were used to measure the significance of the facial shape variation observed among these species. RESULTS: Duck embryos, being more distantly related, differed from the more closely-related chick and quail embryos in the enlargement of their frontonasal prominences. Phenotypic trajectory analyses demonstrated divergence of the three species, most notably, duck. CONCLUSIONS: The results demonstrate that the two more closely related species share similar facial morphologies for a longer time during development, while ducks diverge. This suggests a surprising lability of craniofacial development during early face formation. Developmental Dynamics 244:1158-1167, 2015. © 2015 Wiley Periodicals, Inc.

Facial surface morphology predicts variation in internal skeletal shape.

INTRODUCTION: The regular collection of 3-dimensional (3D) imaging data is critical to the development and implementation of accurate predictive models of facial skeletal growth. However, repeated exposure to x-ray-based modalities such as cone-beam computed tomography has unknown risks that outweigh many potential benefits, especially in pediatric patients. One solution is to make inferences about the facial skeleton from external 3D surface morphology captured using safe nonionizing imaging modalities alone. However, the degree to which external 3D facial shape is an accurate proxy of skeletal morphology has not been previously quantified. As a first step in validating this approach, we tested the hypothesis that population-level variation in the 3D shape of the face and skeleton significantly covaries. METHODS: We retrospectively analyzed 3D surface and skeletal morphology from a previously collected cross-sectional cone-beam computed tomography database of nonsurgical orthodontics patients and used geometric morphometrics and multivariate statistics to test the hypothesis that shape variation in external face and internal skeleton covaries. RESULTS: External facial morphology is highly predictive of variation in internal skeletal shape ([Rv] = 0.56, P <0.0001; partial least squares [PLS] 1-13 = 98.7% covariance, P <0.001) and asymmetry (Rv = 0.34, P <0.0001; PLS 1-5 = 90.2% covariance, P <0.001), whereas age-related (r(2) = 0.84, P <0.001) and size-related (r(2) = 0.67, P <0.001) shape variation was also highly correlated. CONCLUSIONS: Surface morphology is a reliable source of proxy data for the characterization of skeletal shape variation and thus is particularly valuable in research designs where reducing potential long-term risks associated with radiologic imaging methods is warranted. We propose that longitudinal surface morphology from early childhood through late adolescence can be a valuable source of data that will facilitate the development of personalized craniodental and treatment plans and reduce exposure levels to as low as reasonably achievable.

Quantification of shape and cell polarity reveals a novel mechanism underlying malformations resulting from related FGF mutations during facial morphogenesis.

Fibroblast growth factor (FGF) signaling mutations are a frequent contributor to craniofacial malformations including midfacial anomalies and craniosynostosis. FGF signaling has been shown to control cellular mechanisms that contribute to facial morphogenesis and growth such as proliferation, survival, migration and differentiation. We hypothesized that FGF signaling not only controls the magnitude of growth during facial morphogenesis but also regulates the direction of growth via cell polarity. To test this idea, we infected migrating neural crest cells of chicken embryos with  replication-competent avian sarcoma virus expressing either FgfR2(C278F), a receptor mutation found in Crouzon syndrome or the ligand Fgf8. Treated embryos exhibited craniofacial malformations resembling facial dysmorphologies in craniosynostosis syndrome. Consistent with our hypothesis, ectopic activation of FGF signaling resulted in decreased cell proliferation, increased expression of the Sprouty class of FGF signaling inhibitors, and repressed phosphorylation of ERK/MAPK. Furthermore, quantification of cell polarity in facial mesenchymal cells showed that while orientation of the Golgi body matches the direction of facial prominence outgrowth in normal cells, in FGF-treated embryos this direction is randomized, consistent with aberrant growth that we observed. Together, these data demonstrate that FGF signaling regulates cell proliferation and cell polarity and that these cell processes contribute to facial morphogenesis.

Dosage-dependent effects of FGFR2W290R mutation on craniofacial shape and cellular dynamics of the basicranial synchondroses.

Craniosynostosis is a common yet complex birth defect, characterized by premature fusion of the cranial sutures that can be syndromic or nonsyndromic. With over 180 syndromic associations, reaching genetic diagnoses and understanding variations in underlying cellular mechanisms remains a challenge. Variants of FGFR2 are highly associated with craniosynostosis and warrant further investigation. Using the missense mutation FGFR2W290R , an effective mouse model of Crouzon syndrome, craniofacial features were analyzed using geometric morphometrics across developmental time (E10.5-adulthood, n = 665 total). Given the interrelationship between the cranial vault and basicranium in craniosynostosis patients, the basicranium and synchondroses were analyzed in perinates. Embryonic time points showed minimal significant shape differences. However, hetero- and homozygous mutant perinates and adults showed significant differences in shape and size of the cranial vault, face, and basicranium, which were associated with cranial doming and shortening of the basicranium and skull. Although there were also significant shape and size differences associated with the basicranial bones and clear reductions in basicranial ossification in cleared whole-mount samples, there were no significant alterations in chondrocyte cell shape, size, or orientation along the spheno-occipital synchondrosis. Finally, shape differences in the cranial vault and basicranium were interrelated at perinatal stages. These results point toward the possibility that facial shape phenotypes in craniosynostosis may result in part from pleiotropic effects of the causative mutations rather than only from the secondary consequences of the sutural defects, indicating a novel direction of research that may shed light on the etiology of the broad changes in craniofacial morphology observed in craniosynostosis syndromes.

Quantitative analyses link modulation of sonic hedgehog signaling to continuous variation in facial growth and shape.

Variation is an intrinsic feature of biological systems, yet developmental biology does not frequently address population-level phenomena. Sonic hedgehog (SHH) signaling activity in the vertebrate forebrain and face is thought to contribute to continuous variation in the morphology of the upper jaw, but despite its potential explanatory power, this idea has never been quantitatively assessed. Here, we test this hypothesis with an experimental design that is explicitly focused on the generation and measurement of variation in multivariate shape, tissue growth, cellular behavior and gene expression. We show that the majority of upper jaw shape variation can be explained by progressive changes in the spatial organization and mitotic activity of midfacial growth zones controlled by SHH signaling. In addition, nonlinearity between our treatment doses and phenotypic outcomes suggests that threshold effects in SHH signaling may play a role in variability in midfacial malformations such as holoprosencephaly (HPE). Together, these results provide novel insight into the generation of facial morphology, and demonstrate the value of quantifying variation for our understanding of development and disease.

Macroevolutionary diversity of amniote limb proportions predicted by developmental interactions.

Mammals, birds, and reptiles exhibit a remarkable diversity of limb proportions. These evolved differences are thought to reflect selection for biomechanical, postural, and locomotor requirements primarily acting on independent variation in later fetal and postnatal segmental growth. However, earlier conserved developmental events also have the potential to impact the evolvability of limb proportions by limiting or biasing initial variation among segments. Notably, proximo-distal patterning of the amniote limb through activation-inhibition dynamics predicts that initial proportions of segments should exhibit both tradeoffs between stylopod and autopod and a diagnostic reduction in variance of the zeugopod. Here it is demonstrated that this developmental "design rule" predicts patterns of macroevolutionary diversity despite the effects of variation in segmental growth over ontogeny, lineage-specific differences in phylogenetic history, or functional adaptation. These results provide critical comparative evidence of a conserved Turing-like mechanism in proximo-distal limb segmentation, and suggest that development has played a previously unrecognized role in the evolvability of limb proportions in a wide range of amniote taxa.

Development and the evolvability of human limbs.

The long legs and short arms of humans are distinctive for a primate, the result of selection acting in opposite directions on each limb at different points in our evolutionary history. This mosaic pattern challenges our understanding of the relationship of development and evolvability because limbs are serially homologous and genetic correlations should act as a significant constraint on their independent evolution. Here we test a developmental model of limb covariation in anthropoid primates and demonstrate that both humans and apes exhibit significantly reduced integration between limbs when compared to quadrupedal monkeys. This result indicates that fossil hominins likely escaped constraints on independent limb variation via reductions to genetic pleiotropy in an ape-like last common ancestor (LCA). This critical change in integration among hominoids, which is reflected in macroevolutionary differences in the disparity between limb lengths, facilitated selection for modern human limb proportions and demonstrates how development helps shape evolutionary change.

Signaling by SHH rescues facial defects following blockade in the brain.

BACKGROUND: The Frontonasal Ectodermal Zone (FEZ) is a signaling center in the face that expresses Sonic hedgehog (Shh) and regulates patterned growth of the upper jaw. Blocking SHH in the forebrain blocks Shh expression in the FEZ and creates malformations resembling holoprosencephaly (HPE), while inhibition of BMP signaling in the mesenchyme blocks FEZ formation and causes similar dysmorphology. Thus, the brain could regulate FEZ formation by SHH or BMP signaling, and if so, activating one of these pathways in the face might alleviate the effects of repression of SHH in the brain. RESULTS: We blocked SHH signaling in the brain while adding SHH or BMP between the neural and facial ectoderm of the frontonasal process. When applied early, SHH restored Shh expression in the FEZ and significantly improved shape outcomes, which contrasts with our previous experiments that showed later SHH treatments have no effect. BMP-soaked beads introduced early and late caused apoptosis that exacerbated malformations. Finally, removal of Smoothened from neural crest cells did not inhibit Shh expression in the FEZ. CONCLUSIONS: Collectively, this work suggests that a direct, time-sensitive SHH signal from the brain is required for the later induction of Shh in the FEZ. We propose a testable model of FEZ activation and discuss signaling mediators that may regulate these interactions.

The generation of variation and the developmental basis for evolutionary novelty.

Organisms exhibit an incredible diversity of form, a fact that makes the evolution of novelty seemingly self-evident. However, despite the "obvious" case for novelty, defining this concept in evolutionary terms is highly problematic, so much so that some have suggested discarding it altogether. Approaches to this problem tend to take either an adaptation- or development-based perspective, but we argue here that an exclusive focus on either of these misses the original intent of the novelty concept and undermines its practical utility. We propose instead that for a feature to be novel, it must have evolved both by a transition between adaptive peaks on the fitness landscape and that this transition must have overcome a previous developmental constraint. This definition focuses novelty on the explanation of apparently difficult or low-probability evolutionary transitions and highlights how the integration of developmental and functional considerations are necessary to evolutionary explanation. It further reinforces that novelty is a central concern not just of evolutionary developmental biology (i.e., "evo-devo") but of evolutionary biology more generally. We explore this definition of novelty in light of four examples that range from the obvious to subtle.

MusMorph, a database of standardized mouse morphology data for morphometric meta-analyses.

Complex morphological traits are the product of many genes with transient or lasting developmental effects that interact in anatomical context. Mouse models are a key resource for disentangling such effects, because they offer myriad tools for manipulating the genome in a controlled environment. Unfortunately, phenotypic data are often obtained using laboratory-specific protocols, resulting in self-contained datasets that are difficult to relate to one another for larger scale analyses. To enable meta-analyses of morphological variation, particularly in the craniofacial complex and brain, we created MusMorph, a database of standardized mouse morphology data spanning numerous genotypes and developmental stages, including E10.5, E11.5, E14.5, E15.5, E18.5, and adulthood. To standardize data collection, we implemented an atlas-based phenotyping pipeline that combines techniques from image registration, deep learning, and morphometrics. Alongside stage-specific atlases, we provide aligned micro-computed tomography images, dense anatomical landmarks, and segmentations (if available) for each specimen (N = 10,056). Our workflow is open-source to encourage transparency and reproducible data collection. The MusMorph data and scripts are available on FaceBase ( www.facebase.org , https://doi.org/10.25550/3-HXMC ) and GitHub ( https://github.com/jaydevine/MusMorph ).