Nathan Young | University of California, San Francisco (original) (raw)

Papers by Nathan Young

Current Topics in Developmental Biology, 2015

Recent studies have shown how volumetric imaging and morphometrics can add significantly to our u... more Recent studies have shown how volumetric imaging and morphometrics can add significantly to our understanding of morphogenesis, the developmental basis for variation, and the etiology of structural birth defects. On the other hand, the complex questions and diverse imaging data in developmental biology present morphometrics with more complex challenges than applications in virtually any other field. Meeting these challenges is necessary in order to understand the mechanistic basis for variation in complex morphologies. This chapter reviews the methods and theory that enable the application of modern landmark-based morphometrics to developmental biology and craniofacial development, in particular. We discuss the theoretical foundations of morphometrics as applied to development and review the basic approaches to the quantification of morphology. Focusing on geometric morphometrics, we discuss the principal statistical methods for quantifying and comparing morphological variation and covariation structure within and among groups. Finally, we discuss the future directions for morphometrics in developmental biology that will be required for approaches that enable quantitative integration across the genotype-phenotype map.

Current Topics in Developmental Biology, 2015

Morphogenesis of the brain and face is intrinsically linked by a number of factors. These include... more Morphogenesis of the brain and face is intrinsically linked by a number of factors. These include: origins of tissues, adjacency allowing their physical interactions, and molecular cross talk controlling growth. Neural crest cells that form the facial primordia originate on the dorsal neural tube. In the caudal pharyngeal arches, a Homeobox code regulates arch identity. In anterior regions, positional information is acquired locally. Second, the brain is a structural platform that influences positioning of the facial primordia, and brain growth influences the timing of primordia fusion. Third, the brain helps induce a signaling center, the frontonasal ectodermal zone, in the ectoderm, which participates in patterned growth of the upper jaw. Similarly, signals from neural crest cells regulate expression of fibroblast growth factor 8 in the anterior neural ridge, which controls growth of the anterior forebrain. Disruptions to these interactions have significant consequences for normal development of the craniofacial complex, leading to structural malformations and birth defects.

Developmental dynamics : an official publication of the American Association of Anatomists, Jan 22, 2015

How developmental mechanisms generate the phenotypic variation that is the raw material for evolu... more 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. 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. Combined with previous findings, th...

Developmental Dynamics, 2015

Background: Morphological divergence among related species involves changes to developmental proc... more 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.

Development, 2015

The mechanisms of morphogenesis are not well understood, yet shaping structures during developmen... more 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.

We welcome Almécija's critique (1), but his claims of "bias" in our conclusions are unfounded. Al... more We welcome Almécija's critique (1), but his claims of "bias" in our conclusions are unfounded. All available evidence continues to support an African ape-like shoulder and pattern of forelimb use in our last common ancestor (LCA) with chimpanzees and bonobos (Pan). First, controlling for within-group size variation is uncontroversial (2), and neither biases our results nor obscures their biological meaning. Gorilla and Pan shape differences may still be size-related. Specifically, Gorilla's expansive supraspinous fossa is plausibly associated with increased body mass and functional demands to stabilize the joint during knuckle-walking (3). Second, although we note that a "Pan-like" LCA may be favored based on total evidence, a more "Gorilla-like" condition is also plausible; hence, our title states support for an "African apelike" LCA (4). That panin and hominin lineages may have subsequently evolved in different directions along the major axis of variation has no bearing on this conclusion; rather, it suggests joint orientation is highly evolvable. Indeed, although our analyses support homologies between African-ape and human shoulder shape, we also identify evidence for homoplasy in spine orientation among apes, including surprising data suggesting that orangutans retain primitive characteristics of the ancestral hominoid morphotype, consistent with parallelism. Third, the Dikika juvenile (not "baby") preserves the most complete evidence of Australopithecus afarensis shoulder anatomy known. Estimates of adult shape do not substantially differ from the juvenile because growth has only a weak effect on overall scapular shape. As we previously noted (5), Woranso-Mille may differ somewhat from Dikika in joint lateralization, but this does not change our interpretation. Moreover, Australopithecus sediba, which is better preserved, is similarly intermediate, consistent with our interpretation. The proximity of australopithecines to Nasalis is more parsimoniously reconstructed as hominin convergence as a result of lateralization of the shoulder joint rather than independent evolution of a similar blade shape in chimpanzees, gorillas, and hominins. Fourth, there are multiple ways to test alternative hypotheses. Ours was not based on comparing tree lengths but rather on the congruence of the fossils with the model predictions and the number of evolutionary events required to explain them. Fifth, use of equal branch lengths does not force a punctuated model of evolution, while branch lengths based on genetic distances do not alter either our results or conclusions.

Proceedings of the National Academy of Sciences USA, 2015

Reconstructing the behavioral shifts that drove hominin evolution requires knowledge of the timin... more 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 apelike 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.

Journal of Anatomy, 2006

The semi-dominant Br mutation affects presphenoid growth, producing the facial retrognathism and ... more The semi-dominant Br mutation affects presphenoid growth, producing the facial retrognathism and globular neurocranial vault that characterize heterozygotes. We analysed the impact of this mutation on skull shape, comparing heterozygotes to wildtype mice, to determine if the effects are skull-wide or confined to the sphenoid region targeted by the mutation. In addition, we examined patterns of variability of shape for the skull as a whole and for three regions (basicranium, face and neurocranium). We found that the Br mice differed significantly from wildtype mice in skull shape in all three regions as well as in the shape of the skull as a whole. However, the significant increases in variance and fluctuating asymmetry were found only in the basicranium of mutant mice. These results suggest that the mutation has a significant effect on the underlying developmental architecture of the skull, which produces an increase in phenotypic variability that is localized to the anatomical region in which the mean phenotype is most dramatically affected. These results suggest that the same developmental mechanisms that produce the change in phenotypic mean also produce the change in variance.

Evolutionary …, Jan 1, 2009

Evolutionary Biology, 2007

Variability contrasts with variation in that variability describes the potential for variation, n... more Variability contrasts with variation in that variability describes the potential for variation, not simply the expressed variation. The power of studying variability lies in creating a conceptual framework around which the relationship between the genotype and phenotype can be understood. Here, we attempt to demonstrate the importance of phenotypic variability, how it structures variation, and how fundamental developmental processes structure variability. Given the broad scope of this topic, we focus on three widely studied properties of variability: canalization, developmental stability and morphological integration. We have organized the paper to emphasize the importance of differentiating between the theory surrounding these components of phenotypic variability, their measurement and the biological factors surrounding their expression. First, we define these properties of variability, how they relate to each other and to variability as a whole. Second, we summarize the common methods of measurement for canalization, developmental stability and morphological integration and the reasoning behind these methods. Finally, we focus on jaw development as an example of how the basic processes of development affect variability and the resultant variation, with emphasis on how processes at all levels of the organismal hierarchy interact with one another and contribute to phenotypic variability.

Evolutionary Biology, 2009

Mammals exhibit a similar pattern of integration among homologous limb elements, the strength of ... more Mammals exhibit a similar pattern of integration among homologous limb elements, the strength of which is believed to vary in response to selection for functional coordination or similarity. Although integration is hypothesized to primarily reflect the effect of genes intrinsic to limbs, extrinsic genetic or epigenetic factors may also affect the strength of integration through their impact on the magnitude and direction of skeletal variance or covariance. Such factors as neuromuscular coordination or bone-muscle interactions may therefore play a role in both canalization and the structure or magnitude of limb integration. If this were the case, then increased levels of locomotor activity would be predicted to increase canalization and the magnitude of covariation between limbs. To investigate whether postnatal activity levels can have a significant effect on variance within or covariance among homologous limb elements, we compared four groups of male mice from a long-term selective breeding experiment: (1) mice from lines bred for increased voluntary activity on running wheels and allowed free access to a wheel for 8 weeks beginning at weaning (''active''), (2) selected mice that did not have wheel access (''sedentary''), (3) active mice from non-selected control lines, and (4) sedentary control mice. Mice from selected lines that had wheel access ran significantly more than control-line mice. However, when controlled for activity, linetype, and body mass, results indicate few significant differences in means, variance, or covariation structure, and no significant differences in integration between limbs, suggesting that postnatal activity levels do not significantly affect canalization or integration of limb lengths. A possible explanation for this result is that whereas baseline levels of postnatal activity may help to maintain patterns of variance and integration, increased levels of activity do not further increase these measures. Investigations into disrupted epigenetic processes (e.g., via models in which neuromuscular coordination is impaired) are required to further test hypotheses about how canalization or integration of limb variation is affected by epigenetic factors.

A central issue in biology concerns the presence, timing and nature of phylotypic periods of deve... more 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.

Journal of Experimental Zoology Part B: Molecular and Developmental Evolution

Mammals, birds, and reptiles exhibit a remarkable diversity of limb proportions. These evolved di... more 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.

Human Molecular Genetics

FGF signaling mutations are a frequent contributor to craniofacial malformations including midfac... more 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 controls not only 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 RCAS virus expressing both FgfR2C278F, a receptor mutation found in Crouzon syndrome, and 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 together these cell processes contribute to facial morphogenesis.

Craniofacial anomalies can arise from both genetic and environmental factors, including prenatal ... more Craniofacial anomalies can arise from both genetic and environmental factors, including prenatal hypoxia. Recent clinical evidence correlates hypoxia to craniofacial malformations. However, the mechanisms by which hypoxia mediates these defects are not yet understood. We examined the cellular mechanisms underlying malformations induced by hypoxia using a chicken (Gallus gallus) embryo model. Eggs were incubated in either hypoxic (7, 9, 11, 13, 15, 17 or 19% O2) or normoxic (21% O2) conditions. Embryos were photographed for morphological analysis at days 3-6. For analysis of skeletal development, 13-day embryos were cleared and stained with alcian blue and alizarin red for cartilage and bone, respectively. Quantitative analysis of facial shape variation was performed on images of embryos via geometric morphometrics. Early-stage embryos (day 2) were analyzed for apoptosis via whole-mount and section TUNEL staining and immunostaining for cleaved caspase-3, whereas later-stage embryos (days 4-6) were sectioned in paraffin for analysis of cell proliferation (BrdU), apoptosis (TUNEL) and metabolic stress (phospho-AMPK). Results demonstrate that survival is reduced in a dose-dependent manner. Hypoxic embryos displayed a spectrum of craniofacial anomalies, from mild asymmetry and eye defects to more severe frontonasal and cephalic anomalies. Skull bone development was delayed in hypoxic embryos, with some skeletal defects observed. Morphometric analysis showed facial shape variation relative to centroid size and age in hypoxic groups. Hypoxia disrupted cell proliferation and, in early-stage embryos, caused apoptosis of neural crest progenitor cells. Hypoxic embryos also displayed an increased metabolic stress response. These results indicate that hypoxia during early embryonic craniofacial development might induce cellular oxidative stress, leading to apoptosis of the neural crest progenitor cells that are crucial to normal craniofacial morphogenesis.

ABSTRACT Organisms exhibit an incredible diversity of form, a fact that makes the evolution of no... more ABSTRACT 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.

Background: The Frontonasal Ectodermal Zone (FEZ) is a signaling center in the face that expresse... more 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.

genesis

The effect of the brain on the morphology of the face has long been recognized in both evolutiona... more The effect of the brain on the morphology of the face has long been recognized in both evolutionary biology and clinical medicine. In this work, we describe factors that are active between the development of the brain and face and how these might impact craniofacial variation. First, there is the physical influence of the brain, which contributes to overall growth and morphol- ogy of the face through direct structural interactions. Second, there is the molecular influence of the brain, which signals to facial tissues to establish signaling centers that regulate patterned growth. Importantly, subtle alterations to these physical or molecular inter- actions may contribute to both normal and abnormal variation. These interactions are therefore critical to our understanding of how a diversity of facial morphologies can be generated both within species and across evolutionary time.

…, Jan 1, 2010

Variation is an intrinsic feature of biological systems, yet developmental biology does not frequ... more 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.

Current Topics in Developmental Biology, 2015

Recent studies have shown how volumetric imaging and morphometrics can add significantly to our u... more Recent studies have shown how volumetric imaging and morphometrics can add significantly to our understanding of morphogenesis, the developmental basis for variation, and the etiology of structural birth defects. On the other hand, the complex questions and diverse imaging data in developmental biology present morphometrics with more complex challenges than applications in virtually any other field. Meeting these challenges is necessary in order to understand the mechanistic basis for variation in complex morphologies. This chapter reviews the methods and theory that enable the application of modern landmark-based morphometrics to developmental biology and craniofacial development, in particular. We discuss the theoretical foundations of morphometrics as applied to development and review the basic approaches to the quantification of morphology. Focusing on geometric morphometrics, we discuss the principal statistical methods for quantifying and comparing morphological variation and covariation structure within and among groups. Finally, we discuss the future directions for morphometrics in developmental biology that will be required for approaches that enable quantitative integration across the genotype-phenotype map.

Current Topics in Developmental Biology, 2015

Morphogenesis of the brain and face is intrinsically linked by a number of factors. These include... more Morphogenesis of the brain and face is intrinsically linked by a number of factors. These include: origins of tissues, adjacency allowing their physical interactions, and molecular cross talk controlling growth. Neural crest cells that form the facial primordia originate on the dorsal neural tube. In the caudal pharyngeal arches, a Homeobox code regulates arch identity. In anterior regions, positional information is acquired locally. Second, the brain is a structural platform that influences positioning of the facial primordia, and brain growth influences the timing of primordia fusion. Third, the brain helps induce a signaling center, the frontonasal ectodermal zone, in the ectoderm, which participates in patterned growth of the upper jaw. Similarly, signals from neural crest cells regulate expression of fibroblast growth factor 8 in the anterior neural ridge, which controls growth of the anterior forebrain. Disruptions to these interactions have significant consequences for normal development of the craniofacial complex, leading to structural malformations and birth defects.

Developmental dynamics : an official publication of the American Association of Anatomists, Jan 22, 2015

How developmental mechanisms generate the phenotypic variation that is the raw material for evolu... more 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. 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. Combined with previous findings, th...

Developmental Dynamics, 2015

Background: Morphological divergence among related species involves changes to developmental proc... more 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.

Development, 2015

The mechanisms of morphogenesis are not well understood, yet shaping structures during developmen... more 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.

We welcome Almécija's critique (1), but his claims of "bias" in our conclusions are unfounded. Al... more We welcome Almécija's critique (1), but his claims of "bias" in our conclusions are unfounded. All available evidence continues to support an African ape-like shoulder and pattern of forelimb use in our last common ancestor (LCA) with chimpanzees and bonobos (Pan). First, controlling for within-group size variation is uncontroversial (2), and neither biases our results nor obscures their biological meaning. Gorilla and Pan shape differences may still be size-related. Specifically, Gorilla's expansive supraspinous fossa is plausibly associated with increased body mass and functional demands to stabilize the joint during knuckle-walking (3). Second, although we note that a "Pan-like" LCA may be favored based on total evidence, a more "Gorilla-like" condition is also plausible; hence, our title states support for an "African apelike" LCA (4). That panin and hominin lineages may have subsequently evolved in different directions along the major axis of variation has no bearing on this conclusion; rather, it suggests joint orientation is highly evolvable. Indeed, although our analyses support homologies between African-ape and human shoulder shape, we also identify evidence for homoplasy in spine orientation among apes, including surprising data suggesting that orangutans retain primitive characteristics of the ancestral hominoid morphotype, consistent with parallelism. Third, the Dikika juvenile (not "baby") preserves the most complete evidence of Australopithecus afarensis shoulder anatomy known. Estimates of adult shape do not substantially differ from the juvenile because growth has only a weak effect on overall scapular shape. As we previously noted (5), Woranso-Mille may differ somewhat from Dikika in joint lateralization, but this does not change our interpretation. Moreover, Australopithecus sediba, which is better preserved, is similarly intermediate, consistent with our interpretation. The proximity of australopithecines to Nasalis is more parsimoniously reconstructed as hominin convergence as a result of lateralization of the shoulder joint rather than independent evolution of a similar blade shape in chimpanzees, gorillas, and hominins. Fourth, there are multiple ways to test alternative hypotheses. Ours was not based on comparing tree lengths but rather on the congruence of the fossils with the model predictions and the number of evolutionary events required to explain them. Fifth, use of equal branch lengths does not force a punctuated model of evolution, while branch lengths based on genetic distances do not alter either our results or conclusions.

Proceedings of the National Academy of Sciences USA, 2015

Reconstructing the behavioral shifts that drove hominin evolution requires knowledge of the timin... more 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 apelike 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.

Journal of Anatomy, 2006

The semi-dominant Br mutation affects presphenoid growth, producing the facial retrognathism and ... more The semi-dominant Br mutation affects presphenoid growth, producing the facial retrognathism and globular neurocranial vault that characterize heterozygotes. We analysed the impact of this mutation on skull shape, comparing heterozygotes to wildtype mice, to determine if the effects are skull-wide or confined to the sphenoid region targeted by the mutation. In addition, we examined patterns of variability of shape for the skull as a whole and for three regions (basicranium, face and neurocranium). We found that the Br mice differed significantly from wildtype mice in skull shape in all three regions as well as in the shape of the skull as a whole. However, the significant increases in variance and fluctuating asymmetry were found only in the basicranium of mutant mice. These results suggest that the mutation has a significant effect on the underlying developmental architecture of the skull, which produces an increase in phenotypic variability that is localized to the anatomical region in which the mean phenotype is most dramatically affected. These results suggest that the same developmental mechanisms that produce the change in phenotypic mean also produce the change in variance.

Evolutionary …, Jan 1, 2009

Evolutionary Biology, 2007

Variability contrasts with variation in that variability describes the potential for variation, n... more Variability contrasts with variation in that variability describes the potential for variation, not simply the expressed variation. The power of studying variability lies in creating a conceptual framework around which the relationship between the genotype and phenotype can be understood. Here, we attempt to demonstrate the importance of phenotypic variability, how it structures variation, and how fundamental developmental processes structure variability. Given the broad scope of this topic, we focus on three widely studied properties of variability: canalization, developmental stability and morphological integration. We have organized the paper to emphasize the importance of differentiating between the theory surrounding these components of phenotypic variability, their measurement and the biological factors surrounding their expression. First, we define these properties of variability, how they relate to each other and to variability as a whole. Second, we summarize the common methods of measurement for canalization, developmental stability and morphological integration and the reasoning behind these methods. Finally, we focus on jaw development as an example of how the basic processes of development affect variability and the resultant variation, with emphasis on how processes at all levels of the organismal hierarchy interact with one another and contribute to phenotypic variability.

Evolutionary Biology, 2009

Mammals exhibit a similar pattern of integration among homologous limb elements, the strength of ... more Mammals exhibit a similar pattern of integration among homologous limb elements, the strength of which is believed to vary in response to selection for functional coordination or similarity. Although integration is hypothesized to primarily reflect the effect of genes intrinsic to limbs, extrinsic genetic or epigenetic factors may also affect the strength of integration through their impact on the magnitude and direction of skeletal variance or covariance. Such factors as neuromuscular coordination or bone-muscle interactions may therefore play a role in both canalization and the structure or magnitude of limb integration. If this were the case, then increased levels of locomotor activity would be predicted to increase canalization and the magnitude of covariation between limbs. To investigate whether postnatal activity levels can have a significant effect on variance within or covariance among homologous limb elements, we compared four groups of male mice from a long-term selective breeding experiment: (1) mice from lines bred for increased voluntary activity on running wheels and allowed free access to a wheel for 8 weeks beginning at weaning (''active''), (2) selected mice that did not have wheel access (''sedentary''), (3) active mice from non-selected control lines, and (4) sedentary control mice. Mice from selected lines that had wheel access ran significantly more than control-line mice. However, when controlled for activity, linetype, and body mass, results indicate few significant differences in means, variance, or covariation structure, and no significant differences in integration between limbs, suggesting that postnatal activity levels do not significantly affect canalization or integration of limb lengths. A possible explanation for this result is that whereas baseline levels of postnatal activity may help to maintain patterns of variance and integration, increased levels of activity do not further increase these measures. Investigations into disrupted epigenetic processes (e.g., via models in which neuromuscular coordination is impaired) are required to further test hypotheses about how canalization or integration of limb variation is affected by epigenetic factors.

A central issue in biology concerns the presence, timing and nature of phylotypic periods of deve... more 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.

Journal of Experimental Zoology Part B: Molecular and Developmental Evolution

Mammals, birds, and reptiles exhibit a remarkable diversity of limb proportions. These evolved di... more 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.

Human Molecular Genetics

FGF signaling mutations are a frequent contributor to craniofacial malformations including midfac... more 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 controls not only 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 RCAS virus expressing both FgfR2C278F, a receptor mutation found in Crouzon syndrome, and 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 together these cell processes contribute to facial morphogenesis.

Craniofacial anomalies can arise from both genetic and environmental factors, including prenatal ... more Craniofacial anomalies can arise from both genetic and environmental factors, including prenatal hypoxia. Recent clinical evidence correlates hypoxia to craniofacial malformations. However, the mechanisms by which hypoxia mediates these defects are not yet understood. We examined the cellular mechanisms underlying malformations induced by hypoxia using a chicken (Gallus gallus) embryo model. Eggs were incubated in either hypoxic (7, 9, 11, 13, 15, 17 or 19% O2) or normoxic (21% O2) conditions. Embryos were photographed for morphological analysis at days 3-6. For analysis of skeletal development, 13-day embryos were cleared and stained with alcian blue and alizarin red for cartilage and bone, respectively. Quantitative analysis of facial shape variation was performed on images of embryos via geometric morphometrics. Early-stage embryos (day 2) were analyzed for apoptosis via whole-mount and section TUNEL staining and immunostaining for cleaved caspase-3, whereas later-stage embryos (days 4-6) were sectioned in paraffin for analysis of cell proliferation (BrdU), apoptosis (TUNEL) and metabolic stress (phospho-AMPK). Results demonstrate that survival is reduced in a dose-dependent manner. Hypoxic embryos displayed a spectrum of craniofacial anomalies, from mild asymmetry and eye defects to more severe frontonasal and cephalic anomalies. Skull bone development was delayed in hypoxic embryos, with some skeletal defects observed. Morphometric analysis showed facial shape variation relative to centroid size and age in hypoxic groups. Hypoxia disrupted cell proliferation and, in early-stage embryos, caused apoptosis of neural crest progenitor cells. Hypoxic embryos also displayed an increased metabolic stress response. These results indicate that hypoxia during early embryonic craniofacial development might induce cellular oxidative stress, leading to apoptosis of the neural crest progenitor cells that are crucial to normal craniofacial morphogenesis.

ABSTRACT Organisms exhibit an incredible diversity of form, a fact that makes the evolution of no... more ABSTRACT 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.

Background: The Frontonasal Ectodermal Zone (FEZ) is a signaling center in the face that expresse... more 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.

genesis

The effect of the brain on the morphology of the face has long been recognized in both evolutiona... more The effect of the brain on the morphology of the face has long been recognized in both evolutionary biology and clinical medicine. In this work, we describe factors that are active between the development of the brain and face and how these might impact craniofacial variation. First, there is the physical influence of the brain, which contributes to overall growth and morphol- ogy of the face through direct structural interactions. Second, there is the molecular influence of the brain, which signals to facial tissues to establish signaling centers that regulate patterned growth. Importantly, subtle alterations to these physical or molecular inter- actions may contribute to both normal and abnormal variation. These interactions are therefore critical to our understanding of how a diversity of facial morphologies can be generated both within species and across evolutionary time.

…, Jan 1, 2010

Variation is an intrinsic feature of biological systems, yet developmental biology does not frequ... more 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.