Disconnecting bones within the jaw-otic network modules underlies mammalian middle ear evolution.

The origin of the mammalian middle ear ossicles from the craniomandibular articulation of their synapsid ancestors is a key event in the evolution of vertebrates. The richness of the fossil record and the multitude of developmental studies have provided a stepwise reconstruction of this evolutionary innovation, highlighting the homology between the quadrate, articular, pre-articular and angular bones of early synapsids with the incus, malleus, gonial and ectotympanic bones of derived mammals, respectively. There are several aspects involved in this functional exaptation: (i) an increase of the masticatory musculature; (ii) the separation of the quadrate bone from the cranium; and (iii) the disconnection of the post-dentary bones from the dentary. Here, we compared the jaw-otic complex for 43 synapsid taxa using anatomical network analysis, showing that the disconnection of mandibular bones was a key step in the mammalian middle ear evolution, changing the skull anatomical modularity concomitant to the acquisition of new functions. Furthermore, our analysis allows the identification of three types of anatomical modules evolving through five evolutionary stages during the anatomical transformation of the jawbones into middle ear bones, with the ossification and degradation of Meckel's cartilage in mammals as the key ontogenetic event leading the change of anatomical modularity.

Evo-devo mechanisms underlying the continuum between homology and homoplasy.

The different manifestations of equivalence and similarity in structure throughout evolution suggest a continuous and hierarchical process that starts out with the origin of a morphological novelty, unit, or homologue. Once a morphological unit has originated, its properties change subsequently into variants that differ, in magnitude, from the original properties found in the common ancestor. We will look into the nature of morphological units and their degrees of modification, which will provide the starting point for restructuring the concept of "homology," keeping the use of homology as the identity of an anatomical part, and homogeny, as the specific variation of that anatomical part during evolution. We will also show that parallelism has a distinct placement within an evolutionary continuum between homology and homoplasy, whereas the phenomenon of evolutionary convergence is left outside this continuum. We will then provide some epistemological and developmental criteria to justify these distinctions, showing that there is a direct relation between the nature of these concepts and the constraints that developmental mechanisms impose on evolution. Finally, we will propose a hierarchical model that places homology, homogeny, homoplasy, and parallelism, as distinct phenomena within an evolutionary continuum.

Beyond the functional matrix hypothesis: a network null model of human skull growth for the formation of bone articulations.

Craniofacial sutures and synchondroses form the boundaries among bones in the human skull, providing functional, developmental and evolutionary information. Bone articulations in the skull arise due to interactions between genetic regulatory mechanisms and epigenetic factors such as functional matrices (soft tissues and cranial cavities), which mediate bone growth. These matrices are largely acknowledged for their influence on shaping the bones of the skull; however, it is not fully understood to what extent functional matrices mediate the formation of bone articulations. Aiming to identify whether or not functional matrices are key developmental factors guiding the formation of bone articulations, we have built a network null model of the skull that simulates unconstrained bone growth. This null model predicts bone articulations that arise due to a process of bone growth that is uniform in rate, direction and timing. By comparing predicted articulations with the actual bone articulations of the human skull, we have identified which boundaries specifically need the presence of functional matrices for their formation. We show that functional matrices are necessary to connect facial bones, whereas an unconstrained bone growth is sufficient to connect non-facial bones. This finding challenges the role of the brain in the formation of boundaries between bones in the braincase without neglecting its effect on skull shape. Ultimately, our null model suggests where to look for modified developmental mechanisms promoting changes in bone growth patterns that could affect the development and evolution of the head skeleton.

Grist for Riedl's mill: a network model perspective on the integration and modularity of the human skull.

Riedl's concept of burden neatly links development and evolution by ascertaining that structures that show a high degree of developmental co-dependencies with other structures are more constrained in evolution. The human skull can be precisely modeled as an articulated complex system of bones connected by sutures, forming a network of structural co-dependencies. We present a quantitative analysis of the morphological integration, modularity, and hierarchical organization of this human skull network model. Our overall results show that the human skull is a small-world network, with two well-delimited connectivity modules: one facial organized around the ethmoid bone, and one cranial organized around the sphenoid bone. Geometric morphometrics further support this two-module division, stressing the direct relationship between the developmental information enclosed in connectivity patterns and skull shape. Whereas the facial module shows a hierarchy of clustered blocks of bones, the bones of the cranial modules show a regular pattern of connections. We analyze the significance of these arrangements by hypothesizing specific structural roles for the most important bones involved in the formation of both modules, in the context of Riedl's burden. We conclude that it is the morphological integration of each group of bones that defines the semi-hierarchical organization of the human skull, reflecting fundamental differences in the ontogenetic patterns of growth and the structural constraints that generate each module. Our study also demonstrates the adequacy of network analysis as an innovative tool to understand the morphological complexity of anatomical systems.

Notch activity acts as a sensor for extracellular calcium during vertebrate left-right determination.

During vertebrate embryo development, the breaking of the initial bilateral symmetry is translated into asymmetric gene expression around the node and/or in the lateral plate mesoderm. The earliest conserved feature of this asymmetric gene expression cascade is the left-sided expression of Nodal, which depends on the activity of the Notch signalling pathway. Here we present a mathematical model describing the dynamics of the Notch signalling pathway during chick embryo gastrulation, which reveals a complex and highly robust genetic network that locally activates Notch on the left side of Hensen's node. We identify the source of the asymmetric activation of Notch as a transient accumulation of extracellular calcium, which in turn depends on left-right differences in H+/K+-ATPase activity. Our results uncover a mechanism by which the Notch signalling pathway translates asymmetry in epigenetic factors into asymmetric gene expression around the node.

The multiple directions of evolutionary change.

The theory of Punctuated Equilibria challenges the neo-Darwinian tenet that evolution is a uniform process. Recently, an article by Hunt has found that directional change during the evolution of a lineage is relatively small (occurring only in 5% of 250 analyzed traits). Of those traits that were shown to follow a trend, size was more likely to show gradual changes, whereas shape changes were more random. Here, we provide a short view of the nature of evolutionary trends, showing that directional change within lineages and among clades provides valuable evolutionary information about the processes involved in their generation.

A network approach to brain form, cortical topology and human evolution.

Network analysis provides a quantitative tool to investigate the topological properties of a system. In anatomy, it can be employed to investigate the spatial organization of body parts according to their contiguity and patterns of physical contact. In this study, we build a model representing the spatial adjacency of the major regions of the human brain often considered in evolutionary neuroanatomy, to analyse its topological features. Results suggest that the frontal lobe is topologically independent of the posterior regions of the brain, which in turn are more integrated and influenced by reciprocal constraints. The precentral gyrus represents a hinge between the anterior and posterior blocks. The lateral temporal cortex is particularly influenced by the neighbouring regions, while the parietal cortex is minimally constrained by the overall brain organization. Beyond the reciprocal spatial influences among cortical areas, brain form is further constrained by spatial and mechanical influence of the braincase, including bone and connective elements. The anterior fossa and the parietal bones are the elements more sensitive to the brain-braincase spatial organization. These topological properties must be properly considered when making inferences on evolutionary variations and macroscopic differences of the human brain morphology.

Cell lineage transport: a mechanism for molecular gradient formation.

Gradient formation is a fundamental patterning mechanism during embryo development, commonly related to secreted proteins that move along an existing field of cells. Here, we mathematically address the feasibility of gradients of mRNAs and non-secreted proteins. We show that these gradients can arise in growing tissues whereby cells dilute and transport their molecular content as they divide and grow, a mechanism we termed 'cell lineage transport.' We provide an experimental test by unveiling a distal-to-proximal gradient of Hoxd13 in the vertebrate developing limb bud driven by cell lineage transport, corroborating our model. Our study indicates that gradients of non-secreted molecules exhibit a power-law profile and can arise for a wide range of biologically relevant parameter values. Dilution and nonlinear growth confer robustness to the spatial gradient under changes in the cell cycle period, but at the expense of sensitivity in the timing of gradient formation. We expect that gradient formation driven by cell lineage transport will provide future insights into understanding the coordination between growth and patterning during embryonic development.

Bone Fusion in Normal and Pathological Development is Constrained by the Network Architecture of the Human Skull.

Craniosynostosis, the premature fusion of cranial bones, affects the correct development of the skull producing morphological malformations in newborns. To assess the susceptibility of each craniofacial articulation to close prematurely, we used a network model of the skull to quantify the link reliability (an index based on stochastic block models and Bayesian inference) of each articulation. We show that, of the 93 human skull articulations at birth, the few articulations that are associated with non-syndromic craniosynostosis conditions have statistically significant lower reliability scores than the others. In a similar way, articulations that close during the normal postnatal development of the skull have also lower reliability scores than those articulations that persist through adult life. These results indicate a relationship between the architecture of the skull and the specific articulations that close during normal development as well as in pathological conditions. Our findings suggest that the topological arrangement of skull bones might act as a structural constraint, predisposing some articulations to closure, both in normal and pathological development, also affecting the long-term evolution of the skull.

High-resolution episcopic microscopy: a rapid technique for high detailed 3D analysis of gene activity in the context of tissue architecture and morphology.

We describe a new methodology for rapid 2D and 3D computer analysis and visualisation of gene expression and gene product pattern in the context of anatomy and tissue architecture. It is based on episcopic imaging of embryos and tissue samples, as they are physically sectioned, thereby producing inherently aligned digital image series and volume data sets, which immediately permit the generation of 3D computer representations. The technique uses resin as embedding medium, eosin for unspecific tissue staining, and colour reactions (beta-galactosidase/Xgal or BCIP/NBT) for specific labelling of gene activity and mRNA pattern. We tested the potential of the method for producing high-resolution volume data sets of adult human and porcine tissue samples and of specifically and unspecifically stained mouse, chick, quail, frog, and zebrafish embryos. The quality of the episcopic images resembles the quality of digital images of true histological sections with respect to resolution and contrast. Specifically labelled structures can be extracted using simple thresholding algorithms. Thus, the method is capable of quickly and precisely detecting molecular signals simultaneously with anatomical details and tissue architecture. It has no tissue restrictions and can be applied for analysis of human tissue samples as well as for analysis of all developmental stages of embryos of a wide variety of biomedically relevant species.

Evo-Devo insights from pathological networks: exploring craniosynostosis as a developmental mechanism for modularity and complexity in the human skull.

Bone fusion has occurred repeatedly during skull evolution in all tetrapod lineages, leading to a reduction in the number of bones and an increase in their morphological complexity. The ontogeny of the human skull includes also bone fusions as part of its normal developmental process. However, several disruptions might cause premature closure of cranial sutures (craniosynostosis), reducing the number of bones and producing new skull growth patterns that causes shape changes. Here, we compare skull network models of a normal newborn with different craniosynostosis conditions, the normal adult stage, and phylogenetically reconstructed forms of a primitive tetrapod, a synapsid, and a placental mammal. Changes in morphological complexity of newborn-to-synostosed skulls are two to three times less than in newborn-to-adult; and even smaller when we compare them to the increases among the reconstructed ancestors in the evolutionary transitions. In addition, normal, synostosed, and adult human skulls show the same connectivity modules: facial and cranial. Differences arise in the internal structure of these modules. In the adult skull the facial module has an internal hierarchical organization, whereas the cranial module has a regular network organization. However, all newborn forms, normal and synostosed, do not reach such kind of internal organization. We conclude that the subtle changes in skull complexity at the developmental scale can change the modular substructure of the newborn skull to more integrated modules in the adult skull, but is not enough to generate radical changes as it occurs at a macroevolutionary scale. The timing of closure of craniofacial sutures, together with the conserved patterns of morphological modularity, highlights a potential relation between the premature fusion of bones and the evolution of the shape of the skull in hominids.

Anatomical Network Comparison of Human Upper and Lower, Newborn and Adult, and Normal and Abnormal Limbs, with Notes on Development, Pathology and Limb Serial Homology vs. Homoplasy.

How do the various anatomical parts (modules) of the animal body evolve into very different integrated forms (integration) yet still function properly without decreasing the individual's survival? This long-standing question remains unanswered for multiple reasons, including lack of consensus about conceptual definitions and approaches, as well as a reasonable bias toward the study of hard tissues over soft tissues. A major difficulty concerns the non-trivial technical hurdles of addressing this problem, specifically the lack of quantitative tools to quantify and compare variation across multiple disparate anatomical parts and tissue types. In this paper we apply for the first time a powerful new quantitative tool, Anatomical Network Analysis (AnNA), to examine and compare in detail the musculoskeletal modularity and integration of normal and abnormal human upper and lower limbs. In contrast to other morphological methods, the strength of AnNA is that it allows efficient and direct empirical comparisons among body parts with even vastly different architectures (e.g. upper and lower limbs) and diverse or complex tissue composition (e.g. bones, cartilages and muscles), by quantifying the spatial organization of these parts-their topological patterns relative to each other-using tools borrowed from network theory. Our results reveal similarities between the skeletal networks of the normal newborn/adult upper limb vs. lower limb, with exception to the shoulder vs. pelvis. However, when muscles are included, the overall musculoskeletal network organization of the upper limb is strikingly different from that of the lower limb, particularly that of the more proximal structures of each limb. Importantly, the obtained data provide further evidence to be added to the vast amount of paleontological, gross anatomical, developmental, molecular and embryological data recently obtained that contradicts the long-standing dogma that the upper and lower limbs are serial homologues. In addition, the AnNA of the limbs of a trisomy 18 human fetus strongly supports Pere Alberch's ill-named "logic of monsters" hypothesis, and contradicts the commonly accepted idea that birth defects often lead to lower integration (i.e. more parcellation) of anatomical structures.

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.

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.

Network models in anatomical systems.

Network theory has been extensively used to model the underlying structure of biological processes. From genetics to ecology, network thinking is changing our understanding of complex systems, specifically how their internal structure determines their overall behavior. Concepts such as hubs, scale-free or small-world networks, common in the complexity literature, are now used more and more in sociology, neurosciences, as well as other anthropological fields. Even though the use of network models is nowadays so widely applied, few attempts have been carried out to enrich our understanding in the classical morphological sciences such as in comparative anatomy or physical anthropology. The purpose of this article is to introduce the usage of network tools in morphology; specifically by building anatomical networks, dealing with the most common analyses and problems, and interpreting their outcome.

Nodal cilia dynamics and the specification of the left/right axis in early vertebrate embryo development.

Nodal cilia dynamics is a key factor for left/right axis determination in mouse embryos through the induction of a leftward fluid flow. So far it has not been clearly established how such dynamics is able to induce the asymmetric leftward flow within the node. Herein we propose that an asymmetric two-phase nonplanar beating cilia dynamics that involves the bending of the ciliar axoneme is responsible for the leftward fluid flow. We support our proposal with a host of hydrodynamic arguments, in silico experiments and in vivo video microscopy data in wild-type embryos and inv mutants. Our phenomenological modeling approach underscores how the asymmetry and speed of the flow depends on different relevant parameters. In addition, we discuss how the combination of internal and external mechanisms might cause the two-phase beating cilia dynamics.

Theoretical morphology of developmental asymmetries.

Morphospaces are theoretical tools to explore the morphological organization of living and fossil organisms. They have been used mostly by the paleontological community in an effort to get the most out of one of the only pieces of evidence that fossil material usually provide: the morphology of hard parts. The expectation with the establishment of theoretical morphospaces is that, by abstracting and modeling the fundamental parts of form, the multiple processes that generate the phenotypes of embryonic and adult structures will be better understood. In this essay, we suggest that ontogenetic trajectories can be used as the generative functions that build morphospaces, and propose approaches to build theoretical models for the establishment of left-right asymmetries during vertebrate heart embryogenesis.