The overall goal of our research group is to understand the role of molecular, cellular, and tissue-level biomechanics in early embryonic development and morphogenesis. It is increasingly well understood that mechanics plays an important role in controlling cell fate and behavior both in vitro and in vivo, in physiological and disease processes. Much less is known about the role of mechanics in the embryo, but it stands to reason that similar pathways are involved in regulating growth and morphogenesis. In this project we will study the mechanical properties and generation of forces in avian epiblast cells during the critically important morphogenetic process of primitive streak formation, which establishes head-to-tail polarity and acts as the ?organizing center? for gastrulation in amniote embryos. During gastrulation, endodermal and mesodermal precursors in the epiblast gather at the embryonic midline, undergo epithelial-to-mesenchmyal transition (EMT), and become internalized, thus establishing the essential trilaminar vertebrate body plan and setting the stage for the subsequent organogenesis. ?It is not birth, marriage, or death, but gastrulation, which is truly the most important time in your life.? (Lewis Wolpert, 1986)
In the past several decades, many of the molecular signaling pathways required for primitive streak formation have been elucidated, but the underlying cellular and morphogenetic mechanisms are still being debated. Some previously proposed models for primitive streak formation involve increased cell proliferation and directed cell migration or chemotaxis. We hypothesize that actomyosin-generated cell forces drive epiblast cells to condense at the midline, in some ways, similar to contraction of a muscle fiber. A prediction of this hypothesis is that cells in the region of the forming streak region exhibit active stiffening. We will test this hypothesis by using the sensitive atomic force microscopy (AFM) to probe the mechanical properties of intact and isolated epiblast cells in the presence of various biochemical and molecular inhibitors of actomyosin contractility. A further prediction of our hypothesis is that to maintain epithelial integrity or continuity in the epiblast during streak formation, cellular adhesion is upregulated to withstand increased mechanical stresses. To test this hypothesis, we will use a spinning disk adhesion assay that uses controlled application of fluid shear stress acting on arrays of micropatterned epiblast cells. Finally, we will develop a novel in vitro model for studying the effects of substrate mechanical and adhesive properties on isolated epiblast cells or epiblast micro-explants. The results of these studies will further our understanding of mechanics in the earliest stages of embryonic development, and establish mechanical assays that will be useful for identifying and distinguishing morphogenetic mechanisms that may be otherwise difficult to study using conventional biological techniques.
