Genetic randomization reveals functional relationships among morphologic and tissue-quality traits that contribute to bone strength and fragility

We examined femora from adult AXB/BXA recombinant inbred (RI) mouse strains to identify skeletal traits that are functionally related and to determine how functional interactions among these traits contribute to genetic variability in whole-bone stiffness, strength, and toughness. Randomization of A/J and C57BL/6J genomic regions resulted in each adult male and female RI strain building mechanically functional femora by assembling unique sets of morphologic and tissue-quality traits. A correlation analysis was conducted using the mean trait values for each RI strain. A third of the 66 correlations examined were significant, indicating that many bone traits covaried or were functionally related. Path analysis revealed important functional interactions among bone slenderness, cortical thickness, and tissue mineral density. The path coefficients describing these functional relations were similar for both sexes. The causal relationship among these three traits suggested that cellular processes during growth simultaneously regulate bone slenderness, cortical thickness, and tissue mineral density so that the combination of traits is sufficiently stiff and strong to satisfy daily loading demands. A disadvantage of these functional interactions was that increases in tissue mineral density also deleteriously affected tissue ductility. Consequently, slender bones with high mineral density may be stiff and strong but they are also brittle. Thus, genetically randomized mouse strains revealed a basic biological paradigm that allows for flexibility in building bones that are functional for daily activities but that creates preferred sets of traits under extreme loading conditions. Genetic or environmental perturbations that alter these functional interactions during growth would be expected to lead to loss of function and suboptimal adult bone quality.     

Three-dimensional geometric morphometric analysis of the humerus: Comparative postweaning ontogeny between fossorial and semiaquatic water voles (Arvicola)

Different types of locomotion in phylogenetically close rodent species can lead to significantly different growth patterns of certain skeletal structures. In the present study, we compared the allometric and phenotypic trajectories of the humerus in semiaquatic (Arvicola sapidus) and fossorial (Arvicola scherman) water vole taxa, using three-dimensional geometric morphometrics, to investigate the relationships between functional and ontogenetic differences. Results revealed shared humerus traits between A. sapidus and A. scherman, specifically an expansion of the epicondylar and deltopectoral crests along postnatal ontogeny. In both species, the humerus of young specimens is more robust than in adults, possibly as a compensatory response for lower bone stiffness. However, significant interspecific differences were detected in all components of allometric and phenotypic trajectories. Noticeably divergent allometric trajectories were observed, probably as a result of different functional pressures exerted on this bone. Important differences in the form of the adult humerus between taxa were also found, particularly in features located in muscle insertion zones. Furthermore, the allometric regression revealed certain shape variation not associated with size in A. scherman, suggesting mechanical stress produced by the persistent digging activity during adulthood. A. scherman is a chisel-tooth digger that shares several traits in the humerus morphology with scratch-digger rodent species. Nevertheless, these shared characteristics are less pronounced in fossorial water voles, which is congruent with the different implications of the forelimb in the digging activity in these two types of diggers.     

Developmental and genetic origins of murine long bone length variation

If we wish to understand whether development influences the rate or direction of morphological evolution, we must first understand the developmental bases of morphological variation within species. However, quantitative variation in adult morphology is the product of molecular and cellular processes unfolding from embryonic development through juvenile growth to maturity. The Atchley-Hall model provides a useful framework for dissecting complex morphologies into their component parts as a way of determining which developmental processes contribute to variation in adult form. We have examined differences in postnatal allometry and the patterns of genetic correlation between age-specific traits for ten recombinant inbred strains of mice generated from an intercross of LG/J and SM/J. Long bone length is closely tied to body size, but variation in adult morphology is more closely tied to differences in growth rate between 3 and 5 weeks of age. These analyses show that variation generated during early development is overridden by variation generated later in life. To more precisely determine the cellular processes generating this variation we then examined the cellular dynamics of long bone growth plates at the time of maximum elongation rate differences in the parent strains. Our analyses revealed that variation in long bone length is the result of faster elongation rates of the LG/J stain. The developmental bases for these differences in growth rate involve the rate of cell division and chondrocyte hypertrophy in the growth plate.     

Bone Growth Dynamics of the Facial Skeleton and Mandible in <Emphasis Type="Italic">Gorilla gorilla</Emphasis> and <Emphasis Type="Italic">Pan troglodytes</Emphasis>

Adult craniofacial morphology results from complex processes that involve growth by bone modelling and interactions of skeletal components to keep a functional and structural balance. Previous analyses of growth dynamics in humans revealed critical changes during late ontogeny explaining particular morphological features in our species. Data on bone modelling patterns from other primate species could help us to determine whether postnatal changes in the growth dynamics of the craniofacial complex are human specific or are shared with other primates. However, characterizations of bone modelling patterns through ontogeny in non-human hominids are scarce and restricted to isolated data on facial and mandibular regions. In the present study, we analyse the bone modelling patterns in an ontogenetic series of Pan and Gorilla to infer the growth dynamics of their craniofacial complex during postnatal development. Our results show that both Pan troglodytes and Gorilla gorilla are characterized by species-specific bone modelling patterns indicative of a mainly forward growth direction during postnatal development. Both species show minor but consistent ontogenetic changes in the distribution of bone modelling fields in specific regions of the face and mandible, in contrast to other regions which show more constant bone modelling patterns. In addition, we carry out a preliminary integrative study merging histological and geometric morphometric data. Both approaches yield highly complementary data, each analysis providing details on specific growth dynamics unavailable to the other. Moreover, geometric morphometric data show that ontogenetic variation in the modelling pattern of the mandibular ramus may be linked to sexual dimorphism.     

Quantitative trait loci affecting phenotypic plasticity and the allometric relationship of ovariole number and thorax length in Drosophila melanogaster

Environmental factors during juvenile growth such as temperature and nutrition have major effects on adult morphology and life-history traits. In Drosophila melanogaster, ovary size, measured as ovariole number, and body size, measured as thorax length, are developmentally plastic traits with respect to larval nutrition. Herein we investigated the genetic basis for plasticity of ovariole number and body size, as well the genetic basis for their allometric relationship using recombinant inbred lines (RILs) derived from a natural population in Winters, California. We reared 196 RILs in four yeast concentrations and measured ovariole number and body size. The genetic correlation between ovariole number and thorax length was positive, but the strength of this correlation decreased with increasing yeast concentration. Genetic variation and genotype-by-environment (G x E) interactions were observed for both traits. We identified quantitative trait loci (QTL), epistatic, QTL-by-environment, and epistatic-by-environment interactions for both traits and their scaling relationships. The results are discussed in the context of multivariate trait evolution.     

Fetal and early post-natal mineralization of the tympanic bulla in fin whales may reveal a Hitherto undiscovered evolutionary trait

The evolution of the cetacean skeleton followed a path that differentiated this group from other terrestrial mammals about 50 million years ago [1], and debate is still going on about the relationships between Cetacea and Artiodactyla [2], [3], [4]. Some skeletal traits of the basilosaurids (the more advanced forms of Archaeocetes), such as the expansion of the peribullary air sinuses, dental modification and vertebral size uniformity [5] are maintained and further emphasized also in contemporary odontocetes and mysticetes. Using Dual-Energy X-Ray Absorptiometry here we report that the deposition of bone mineral in fetal and newborn specimens of the fin whale Balaenoptera physalus is remarkably higher in the bulla tympanica than in the adjacent basal skull or in the rest of the skeleton. Ossification of the tympanic bulla in fetal Artiodactyla (bovine, hippopotamus) is minimal, becomes sensible after birth and then progresses during growth, contrarily to the precocious mineralization that we observed in fin whales. Given the importance of the ear bones for the precise identification of phylogenetic relationship in therian evolution [6], this feature may indicate a specific evolutionary trait of fin whales and possibly other cetacean species or families. Early mineralization of the tympanic bulla allows immediate sound conduction in the aquatic medium and consequently holds potential importance for mother-calf relationship and postnatal survival.     

Microcomputed tomography imaging of skeletal development and growth

Skeletogenesis is an exquisitely orchestrated and dynamic process, culminating in the formation of highly variable and complex mineralized structures that are optimized for their function. While cellular and molecular biology studies have provided tremendous recent progress toward understanding how patterns of bone formation are regulated, high resolution imaging techniques such as microcomputed tomography (micro-CT) can provide complementary quantitative information about the progressive changes in three-dimensional (3-D) skeletal morphology and density that occur during early skeletal development and postnatal growth. Furthermore, recently developed in vivo micro-CT systems promise to be a powerful and efficient tool for noninvasively monitoring normal skeletogenesis, as well as for evaluating the effects of genetic or environmental manipulation. This review focuses on the use of micro-CT imaging and analysis to better understand normal and abnormal skeletal development and growth.     

Common genetic and environmental factors among craniofacial traits in Belgian nuclear families: Comparing skeletal and soft-tissue related phenotypes

The major objective of this study was to determine the possible effects of common genetic and environmental factors among 18 craniofacial anthropometric traits, with special attention to the differences between skeletal and soft-tissue related phenotypes. The studied sample consisted of 122 nuclear families living in Brussels and included 251 males and 258 females aged from 13 to 72 years. Univariate and bivariate quantitative genetic analyses were performed using a variance components procedure implemented in SOLAR software.All phenotypes were significantly influenced by additive genetic factors with heritability estimates ranging from 0.46 (nose height) to 0.72 (external biocular breadth). Sex, age and their interactions explained 7-46% of the total phenotypic variance of the traits. Bivariate analysis revealed that several traits share a common genetic and/or environmental basis while other traits show genetic and environmental independence from one another. More and greater genetic and environmental correlations were observed among skeletal phenotypes, than among soft-tissue traits and between both categories. Apart from the tissue composition, other characteristics of the craniofacial morphology such as the orientation (e.g. heights, breadths) have shown to be important factors in determining pleiotropy and common environmental effects between some pairs of traits. In conclusion, the results confirm that overall head configuration is largely determined by additive genetic effects, and that common genetic and environmental factors affecting craniofacial size and shape are stronger for the skeletal traits than for the soft-tissue traits.     

Evolution of mechanoregulation of bone growth will lead to non-optimal bone phenotypes

Mechanical forces acting on the bones during growth affect their final shape and strength. Mechanoregulation of bone growth may be recognized in embryogenesis, and also in the adaptation of the adult skeleton to changes in mechanical loading. Mechanoregulatory responses for tissues have arisen during evolution, but does evolution give rise to responses that produce optimal skeletal phenotypes? In this paper, we investigate the emergence of an optimal mechanoregulation response in a population. By combining equations describing long bone growth with a genetic algorithm to describe evolutionary change, we created a computational model to simulate the evolution of mechanoregulation in bone growth. A population of individuals is created where each individual is assigned a diploid gene set which controls the growth and remodelling of the bone. At maturity, each bone is assessed and its 'fitness' calculated; fitness is quantified as bone strength relative to bone mass. The simulation continues for many generations, and includes mutations and a varying environment. The genes present in the population are tracked and the evolution of parameters governing mechanoregulation is calculated. The results indicate that a population may converge to one bone growth algorithm but, more usually, a range of mechanoregulation algorithms for different individuals will persist after many generations. Even if the population converges to one mechanoregulation law, convergence to the 'optimum' bone was never found. Although many researchers propose that natural selection has pushed skeletal structure towards an optimum, our computational model suggests that this is unlikely to be the case.     

A thyrotoxic skeletal phenotype of advanced bone formation in mice with resistance to thyroid hormone

Thyroid hormone (T3) regulates bone turnover and mineralization in adults and is essential for skeletal development during childhood. Hyperthyroidism is an established risk factor for osteoporosis. Nevertheless, T3 actions in bone remain poorly understood. Patients with resistance to thyroid hormone, due to mutations of the T3-receptor beta (TRbeta) gene, display variable phenotypic abnormalities, particularly in the skeleton. To investigate the actions of T3 during bone development, we characterized the skeleton in TRbetaPV mutant mice. TRbetaPV mice harbor a targeted resistance to thyroid hormone mutation in TRbeta and recapitulate the human condition. A severe phenotype, which includes shortened body length, was evident in homozygous TRbetaPV/PV animals. Accelerated growth in utero was associated with advanced endochondral and intramembranous ossification. Advanced bone formation resulted in postnatal growth retardation, premature quiescence of the growth plates, and shortened bone length, together with increased bone mineralization and craniosynostosis. In situ hybridization demonstrated increased expression of fibroblast growth factor receptor-1, a T3-regulated gene in bone, in TRbetaPV/PV perichondrium, growth plate chondrocytes, and osteoblasts. Thus, the skeleton in TRbetaPV/PV mice is thyrotoxic and displays phenotypic features typical of juvenile hyperthyroidism.     

Bone calcification rate as a factor of craniofacial transformations in salmonid fish: Insights from an experiment with hormonal treatment of calcium metabolism

Adaptation to different environments can be achieved by physiological shifts throughout development. Hormonal regulators shape the physiological and morphological traits of the evolving animals making them fit for the particular ecological surroundings. We hypothesized that the artificially induced hypersynthesis of calcitonin and parathyroid hormone mutually influencing calcium metabolism could affect bone formation during early ontogeny in fish imitating the heterochrony in craniofacial ossification in natural adaptive morphs. Conducting an experiment, we found that the long-standing treatment of salmonid juveniles with high doses of both hormones irreversibly shifts the corresponding hormone status for a period well beyond the time scale for total degradation of the injected hormone. The hormones program the ossification of the jaw suspension bones and neurocranial elements in a specific manner affecting the jaws position and pharingo-branchial area stretching. These morphological shifts resemble the adaptive variants found in sympatric pelagic and demersal morphs of salmonids. We conclude that solitary deviations in the regulators of calcium metabolism could determine functional morphological traits via transformations in skeletal development.     

Estrogen, exercise, and the skeleton

Patterns of variation in bone size and shape provide crucial data for reconstructing hominin paleobiology, including ecogeographic adaptation, life history, and functional morphology. Measures of bone strength, including robusticity (diaphyseal thickness relative to length) and cross-sectional geometric properties such as moments of area, are particularly useful for inferring behavior because bone tissue adapts to its mechanical environment. Particularly during skeletal growth, exercise-induced strains can stimulate periosteal modeling so that, to some extent, bone thickness reflects individual behavior. Thus, patterns of skeletal robusticity have been used to identify gender-based activity differences, temporal shifts in mobility, and changing subsistence strategies. Although there is no doubt that mechanical loading leaves its mark on the skeleton, less is known about whether individuals differ in their skeletal responses to exercise. For example, the potential effects of hormones or growth factors on bone-strain interactions are largely unexplored. If the hormonal background can increase or decrease the effects of exercise on skeletal robusticity, then the same mechanical loads might cause different degrees of bone response in different individuals. Here I focus on the role of the hormone estrogen in modulating exercise-induced changes in human bone thickness.     

The ontogeny of Holocene and Late Pleistocene human postcranial strength

While a wide variety of studies have focused on population variation in adult cross‐sectional properties, relatively little is known about population variation in postcranial robusticity in immature individuals. Furthermore, the age at which the population differences readily detected in adults manifest during growth is also unknown. This research addresses these gaps in our current understanding through the analysis of immature humeral and femoral long bone strength. Cross‐sectional geometry was used to compare the developmental trajectories of diaphyseal strength in Late Pleistocene Neandertal and modern human subadults to a sample of immature humans from seven geographically diverse Holocene populations. Population differences in size‐standardized cross‐sectional properties appear to be systemic and develop very early in ontogeny in the Holocene sample. In many cases, these differences are present before one year of age. In general, the Late Pleistocene fossil samples fit within the range of recent human variation in long bone strength. Population differences detected here are likely related to a combination of factors including activity patterns, genetic propensities, and nutritional status. These results highlight the complex mosaic of processes that result in adult postcranial robusticity, and suggest that further exploration of the developmental interplay between intrinsic and extrinsic influences on skeletal robusticity will likely enhance our understanding of adult postcranial morphology. Am J Phys Anthropol 2010. © 2009 Wiley‐Liss, Inc.     

Patterns of growth of the mandibular corpus in spotted hyenas (Crocuta crocuta) and cougars (Puma concolor)

Differences in jaw morphology among adult carnivorans are well established, but the ontogenetic mechanisms by which these differences arise are largely unexplored. Mandibular ontogeny in Crocuta crocuta and Puma concolor is analysed biomechanically using principles of beam theory. In each species, the development of cross-sectional properties of the mandibular corpus associated with rigidity under loading follows a biphasic pattern of growth. In early postnatal growth, deposition of cortical bone appears to be constrained by the overall weaker tissue with which juvenile skeletons are constructed and by the need to volumelrically accommodate the developing teeth within their bony crypts. Thus, this stage of growth is characterized by a net periosteal deposition of bone and a swelling of the medullary cavity. In late postnatal growth, the constraints on endosteal deposition of bone are relieved as the permanent teeth erupt; thus, cortical thicknesses increase sharply by periosteal expansion as well as medullary contraction. Finally, it is noted that basic differences in jaw construction between Crocuta and Puma appear to develop prenatally as they are largely in place at birth. Hence, postnatal development enhances, but does not soley contribute to, the biomechanical differences in the jaws of these species.     

Myogenin regulates a distinct genetic program in adult muscle stem cells

In contrast to the detailed understanding we have for the regulation of skeletal muscle gene expression in embryos, similar insights into postnatal muscle growth and regeneration are largely inferential or do not directly address gene regulatory mechanisms. Muscle stem cells (satellite cells) are chiefly responsible for providing new muscle during postnatal and adult life. The purpose of this study was to determine the role that the myogenic basic helix-loop-helix regulatory factor myogenin has in postnatal muscle growth and adult muscle stem cell gene expression. We found that myogenin is absolutely required for skeletal muscle development and survival until birth, but it is dispensable for postnatal life. However, Myog deletion after birth led to reduced body size implying a role for myogenin in regulating body homeostasis. Despite a lack of skeletal muscle defects in Myog-deleted mice during postnatal life and the efficient differentiation of cultured Myog-deleted adult muscle stem cells, the loss of myogenin profoundly altered the pattern of gene expression in cultured muscle stem cells and adult skeletal muscle. Remarkably, these changes in gene expression were distinct from those found in Myog-null embryonic skeletal muscle, indicating that myogenin has separate functions during postnatal life.     

Pleiotropic patterns of quantitative trait loci for 70 murine skeletal traits

Quantitative trait locus (QTL) studies of a skeletal trait or a few related skeletal components are becoming commonplace, but as yet there has been no investigation of pleiotropic patterns throughout the skeleton. We present a comprehensive survey of pleiotropic patterns affecting mouse skeletal morphology in an intercross of LG/J and SM/J inbred strains (N = 1040), using QTL analysis on 70 skeletal traits. We identify 798 single-trait QTL, coalescing to 105 loci that affect on average 7-8 traits each. The number of traits affected per locus ranges from only 1 trait to 30 traits. Individual traits average 11 QTL each, ranging from 4 to 20. Skeletal traits are affected by many, small-effect loci. Significant additive genotypic values average 0.23 standard deviation (SD) units. Fifty percent of loci show codominance with heterozygotes having intermediate phenotypic values. When dominance does occur, the LG/J allele tends to be dominant to the SM/J allele (30% vs. 8%). Over- and underdominance are relatively rare (12%). Approximately one-fifth of QTL are sex specific, including many for pelvic traits. Evaluating the pleiotropic relationships of skeletal traits is important in understanding the role of genetic variation in the growth and development of the skeleton.     

Genetic Analysis of Size-Scaling Patterns in the Mouse Mandible

The relationship between multidimensional form of the adult mouse mandible and body size is examined from an ontogenetic perspective. The origin and ontogeny of phenotypic correlations are described in terms of genetic and environmental covariance patterns between adult skeletal morphology and growth in body weight. Different ontogenetic patterns are observed in the genetic correlations, and these can be related to the developmental as well as the functional aspects of mandibular form. The quantitative genetic aspects of craniomandibular growth and morphogenesis are explored, together with an examination of the impact of ontogenetic changes in the genetic variance-covariance structure on morphogenetic integration and evolution by selection.     

MULTIVARIATE ANALYSIS OF SELECTION IN EXPERIMENTAL POPULATIONS DERIVED FROM HYBRIDIZATION OF TWO ECOTYPES OF THE ANNUAL PLANT DIODIA TERES W. (RUBIACEAE)

Morphologically variable F₂ genotypes derived from hybridization of coastal and inland ecotypes of the annual plant Diodia teres were used to identify selection on morphological traits in the natural habitat of each ecotype. These ecotypes occur in very different habitats, and have evolved pronounced morphological differentiation. Selection analysis can suggest whether present patterns of selection can explain morphological differences between ecotypes. F₂ genotypes were characterized morphologically, clonally replicated, and transplanted into the habitat of each ecotype. Selection was measured on six morphological traits. Directional and stabilizing selection occurred on many traits; direction and strength of selection varied sharply at different stages of growth, as revealed by a path-analysis approach that divided selection into a set of independent components. Directional selection favored traits of the native population at the coastal habitat, but less so at the inland habitat. Selection was of sufficient strength to create the observed morphological differences between ecotypes in 25–100 generations, given constant selection and sufficient genetic variation. In effects on fitness, most traits were neither independent nor consistently interactive with other traits. Rather, many traits entered into strong but evanescent interactions affecting particular components of fitness. Observed interactions did not support the hypothesis that the morphology of each ecotype was functionally integrated to a high degree.     

Developmental regulation of skull morphology. I. Ontogenetic dynamics of variance

In the absence of processes regulating morphogenesis and growth, phenotypic variance of a population experiencing no selective mortality should increase throughout ontogeny. To determine whether it does, we measure variance of skull shape using geometric morphometrics and examine its ontogenetic dynamics in the precocial cotton rat (Sigmodon fulviventer) and the altricial house mouse (Mus musculus domesticus). In both species, variance of shape halves between the two youngest samples measured (between 1 and 10 days postnatal and 10 and 15 days postnatal, respectively) and thereafter is nearly constant. The reduction in variance did not appear to result from a general regulation of skull size or developmental timing, although skull size may also be regulated and developmental timing is an important component of the variation in skull shape of young house mice. The ontogenetic dynamics of variance suggest two possible scenarios. First, variation generated during fetal or early postnatal growth is not immediately compensated and therefore accumulates, whereas later in growth, variation is continually generated and rapidly compensated. Second, variation generated during fetal and early postnatal growth is rapidly compensated, after which no new variance is produced. Based on a general model for bone growth, we hypothesize that variance is generated when bone grows under the direction of disorganized muscular movements and decreases with increasing neuromuscular control. Additionally, increasing coherence of signals transmitted by the growing brain and sensory organs, which exert tensile forces on bone, may also canalize skull shape.     

Ecological interactions drive evolutionary loss of traits

Loss of traits can dramatically alter the fate of species. Evidence is rapidly accumulating that the prevalence of trait loss is grossly underestimated. New findings demonstrate that traits can be lost without affecting the external phenotype, provided the lost function is compensated for by species interactions. This is important because trait loss can tighten the ecological relationship between partners, affecting the maintenance of species interactions. Here, we develop a new perspective on so-called `compensated trait loss' and how this type of trait loss may affect the evolutionary dynamics between interacting organisms. We argue that: (1) the frequency of compensated trait loss is currently underestimated because it can go unnoticed as long as ecological interactions are maintained; (2) by analysing known cases of trait loss, specific factors promoting compensated trait loss can be identified and (3) genomic sequencing is a key way forwards in detecting compensated trait loss. We present a comprehensive literature survey showing that compensated trait loss is taxonomically widespread, can involve essential traits, and often occurs as replicated evolutionary events. Despite its hidden nature, compensated trait loss is important in directing evolutionary dynamics of ecological relationships and has the potential to change facultative ecological interactions into obligatory ones.