Julia C. Boughner

Deciphering the Palimpsest: Studying the Relationship Between Morphological Integration and Phenotypic Covariation

Organisms represent a complex arrangement of anatomical structures and individuated parts that must maintain functional associations through development. This integration of variation between functionally related body parts and the modular organization of development are fundamental determinants of their evolvability. This is because integration results in the expression of coordinated variation that can create preferred directions for evolutionary change, while modularity enables variation in a group of traits or regions to accumulate without deleterious effects on other aspects of the organism. Using our own work on both model systems (e.g., lab mice, avians) and natural populations of rodents and primates, we explore in this paper the relationship between patterns of phenotypic covariation and the developmental determinants of integration that those patterns are assumed to reflect. We show that integration cannot be reliably studied through phenotypic covariance patterns alone and argue that the relationship between phenotypic covariation and integration is obscured in two ways. One is the superimposition of multiple determinants of covariance in complex systems and the other is the dependence of covariation structure on variances in covariance-generating processes. As a consequence, we argue that the direct study of the developmental determinants of integration in model systems is necessary to fully interpret patterns of covariation in natural populations, to link covariation patterns to the processes that generate them, and to understand their significance for evolutionary explanation.

Epigenetic integration of the developing brain and face

The integration of the brain and face and to what extent this relationship constrains or enables evolutionary change in the craniofacial complex is an issue of long‐standing interest in vertebrate evolution. To investigate brain‐face integration, we studied the covariation between the forebrain and midface at gestational days 10–10.5 in four strains of laboratory mice. We found that phenotypic variation in the forebrain is highly correlated with that of the face during face formation such that variation in the size of the forebrain correlates with the degree of prognathism and orientation of the facial prominences. This suggests strongly that the integration of the brain and face is relevant to the etiology of midfacial malformations such as orofacial clefts. This axis of integration also has important implications for the evolutionary developmental biology of the mammalian craniofacial complex. Developmental Dynamics 240:2233–2244, 2011.

Short‐faced mice and developmental interactions between the brain and the face

The length of the face represents an important axis of variation in mammals and especially in primates. Mice with mutations that produce variation along this axis present an opportunity to study the developmental factors that may underlie evolutionary change in facial length. The Crf4 mutant, obtained from the C57BL/6J (wt/wt) background by chemical mutagenesis by the Baylor Mouse Mutagenesis Resource, is reported to have a short‐faced phenotype. As an initial step towards developing this model, we performed 3D geometric morphometric comparisons of Crf4 mice to C57BL/6J wild‐type mice focusing on three stages of face development and morphology – embryonic (GD 9.5–12), neonatal, and adult. Morphometric analysis of adult Crf4 mutants revealed that in addition to a shortened face, these mice exhibit a significant reduction in brain size and basicranial length. These same features also differ at the neonatal stage. During embryonic face formation, only dimensions related to brain growth were smaller, whereas the Crf4 face actually appeared advanced relative to the wild‐type at the same somite stage. These results show that aspects of the Crf4 phenotype are evident as early as embryonic face formation. Based on our anatomical findings we hypothesize that the reduction in facial growth in Crf4 mice is a secondary consequence of reduction in the growth of the brain. If correct, the Crf4 mutant will be a useful model for studying the role of epigenetic interactions between the brain and face in the evolutionary developmental biology of the mammalian craniofacial complex as well as human craniofacial dysmorphology.

Rediscovering Waddington in the post‐genomic age

Conrad Hal Waddington was a revolutionary interdisciplinary thinker well ahead of his time. Many of his ideas have been subsumed into our current understanding of developmental biology [1]. His pioneering theories, first published in the mid-20th century, continue to find validation 50 years later in the molecular era of developmental genetics [2]. Among his many contributions, Waddington [3] introduced the term epigenetics to describe the full variety of emergent developmental phenomena above the level of the genome, and elegantly expressed these ideas in the form of his widely recognised and explicitly evolutionary epigenetic landscape metaphor [3]. These emergent phenomena bridge the gap between genotype and phenotype, and comprise the epigenotype [5]. Because of this close relationship between development and evolution, it is important to grasp how such epigenetic mechanisms function. The diverse use of the term epigenetics in the subsequent literature has led to substantial disagreement about what exactly is being discussed, and at which level(s) of inquiry, despite several attempts to achieve consensus [6, 7]. In most contemporary biological contexts, epigenetics refers to chromatin modification [8]. Not only does Waddington’s more inclusive definition appear to have been largely abandoned, also the different uses of his term have coincided with the near disappearance of the original concept of epigenetics from models of evolutionary change [9]. We see this as a potentially significant problem for evolutionary biologists. In this essay our focus is on the theoretical concepts originally specified by Waddington’s epigenetics. We argue that, in this age of powerful postgenomic laboratory and bioinformatics tools, epigenetics sensu Waddington is more informative and instructive than it has been for decades. Waddington’s epigenetics has the potential to shed new light on the means by which both selectable variation and innovation, two key features of evolutionary theory, are

Biological spacetime and the temporal integration of functional modules: A case study of dento–gnathic developmental timing

For the individual, coordination between tooth and jaw development is important to proper food acquisition and ingestion later in life. Among and within species, variation in dental and gnathic size, shape, and, in the case of teeth, number, must be mutually accommodating and functionally compatible. For these reasons, the development and evolution of these two systems should be closely integrated. Furthermore, the timing of dental development correlates tightly with life history events such as weaning. This correlation hints at a central regulation of the developmental timing of multiple systems that have tandem effects on physiology and behaviour. Important work on embryonic oral development continues to tease apart the molecular mechanisms that pattern jaw identity and establish tooth morphology and position in the alveolar bone. Still very poorly understood is what underlies rates and periods of gene activity such that pre‐ and postnatal tooth and jaw development are coordinated. Recent literature suggests at least some level of autonomy between permanent tooth and mandibular ontogenetic timing. However, whether the timing of these various signaling pathways is directly regulated or is an outcome of the pathways themselves is untested. Here, we review what is currently known about the embryonic signaling pathways that regulate tooth and jaw development in the context of time rather than space, as has been traditional. We hypothesize that the timing of mandibular and dental development is not directly mediated by a common factor but is an indirect outcome of strong selection for coordinated molecular pathways and growth trajectories. The mandible and lower jaw dentition is a powerful model with which to investigate the mechanisms that facilitate morphological change—in this case, the development and evolution—of organs that are closely integrated in terms of function, space and time. Developmental Dynamics 237:1–17, 2008.

Anatomical networks reveal the musculoskeletal modularity of the human head

Mosaic evolution is a key mechanism that promotes robustness and evolvability in living beings. For the human head, to have a modular organization would imply that each phenotypic module could grow and function semi-independently. Delimiting the boundaries of head modules, and even assessing their existence, is essential to understand human evolution. Here we provide the first study of the human head using anatomical network analysis (AnNA), offering the most complete overview of the modularity of the head to date. Our analysis integrates the many biological dependences that tie hard and soft tissues together, arising as a consequence of development, growth, stresses and loads, and motion. We created an anatomical network model of the human head, where nodes represent anatomical units and links represent their physical articulations. The analysis of the human head network uncovers the presence of 10 musculoskeletal modules, deep-rooted in these biological dependences, of developmental and evolutionary significance. In sum, this study uncovers new anatomical and functional modules of the human head using a novel quantitative method that enables a more comprehensive understanding of the evolutionary anatomy of our lineage, including the evolution of facial expression and facial asymmetry.

Anatomical Network Analysis Shows Decoupling of Modular Lability and Complexity in the Evolution of the Primate Skull

Modularity and complexity go hand in hand in the evolution of the skull of primates. Because analyses of these two parameters often use different approaches, we do not know yet how modularity evolves within, or as a consequence of, an also-evolving complex organization. Here we use a novel network theory-based approach (Anatomical Network Analysis) to assess how the organization of skull bones constrains the co-evolution of modularity and complexity among primates. We used the pattern of bone contacts modeled as networks to identify connectivity modules and quantify morphological complexity. We analyzed whether modularity and complexity evolved coordinately in the skull of primates. Specifically, we tested Herbert Simon’s general theory of near-decomposability, which states that modularity promotes the evolution of complexity. We found that the skulls of extant primates divide into one conserved cranial module and up to three labile facial modules, whose composition varies among primates. Despite changes in modularity, statistical analyses reject a positive feedback between modularity and complexity. Our results suggest a decoupling of complexity and modularity that translates to varying levels of constraint on the morphological evolvability of the primate skull. This study has methodological and conceptual implications for grasping the constraints that underlie the developmental and functional integration of the skull of humans and other primates.

4D atlas of the mouse embryo for precise morphological staging

After more than a century of research, the mouse remains the gold-standard model system, for it recapitulates human development and disease and is quickly and highly tractable to genetic manipulations. Fundamental to the power and success of using a mouse model is the ability to stage embryonic mouse development accurately. Past staging systems were limited by the technologies of the day, such that only surface features, visible with a light microscope, could be recognized and used to define stages. With the advent of high-throughput 3D imaging tools that capture embryo morphology in microscopic detail, we now present the first 4D atlas staging system for mouse embryonic development using optical projection tomography and image registration methods. By tracking 3D trajectories of every anatomical point in the mouse embryo from E11.5 to E14.0, we established the first 4D atlas compiled from ex vivo 3D mouse embryo reference images. The resulting 4D atlas comprises 51 interpolated 3D images in this gestational range, resulting in a temporal resolution of 72 min. From this 4D atlas, any mouse embryo image can be subsequently compared and staged at the global, voxel and/or structural level. Assigning an embryonic stage to each point in anatomy allows for unprecedented quantitative analysis of developmental asynchrony among different anatomical structures in the same mouse embryo. This comprehensive developmental data set offers developmental biologists a new, powerful staging system that can identify and compare differences in developmental timing in wild-type embryos and shows promise for localizing deviations in mutant development. Summary: This comprehensive developmental dataset, which combines optical projection tomography and image registration methods, provides a new and powerful staging system for mouse embryos.

Geometric Morphometrics and the Study of Development

Even though developmental biology seeks to provide developmental explanations for morphological variation, the quantification of morphological variation has been regarded as peripheral to the mechanistic study of development. In this chapter, we argue that this is now changing because the rapidly advancing knowledge of development in post-genomic biology is creating a need for more refined measurements of the morphological changes produced by genetic perturbations or treatments. This need, in turn, is driving the development of new morphometric methods that allow the rapid and meaningful integration of molecular, cellular and morphometric data. We predict that such integration will offer new ways of looking at development, which will lead to significant advances in the study of dysmorphology and also the relationship between the generation of variation through development and its transformation through evolutionary history.

Technique: Imaging Earliest Tooth Development in 3D Using a Silver-Based Tissue Contrast Agent

Looking in microscopic detail at the 3D organization of initiating teeth within the embryonic jaw has long‐proved technologically challenging because of the radio‐translucency of these tiny un‐mineralized oral tissues. Yet 3D image data showing changes in the physical relationships among developing tooth and jaw tissues are vital to understand the coordinated morphogenesis of vertebrate teeth and jaws as an animal grows and as species evolve. Here, we present a new synchrotron‐based scanning solution to image odontogenesis in 3D and in histological detail using a silver‐based contrast agent. We stained fixed, intact wild‐type mice aged embryonic (E) day 10 to birth with 1% Protargol‐S at 37°C for 12–32 hr. Specimens were scanned at 4–10 µm pixel size at 28 keV, just above the silver K‐edge, using micro‐computed tomography (µCT) at the Canadian Light Source synchrotron. Synchrotron µCT scans of silver‐stained embryos showed even the earliest visible stages of tooth initiation, as well as many other tissue types and structures, in histological detail. Silver stain penetration was optimal for imaging structures in intact embryos E15 and younger. This silver stain method offers a powerful yet straightforward approach to visualize at high‐resolution and in 3D the earliest stages of odontogenesis in situ, and demonstrates the important of studying the tooth organ in all three planes of view. Anat Rec, 297:222–233, 2014.