IRFP: Single cell analysis of the segmentation clock

Webb Alexis B, Dresden

Investigators

Abstract

The International Research Fellowship Program enables U.S. scientists and engineers to conduct nine to twenty-four months of research abroad. The program's awards provide opportunities for joint research, and the use of unique or complementary facilities, expertise and experimental conditions abroad. This award will support a twenty-four month postdoctoral research fellowship by Dr. Alexis Webb in the laboratory of Dr. Andrew Oates at the Max Planck Institute for Molecular Cell Biology and Genetics in Dresden, Germany. The periodic formation of somites along the vertebrate body axis is evidence of an oscillator in the developing embryo. In zebrafish this cellular segmentation clock consists of members of the her/hes transcription factor family, which are thought to form a negative feedback loop through transcriptional repression. Static gene expression patterns in presomitic mesoderm (PSM) tissue provide an overview of the process, but are incomplete because oscillations occur in single cells and are dynamic. The host lab has developed live transgenic reporter lines for the cyclic genes her1 and her7 that accurately recapitulate the spatiotemporal phenomena, opening the door to dynamic analysis of the segmentation clock. This reporter line allows continuous monitoring in both the developing zebrafish embryo in vivo and isolated PSM cells in vitro. A tissue and cellular-level analysis of these embryo lines is ongoing, but in order to understand the fundamental properties of the clock, it is necessary to know the behavior of the individual pieces, i.e. the isolated oscillating cells. This research studies current models of the segmentation clock for the first time at the single cell level. The first objective of the project is measurement of dynamic properties of gene expression in single PSM cells dispersed in culture such as frequency, amplitude, relative phase to other cells, and duration of oscillation to determine whether individual PSM cells continue to function as autonomous oscillators in vitro or if additional factors are needed to maintain clock behavior. Second, mutations to the genetic oscillator or manipulations to signaling pathways between cells can further test the necessary and sufficient components within current segmentation clock models. Beyond the project's intellectual merit, this fellowship funds training for a young US researcher to expand her research background into developmental biology, and specifically, zebrafish as a model organism. The host lab will provide an outstanding mentoring environment in a world-renowned research institution. Such knowledge will prove critical as the PI moves forward in her professional development and begins a teaching and research career at small liberal arts college, where model organisms fit into well-rounded science training for undergraduates. The collaborative relationships developed between the PI and international research colleagues during the funding period will further benefit her future research endeavors.