The role of the apical extracellular matrix (aECM) as an important mediator of animal development is only just beginning to be recognized and explored, and few studies have used intact systems to examine how the aECM affects the response of cells and tissues to mechanical forces. This knowledge gap limits the understanding of fundamental biological processes, which when misregulated can lead to diverse developmental and adult- onset diseases. Recent studies uncovered mechanical forces acting on epidermal cells adjacent to the devel- oping anterior foregut in C. elegans embryos. These studies also identified highly conserved proteins that are required to mediate the response to this force, including FBN-1, a fibrillin-related aECM protein; MEC-8, a splicing factor that regulates FBN-1; and SYM-3 and SYM-4, candidate RAB-11?associated factors implicated in apical protein trafficking. The principal objectives of this application are to understand how the epidermal aECM is generated and how the aECM facilitates morphogenesis, cell organization, and resistance to mechan- ical stress. These objectives will be met by pursuing three specific aims.
Aim 1 will identify and further charac- terize key components of the aECM, including FBN-1, and will determine how the aECM is physically attached to the underlying epidermis.
Aim 2 will test the hypothesis that SYM-3 and SYM-4 are RAB-11 cofactors and will determine the molecular functions of these proteins and other conserved trafficking components in the transport of protein cargos to the apical membrane.
Aim 3 will extend preliminary studies to understand the mechanisms by which diverse embryonic epithelial cells are organized into a rosette structure that must resist mechanical forces to form a normal foregut lumen. We will test the hypothesis that the aECM, acting with the cytoskeleton and adherens junction proteins, maintains the structural integrity of the rosette, a widespread but little-studied embryonic structure. In addition, these studies will reveal the locations, magnitude, and dynamics of mechanical forces acting at cell?cell and cell?ECM junctions and within the cytoskeletal network during C. elegans embryonic development. This research is significant because it will (1) characterize physical forces in an intact developing system and reveal mechanisms governing how cells and tissues respond to mechanical stress; (2) lead to the discovery of novel aECM components and regulators of apical protein trafficking; (3) pro- vide insights into how diverse epithelial cells underlying the aECM organize into rosette structures and resolve to form lumens; and (4) identify genetic modifiers of a fibrillin-related protein, which are clinically important in Marfan syndrome but have not been previously characterized. This research is innovative because (1) it is based on the discovery of a previously unrecognized biomechanical force in C. elegans embryos; (2) it is the first study to directly visualize mechanical forces in developing C. elegans embryos using FRET-based tension sensors; and (3) the phenotypes under study are generally penetrant only when two or more genes have been inactivated, thereby serving as a model for redundant control mechanisms and complex disease traits.
The proposed research is relevant to public health because knowledge of how cells and tissues respond to biomechanical forces during development is critical to understanding the underlying basis for many birth defects in humans. Furthermore, the genes, proteins, and processes that will be studied all have direct correlates in human biology and development and in many cases have been implicated in human diseases such as Marfan syndrome, cardiovascular disease and cancer. The overriding goal is that the fundamental knowledge acquired through these studies may ultimately be applied towards preventing or treating developmental disabilities thereby improving the public health.
