Stephanie Woo, Ph.D. - US grants

DESCRIPTION (provided by applicant): The goal of this study is to identify the mechanisms regulating early endodermal cell migration and understand how cell movements contribute to the overall formation of the gastrointestinal tract. Although much progress has been made in identifying the transcription factors that specify endodermal cells, much less is known about the cell biological processes that occur subsequently, including the onset of cell motility. Shortly after specification, endodermal cells internalize to form the inner layer of the body during a process known as gastrulation. Using the zebrafish embryo as a model system, I will examine two aspects of endodermal cell migration - random directionality and contact-dependent repulsion. For these studies, I will use high-resolution fluorescence imaging of live embryos and a novel transgenic line that I developed that fluorescently labels the actin cytoskeleton. Preliminary evidence suggests that Nodal, a signaling protein already known to induce endodermal fate, can promote endodermal migration in a directionally random manner. I will characterize the effects of Nodal signaling on the actin cytoskeleton within endodermal cells, and I will identify the actin regulatory proteins that function to promote Nodal-induced motility. In the second specific aim, I will examine the mechanisms regulating contact-dependent repulsion. This is a process whereby migrating endodermal cells are repelled away from each other after making brief contact. Using both candidate and microarray- based approaches, I will attempt to identify the cell surface receptors that initiate contact repulsion as well as the cytoplasmic signaling proteins that act downstream of these receptors. Together, directionally random motility and contact repulsion appear to drive the dispersal of endodermal cells across the surface of the early embryo, after which these cells coalesce into a single gut tube. To understand the significance of this initial dispersal, I will use gain and loss of function techniques to interfere with normal endodermal migratory behavior and assess the effects on subsequent development of the gastrointestinal tract. In the course of this study, I will gain further expertise in vertebrate gut development, whole-embryo time-lapse imaging, and quantitative methods for examining in vivo cell migration. My primary mentor, Dr. Didier Stainier, and my co-mentor, Dr. Orion Weiner, will provide additional training in these areas to help me achieve my research goals. This study will shed light on a poorly appreciated aspect of gastrointestinal development and will form the basis for a future R-level application as I continue to explore the relationship between cell migration and organ development. PUBLIC HEALTH RELEVANCE: Cell migration is important for many processes such as embryonic development, immune function, tissue repair, inflammation, and tumor metastasis. In particular for the gastrointestinal system, defects in endodermal cell migration during early embryogenesis have been reported to cause various malformations of the gastrointestinal tract. This study will advance our knowledge of how endodermal cell migration is regulated, which will contribute to a better understanding of digestive diseases, many of which are precipitated by congenital defects, as well as other conditions in which cell migration plays a prominent role.

? DESCRIPTION (provided by applicant): The goal of this study is to investigate the cell biological mechanisms that drive the formation of the gastrointestinal epithelium. Although much progress has been made in understanding in the molecular genetics of gastrointestinal tract development and disease, the dynamic cell behaviors that contribute to organ shape and function are less well understood. During embryonic development, endodermal cells initially undergo a phase of highly dynamic single-cell migration but then later converge and adhere together into a coherent endodermal sheet, which ultimately gives rise to the epithelial lining of the gut tube. For this study, I will use high-resolution fluorescence imaging of live zebrafish embryos to investigate the transition from single- cell migration to epithelium formation. Preliminary experiments suggest that endodermal cells initiate epithelium formation by upregulating cell junction molecules to facilitate adhesion between cells while also spatially regulating actin polymerization and membrane protrusion to close gaps in the newly forming sheet. In the first aim of this study, I will determine the mechanisms by which cells identify cell free areas and extend their membrane across these gaps. Concurrently, I will define the progression of cell-cell adhesion by monitoring the dynamics of fluorescently labeled cell junction components alpha-catenin, E-cadherin, and ZO- 1. Finally, I will use RNA-Seq transcriptome profiling to identify new factors in endodermal sheet formation and will test the function of these new candidate genes by generating mutants with CRISPR/Cas9 technology. In Aim2, I will explore the role of the cytoskeletal gene septin9a (sept9a) in endodermal sheet formation. I had previously identified sept9a as gene that is upregulated specifically in the endoderm at the onset of sheet formation. Septins are known to regulate changes in cell shape and cortex tension, which may provide the necessary structural integrity to form a coherent epithelium. In this aim, I will characterize the effects of sept9a loss-of-function mutations on endodermal cell motility, cell-cell interactions, and the initiation and maintenance of cell-cell adhesion. Proper formation of the gastrointestinal epithelium is required for the tissue to perform its functions in barrier protection, digestion, and nutrient absorption, and defects in epithelial structure may lead to diseases such as inflammatory bowel disease. However, much of our current understanding of epithelium formation has come from in vitro cell culture systems that may not accurately represent epithelial differentiation as occurs in a developing embryo. Thus, the early zebrafish endoderm may be a much-needed in vivo model of de novo epithelium formation. This model could be used in future studies exploring fundamental aspects of epithelial biology as well as the cell biological mechanisms underlying gastrointestinal disorders and diseases.

PROJECT SUMMARY Protein labeling by fusion with genetically encoded fluorescent proteins has been a powerful tool for investigating biological processes, allowing scientists to observe and analyze protein expression, localization, and dynamics in living cells. However, traditional approaches for expressing fluorescent fusion proteins possess drawbacks including potential overexpression artifacts, and new methods are needed, especially for in vivo studies. Our lab studies vertebrate organ formation using the zebrafish (Danio rerio) model system. In zebrafish and many other model organisms, expression of fluorescent fusion proteins is often achieved by injection of in vitro transcribed mRNA, which provides ubiquitous expression, or by transgenesis, which utilizes gene regulatory elements to drive spatially and/or temporally restricted expression. However, both approaches run the risk of producing overexpression artifacts. An alternative approach is to ?knock-in? fluorescent coding sequences into the genetic locus for the protein of interest. Although this approach preserves endogenous regulation of expression, targeted insertion can be technically difficult to achieve. Moreover, many proteins are expressed quite broadly, and fluorescent protein tagging at the endogenous locus does not allow one to study the tissue- specific roles of such proteins. To overcome these limitations, we propose using a split fluorescent protein approach to achieve tissue-specific and endogenous protein labeling. Split fluorescent proteins consist of protein fragments that are expressed independently and possess little to no fluorescence on their own. However, when present in the same cell, the fragments self-assemble into a fluorescent complex. In this proposal, we will use a recently developed two- component system based on the green fluorescent protein mNeonGreen2 (split-NG). By expressing one component of the split-NG pair under a tissue specific promoter while fusing the second component to a protein of interest via genomic ?knock-in?, our technique will enable tissue-specific examination of broadly expressed proteins. This technique has the potential to open new lines of inquiry in many fields of biological and biomedical research.