Mouse Models and the Evolutionary Developmental Biology of the Skull

Understanding development is relevant to understanding evolution because developmental processes structure the expression of phenotypic variation upon which natural selection acts. Advances in developmental biology are fueling a new synthesis of developmental and evolutionary biology, but it remains unclear how to use developmental information that largely derives from a few model organisms to test hypotheses about the evolutionary developmental biology of taxa such as humans and other primates that have not been or are not amenable to direct study through experimental developmental biology. In this article, we discuss how and when model organisms like mice are useful for studying the evolutionary developmental biology of even rather distantly related and morphologically different groups like primates. A productive approach is to focus on processes that are likely to play key roles in producing evolutionarily significant phenotypic variation across a large phylogenetic range. We illustrate this approach by applying the analysis of craniofacial variation in mouse mutant models to primate and human evolution.Anthropolog

The Interaction of Genetic Background and Mutational Effects in Regulation of Mouse Craniofacial Shape.

Inbred genetic background significantly influences the expression of phenotypes associated with known genetic perturbations and can underlie variation in disease severity between individuals with the same mutation. However, the effect of epistatic interactions on the development of complex traits, such as craniofacial morphology, is poorly understood. Here, we investigated the effect of three inbred backgrounds (129X1/SvJ, C57BL/6J, and FVB/NJ) on the expression of craniofacial dysmorphology in mice (Mus musculus) with loss of function in three members of the Sprouty family of growth factor negative regulators (Spry1, Spry2, or Spry4) in order to explore the impact of epistatic interactions on skull morphology. We found that the interaction of inbred background and the Sprouty genotype explains as much craniofacial shape variation as the Sprouty genotype alone. The most severely affected genotypes display a relatively short and wide skull, a rounded cranial vault, and a more highly angled inferior profile. Our results suggest that the FVB background is more resilient to Sprouty loss of function than either C57 or 129, and that Spry4 loss is generally less severe than loss of Spry1 or Spry2 While the specific modifier genes responsible for these significant background effects remain unknown, our results highlight the value of intercrossing mice of multiple inbred backgrounds to identify the genes and developmental interactions that modulate the severity of craniofacial dysmorphology. Our quantitative results represent an important first step toward elucidating genetic interactions underlying variation in robustness to known genetic perturbations in mice

Craniofacial diversification in the domestic pigeon and the evolution of the avian skull.

A central question in evolutionary developmental biology is how highly conserved developmental systems can generate the remarkable phenotypic diversity observed among distantly related species. In part, this paradox reflects our limited knowledge about the potential for species to both respond to selection and generate novel variation. Consequently, the developmental links between small-scale microevolutionary variations within populations to larger macroevolutionary patterns among species remain unbridged. Domesticated species, such as the pigeon, are unique resources for addressing this question, because a history of strong artificial selection has significantly increased morphological diversity, offering a direct comparison of the developmental potential of a single species to broader evolutionary patterns. Here, we demonstrate that patterns of variation and covariation within and between the face and braincase in domesticated breeds of the pigeon are predictive of avian cranial evolution. These results indicate that selection on variation generated by a conserved developmental system is sufficient to explain the evolution of crania as different in shape as the albatross or eagle, parakeet or hummingbird. These 'rules' of cranio-facial variation are a common pattern in the evolution of a broad diversity of vertebrate species and may ultimately reflect structural limitations of a shared embryonic bauplan on functional variation

Microarchitecture, but Not Bone Mechanical Properties, Is Rescued with Growth Hormone Treatment in a Mouse Model of Growth Hormone Deficiency

Growth hormone (GH) deficiency is related to an increased fracture risk although it is not clear if this is due to compromised bone quality or a small bone size. We investigated the relationship between bone macrostructure, microarchitecture and mechanical properties in a GH-deficient (GHD) mouse model undergoing GH treatment commencing at an early (prepubertal) or late (postpubertal) time point. Microcomputed tomography images of the femur and L4 vertebra were obtained to quantify macrostructure and vertebral trabecular microarchitecture, and mechanical properties were determined using finite element analyses. In the GHD animals, bone macrostructure was 25 to 43% smaller as compared to the GH-sufficient (GHS) controls (P < 0.001). GHD animals had 20% and 19% reductions in bone volume ratio (BV/TV) and trabecular thickness (Tb.Th), respectively. Whole bone mechanical properties of the GHD mice were lower at the femur and vertebra (67% and 45% resp.) than the GHS controls (P < 0.001). Both early and late GH treatment partially recovered the bone macrostructure (15 to 32 % smaller than GHS controls) and the whole bone mechanical properties (24 to 43% larger than GHD animals) although there remained a sustained 27–52% net deficit compared to normal mice (P < 0.05). Importantly, early treatment with GH led to a recovery of BV/TV and Tb.Th with a concomitant improvement of trabecular mechanical properties. Therefore, the results suggest that GH treatment should start early, and that measurements of microarchitecture should be considered in the management of GHD

Canalization and developmental instability of the fetal skull in a mouse model of maternal nutritional stress

Nutritional imbalance is one of the main sources of stress in both extant and extinct human populations. Restricted availability of nutrients is thought to disrupt the buffering mechanisms that contribute to developmental stability and canalization, resulting in increased levels of fluctuating asymmetry (FA) and phenotypic variance among individuals. However, the literature is contradictory in this regard. This study assesses the effect of prenatal nutritional stress on FA and among-individual variance in cranial shape and size using a mouse model of maternal protein restriction. Two sets of landmark coordinates were digitized in three dimensions from skulls of control and protein restricted specimens at E17.5 and E18.5. We found that, by the end of gestation, maternal protein restriction resulted in a significant reduction of skull size. Fluctuating asymmetry in size and shape exceeded the amount of measurement error in all groups, but no significant differences in the magnitude of FA were found between treatments. Convsersely, the pattern of shape asymmetry was affected by the environmental perturbation since the angles between the first eigenvectors extracted from the covariance matrix of shape asymmetric component of protein restricted and control groups were not significantly different from the expected for random vectors. In addition, among-individual variance in cranial shape was significanlty higher in the protein restricted than the control group at E18.5. Overall, the results obtained from a controlled experiment do not support the view of fluctuating asymmetry of cranial structures as a reliable index for inferring nutritional stress in human populations.Instituto de Genética VeterinariaFacultad de Ciencias Naturales y Muse

Developmental plasticity in covariance structure of the skull: effects of prenatal stress

Environmental perturbations of many kinds influence growth and development. Little is known, however, about the influence of environmental factors on the patterns of phenotypic integration observed in complex morphological traits. We analyze the changes in phenotypic variance–covariance structure of the rat skull throughout the early postnatal ontogeny (from birth to weaning) and evaluate the effect of intrauterine growth retardation (IUGR) on this structure. Using 2D coordinates taken from lateral radiographs obtained every 4 days, from birth to 21 days old, we show that the pattern of covariance is temporally dynamic from birth to 21 days. The environmental perturbation provoked during pregnancy altered the skull growth, and reduced the mean size of the IUGR group. These environmental effects persisted throughout lactancy, when the mothers of both groups received a standard diet. More strikingly, the effect grew larger beyond this point. Altering environmental conditions did not affect all traits equally, as revealed by the low correlations between covariance matrices of treatments at the same age. Finally, we found that the IUGR treatment increased morphological integration as measured by the scaled variance of eigenvalues. This increase coincided and is likely related to an increase in morphological variance in this group. This result is expected if somatic growth is a major determinant of covariance structure of the skull. In summary, our findings suggest that environmental perturbations experienced in early ontogeny alter fundamental developmental processes and are an important factor in shaping the variance–covariance structure of complex phenotypic traits.Facultad de Ciencias Naturales y MuseoFacultad de Ciencias VeterinariasInstituto de Genética Veterinari

Genetics of murine craniofacial morphology: diallel analysis of the eight founders of the Collaborative Cross

Using eight inbred founder strains of the mouse Collaborative Cross (CC) project and their reciprocal F1 hybrids, we quantified variation in craniofacial morphology across mouse strains, explored genetic contributions to craniofacial variation that distinguish the founder strains, and tested whether specific or summary measures of craniofacial shape display stronger additive genetic contributions. This study thus provides critical information about phenotypic diversity among CC founder strains and about the genetic contributions to this phenotypic diversity, which is relevant to understanding the basis of variation in standard laboratory strains and natural populations. Craniofacial shape was quantified as a series of size-adjusted linear dimensions (RDs) and by principal components (PC) analysis of morphological landmarks captured from computed tomography images from 62 out of the 64 reciprocal crosses of the CC founder strains. We first identified aspects of skull morphology that vary between these phenotypically ‘normal’ founder strains and that are defining characteristics of these strains. We estimated the contributions of additive and various non-additive genetic factors to phenotypic variation using diallel analyses of a subset of these strongly differing RDs and the first 8 PCs of skull shape variation. We find little difference in the genetic contributions to RD measures and PC scores, suggesting fundamental similarities in the magnitude of genetic contributions to both specific and summary measures of craniofacial phenotypes. Our results indicate that there are stronger additive genetic effects associated with defining phenotypic characteristics of specific founder strains, suggesting these distinguishing measures are good candidates for use in genotype-phenotype association studies of CC mice. Our results add significantly to understanding of genotype-phenotype associations in the skull, which serve as a foundation for modeling the origins of medically and evolutionarily relevant variation

Estimating Cell Count and Distribution in Labeled Histological Samples Using Incremental Cell Search

Cell proliferation is critical to the outgrowth of biological structures including the face and limbs. This cellular process has traditionally been studied via sequential histological sampling of these tissues. The length and tedium of traditional sampling is a major impediment to analyzing the large datasets required to accurately model cellular processes. Computerized cell localization and quantification is critical for high-throughput morphometric analysis of developing embryonic tissues. We have developed the Incremental Cell Search (ICS), a novel software tool that expedites the analysis of relationships between morphological outgrowth and cell proliferation in embryonic tissues. Based on an estimated average cell size and stain color, ICS rapidly indicates the approximate location and amount of cells in histological images of labeled embryonic tissue and provides estimates of cell counts in regions with saturated fluorescence and blurred cell boundaries. This capacity opens the door to high-throughput 3D and 4D quantitative analyses of developmental patterns

Estimating Cell Count and Distribution in Labeled Histological Samples Using Incremental Cell Search

Cell proliferation is critical to the outgrowth of biological structures including the face and limbs. This cellular process has traditionally been studied via sequential histological sampling of these tissues. The length and tedium of traditional sampling is a major impediment to analyzing the large datasets required to accurately model cellular processes. Computerized cell localization and quantification is critical for high-throughput morphometric analysis of developing embryonic tissues. We have developed the Incremental Cell Search (ICS), a novel software tool that expedites the analysis of relationships between morphological outgrowth and cell proliferation in embryonic tissues. Based on an estimated average cell size and stain color, ICS rapidly indicates the approximate location and amount of cells in histological images of labeled embryonic tissue and provides estimates of cell counts in regions with saturated fluorescence and blurred cell boundaries. This capacity opens the door to high-throughput 3D and 4D quantitative analyses of developmental patterns

Micro-computed tomography-based phenotypic approaches in embryology: procedural artifacts on assessments of embryonic craniofacial growth and development

<p>Abstract</p> <p>Background</p> <p>Growing demand for three dimensional (3D) digital images of embryos for purposes of phenotypic assessment drives implementation of new histological and imaging techniques. Among these micro-computed tomography (μCT) has recently been utilized as an effective and practical method for generating images at resolutions permitting 3D quantitative analysis of gross morphological attributes of developing tissues and organs in embryonic mice. However, histological processing in preparation for μCT scanning induces changes in organ size and shape. Establishing normative expectations for experimentally induced changes in size and shape will be an important feature of 3D μCT-based phenotypic assessments, especially if quantifying differences in the values of those parameters between comparison sets of developing embryos is a primary aim. Toward that end, we assessed the nature and degree of morphological artifacts attending μCT scanning following use of common fixatives, using a two dimensional (2D) landmark geometric morphometric approach to track the accumulation of distortions affecting the embryonic head from the native, uterine state through to fixation and subsequent scanning.</p> <p>Results</p> <p>Bouin's fixation reduced average centroid sizes of embryonic mouse crania by approximately 30% and substantially altered the morphometric shape, as measured by the shift in Procrustes distance, from the unfixed state, after the data were normalized for naturally occurring shape variation. Subsequent μCT scanning produced negligible changes in size but did appear to reduce or even reverse fixation-induced random shape changes. Mixtures of paraformaldehyde + glutaraldehyde reduced average centroid sizes by 2-3%. Changes in craniofacial shape progressively increased post-fixation.</p> <p>Conclusions</p> <p>The degree to which artifacts are introduced in the generation of random craniofacial shape variation relates to the degree of specimen dehydration during the initial fixation. Fixation methods that better maintain original craniofacial dimensions at reduced levels of dehydration and tissue shrinkage lead to the progressive accumulation of random shape variation during handling and data acquisition. In general, to the degree that embryonic organ size and shape factor into μCT-based phenotypic assessments, procedurally induced artifacts associated with fixation and scanning will influence results. Experimental designs will need to address these significant effects, either by employing alternative methods that minimize artifacts in the region of focus or in the interpretation of statistical patterns.</p