Benedikt Hallgrimsson, PhD - US grants

The broad long-term research objective of this project is to understand the causes and clinical significance of variation in developmental stability among individuals. Variation in developmental stability is correlated with stress, inbreeding, teratologies and a growing number of other developmentally based pathologies. An important step in understanding how developmental stability relates to abnormal development is to understand the causes of normal variation in developmental stability. The most common measure of developmental stability, fluctuation asymmetry (FA), refers to normally distributed deviations across planes of organismal symmetry. This question is approached by studying patterns of variation in FA of skeletal structures during prenatal development of the mouse limb. Comparisons are also made with variation in adult skeletal structures. This project has four specific aims. 1) The first will determine how FA of limb skeletal elements changes during prenatal development. This aim addresses the question of whether imprecision in development arises through the cumulative effects of minor perturbations on an initially precise substrate or whether it represents the degree to which regulatory mechanism guide an initially sloppy process towards a precise end. 2) The second specific aim will determine whether postnatal patterns of variation in developmental instability among skeletal structures can be embryologically determined. 3) The third specific aim will determine whether variation in developmental rate is related to developmental instability. Through sub-hypotheses that address the significance of overall developmental rate, overall coordination of rates, and character specific variation in developmental rate, this aim will tet the proposition that developmental instability derives from breakdown in the internal coordination of developmental rates. Since developmental disorders linked to developmental disorders linked to developmental instability (e.g. cleft palate) may involve breakdown in the timing-coordination of related developmental processes, this proposition may bridge the gap between the association of developmental stability time or rate and its association with abnormal development. 4) The fourth specific aim examines the effect of specific mutations on FA in limb skeletal structures. If breakdown in the internal coordination of developmental rates causes developmental instability, then mutations that affect overall and region-specific growth rates should increase FA. Production of double-mutants with both regional and global defects will determine whether these effects are additive.

DESCRIPTION (provided by applicant): Phenotypic variation is a hallmark of craniofacial birth defects, but understanding the relationship between variable morphology and disease remains elusive. The difficulty in defining the basis for variable morphology occurs because of the multitude of genetic factors that influence facial morphology. However, the molecular pathways that regulate facial form are likely to converge on a smaller set of cellular processes. In this work, we focus on two cellular processes that control growth of the facial primordia, because differences in growth contribute to variation in phenotype. We hypothesize that by discretely altering molecular signaling pathways that regulate patterning of the major axes of the upper jaw continuous phenotypic variation will be produced due to altered patterns of gene expression that ultimately control cell proliferation and apoptosis. In each Aim we will focus on the relationship among cell proliferation, signaling by specific molecular pathways, and morphology. In the Third Specific Aim we will turn our attention to examine the relationship among cell death, cell survival, signaling, and morphology. In Aim 1 we will disrupt signals form the brain that control proliferation of neural crest cells. In Aim 2 we will disrupt signals within the neural crest mesenchyme that regulate cell proliferation. In Aim 3 we will disrupt signals from the brain and within the neural crest that regulate apopotosis and cell proliferation. In each Aim we will use 3-D and 2-D morphometrics to quantify morphologic changes in the brain and face, and we will correlate these changes with activation of specific molecular pathways, expression of signaling molecules, receptors, and transcription factors that control cell proliferation and survival, and cell proliferation and cell death. In each aim, we propose biochemical or cell- based experiments to ameliorate the phenotypic changes induced by our treatments and directly test the mechanisms that underlie production of altered morphology. These experiments will be evaluated using morphometric analysis, because this approach allows us to objectively and systematically evaluate our interventions. Overall, this work will allow us to quantitatively assess the role of growth in production of morphologic variation during development of the face. This approach will allow investigators to bridge work on specific genetic disruptions with molecular changes and cellular processes that regulate facial form. Further, with the advent of high resolution in utero imaging methods, our research will create a basis for developing parameters that allow earlier detection of facial malformations and may lead the way to in utero treatments. PUBLIC HEALTH RELEVANCE: Structural malformations of the face are common and often exhibit a large degree of morphologic variation; however, the mechanisms underlying variation are not known. Our objective is to examine cellular processes that affect growth in order to assess morphologic variation in normal and diseased populations. This basic research will help develop metrics for better in utero diagnostics and eventual treatments of structural birth defects of the face.

DESCRIPTION (provided by applicant): Phenotypic variation is a hallmark of craniofacial birth defects, but understanding the relationship between variable morphology and disease remains elusive. In this work we propose to test a model that may explain phenotypic variation within similar genotypes. This model is based on our preliminary data showing a nonlinear relationship between Sonic hedgehog (SHH) signaling and continuous phenotypic variation in the face. We hypothesize that small changes in SHH pathway activity produce large phenotypic changes that have increased variance due to heterogeneous cellular responses to compromised pathway activity. In the first Specific Aim of this grant we will focus on examining the extent to which nonlinear SHH signaling contributes to variation in cellular response. This will be done at the population and the individual cell level. In the Second and Third Specific Aims we will turn our attention to in vivo genetic models of graded SHH signaling. We will examine the phenotype of resulting embryos and we will determine the variance of the phenotypes. Our model predicts that as the nonlinearity in SHH signaling increases the potential to produce large variance also increases. The experiments designed in this application will directly test this prediction and will illuminate a potentially important mechanism that destabilizes complex developmental systems. The use of quantitative analyses such as, geometric morphometrics coupled with quantitative cellular assays and quantitative PCR, throughout this grant gives us the power to perform the analyses proposed in this application.

DESCRIPTION (provided by applicant): Dysmorphology is the branch of pediatrics and clinical genetics concerned with structural birth defects and delineation of syndromes. More than 1500 syndromes that include orofacial dysmorphia have been described. Today, dysmorphology remains largely descriptive, with diagnoses based on subjective or semi-quantitative clinical impressions of facial and other anatomic features. Over the past decade, dramatic technological advances in imaging, quantification, and analysis of variation in complex three-dimensional (3D) shape have revolutionized the assessment of morphologic variation, permitting robust definition of quantitative morphometric phenotypes that can distinguish patients from controls in a variety of syndromes. The goal of this application is to develop systems that will enable diagnostic application of craniofacial 3D morphometrics in clinical practice. We aim to define specific quantitative measures that characterize the aberrant facial shapes in a large number of human dysmorphic syndromes. Specifically, we aim to build a broad and deep 3D morphometric facial scan library of defined craniofacial dysmorphic syndromes, a resource that can be shared with approved investigators for research purposes via the NIDCR FaceBase Hub; to develop 3D geometric morphometric (GM) and dense surface modeling (DSM) analytical tools to systematically analyze and distinguish dysmorphic syndromes from unaffected individuals and from each other; and finally to develop a functional, automated, prototype clinical tool that is capable of simultaneously distinguishing a large number of syndromes, and that thereby can assist real-time diagnosis of syndromes in the clinical setting. We anticipate that 3D photomorphometric deep-phenotyping, in conjunction with the rapid advent of exome and genome sequencing in clinical medicine, will transform dysmorphology from a clinical art into a medical science.

Summary: Morphogenesis of the face is a complex process that is regulated by a series of cellular processes that control directional growth and fusion of the facial primordia. The facial primordia exhibited highly stereotyped patterns of directional growth that allow the primordia to grow together and fuse to form the face, but the mechanisms underlying the directional outgrowth remain largely unknown. Classic models of directional growth of the face and limb bud rely on cell and tissue displacement by regionalized areas of cell proliferation. However, regional proliferation is likely to operate in concert with other processes to regulate the pattern of growth. For example, polarized cellular behaviors may contribute to directional growth of the facial primordia, as in the limb bud. In our previous research, we observed that neural crest mesenchyme is polarized and activation of signaling by Fibroblast growth factor disrupts this polarity, which is associated with altered outgrowth of the facial primordia, and this may be due to alterations in directional cell movements within the developing facial complex. As cells move, they generate forces that are transmitted along the extracellular matrix (ECM) within the mesenchyme and the basement membrane (BM) underlying the surface epithelium. In humans, mutations in genes comprising the ECM and BM have been implicated in orofacial clefting, but the mechanism is unknown. Here, we propose that Fgf signaling produces variation in the distribution of ECM and BM molecules, which create regions of compliance and resistance that allow different regions of the face to resist deformation (nasal pit) or to allow deformation to occur (globular process). We will test this idea in two specific aims. First, we will assess the effect of Fgf on the distribution of components of the ECM and the BM, and we will assess cellular changes in the surface epithelium. Second, we will use atomic force microscopy to map the tissue material properties across regions of the developing upper jaw in order to determine the extent to which the distribution of the ECM and BM components is associated with changes in stiffness of the developing facial regions that either resist or undergo deformation. Physical forces are involved in morphogenesis and this work will be the first to directly measure and map the forces that are generated during directional outgrowth of the developing upper jaw. We chose to examine the developing upper jaw, because disruption to directional growth in this region contributes to craniofacial birth defects, such as cleft lip and palate. Hence, understanding how directional growth is controlled in this region is important for understanding the etiology of birth defects in this region of the embryo.  

SUMMARY/ABSTRACT Normal facial morphogenesis involves the precise spatiotemporal choreography of independent facial prominences that together must grow, contact, and fuse to form a functional upper jaw. The complexity of this process yields multiple ways in which it may go awry, so it is unsurprising that cleft lip (CL) has both a diverse etiology, and is also one of the most common human birth defects (~1:500-2500 births). Of the potential causes of CL, those that impact facial prominence growth are thought to play an outsized role since later events like contact and fusion are critically dependent on its success. That said, modeling growth has proven to be difficult because of the complex nature of tissue movements in space and time. In this grant we take a novel approach to the challenge of modeling facial prominence growth by combining innovative imaging protocols with three-dimensional geometric morphometric analyses of shape. With these tools we build novel ?developmental morphospace? model of 3D embryonic craniofacial morphogenesis in the mouse and chick and use it to generate in silico predictions of how growth variation impacts the phenotypic landscape of contact and fusion events, both normal and abnormal. We next experimentally modulate facial prominence and brain growth to directly test these model predictions in vivo. Support of DM predictions would validate a priori predictions of the effect of heterogeneous genetic mutational or environmental effects on CL-liability. Moreover, the DM would provide a generalized model for predicting how perturbations to facial prominence shape variability, growth trajectory, and brain size can combine to impact a range of contact and fusion events.