To decipher the causes developmental defects and to improve methods of identifying potential teratogens, we need to understand the processes that buffer development against environmental stresses. Temperature affects all biochemical processes, so the mechanisms that allow coordinated development across a broad range of temperatures are likely to buffer against other types of perturbations as well. Furthermore heat stress, whether due to fever or other environmental exposures, is a significant risk factor in many birth defects (Milunsky et al., 1992;Chambers et al., 1998;Acs et al., 2005;Edwards, 2006). Development occurs successfully despite many environmental and genetic stressors, yet it can fail dramatically in response to other stresses, or to stresses above a threshold (Hamdoun and Epel, 2007). Here we propose a novel biophysical study of the mechanisms that buffer morphogenesis against perturbations. Morphogenesis is a mechanical process in that cell-generated forces move and deform tissues to drive shape change, and these forces are resisted by the viscoelastic properties of the developing tissues (Koehl, 1990;Hutson et al., 2003). The mechanics of embryo is likely to be a readout of the expression, localization and activity of a very large network of interacting gene products, including actin, myosin, regulatory proteins, and many others, most of which are highly conserved among animal species (Davidson et al., 2009). Hence we expect embryo mechanical properties to be sensitive reporters of potential teratogenicity and other effects relevant to human health. Temperature-dependence of the speed of morphogenetic movements (Bachmann, 1969), and temperature-dependence of cell mechanics (Sunyer et al., 2009b) strongly suggest that temperature- dependence of cellular force generation and of the mechanical resistance to those forces must have a role in the temperature-dependence of development. To determine how the mechanics of the embryo is regulated in the face of temperature variation we propose the following two aims:
Aim 1 : Identify biophysical contributions to developmental buffering. We will test whether tissue viscoelasticity and force generation exhibit coupled responses to a broad-spectrum environmental perturbation, temperature. Morphogenetic rates increase rapidly with temperature in ectothermic animals. We will test whether temperature-dependence of force generation and viscoelastic resistance can explain the temperature- dependence of morphogenetic movements. We will then test whether temperature affects the sensitivity of morphogenesis to perturbations in cytoskeletal function.
Aim 2 : Identify the role of specific cytoskeletal proteins in the temperature dependence of viscoelasticity and contractility. We will use cytoplasmic extracts from Xenopus eggs to investigate the contribution of the cytoskeleton - in the absence of transcription, and cell-cell or cell-substrate interactions - to the temperature dependence of viscoelasticity and force generation. We will use in vitro and in vivo experiments to test the contribution of myosin, actin crosslinking, and F-actin polymerization state to the temperature dependence of force and resistance. Together these aims constitute a novel biophysical approach to understanding how cells and embryos are buffered against environmental perturbations.
These aims will help in understanding the contributions of the cytoskeleton and cellular biophysics to birth defects. In particular it will aid in understanding the mechanisms by which hyperthermia causes birth defects. Since temperature effects all biochemical reactions, these same mechanisms are likely to contribute to developmental defects caused by other stresses.
These aims will also help us design novel bioassays based on the mechanical behavior of embryos to identify potential environmental health hazards. This approach complements the extensive body of existing studies on the genetics and molecular pathways involved morphogenesis and birth defects.
The goal of this proposal is to decipher the causes developmental defects and to improve methods of identifying potential teratogens. To achieve this goal we need to understand the processes that buffer development against environmental stresses such as temperature by identifying biophysical contributions to developmental buffering and identifying the role of specific cytoskeletal proteins in the temperature dependence of viscoelasticity and contractility. The significance of this proposal is the mechanistic analysis of birth defects from physical principles and understanding the incidence of birth defects in terms of population variation and interaction of cell- and cytoskeletal-mechanics with external risk factors such as maternal fever.
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