Every animal begins as a single cell. How this cell ultimately gives rise to a vertebrate animal possessing millions to trillions of cells, each specialized to perform specific functions in a variety of organ systems, remains a fundamental question in biology. Until now, in toto capture of the collective cellular behaviors that generate the vertebrate body plan has not been possible. We have assembled a team with complementary expertise in developmental genetics, tissue patterning, imaging, and quantitative biology that will synergize to take lineage analysis in vertebrates to an entirely new level. We will utilize the unique strengths of the zebrafish, combined with recent advances in genetic fate mapping techniques, high-resolution, whole-animal 4D microscopy, and computational methodologies to derive its complete lineage map. Our proposed approaches are precise, enabling prospective and retrospective lineage analyses, and they are visual, enabling direct identification and quantification of cell behaviors. We have developed indelible marking systems to laser-target single cells in the embryo and permanently activate reporter gene expression. Clonal analysis beyond the embryo will involve inducible Cre recombinases and single/multicolor Cre-releasable reporter transgenes. Our toolset will allow any cell of interest and its progeny to be followed for life. By deriving a library of thousands of labeled animals, we will create unprecedented access to the biography of each organ. To complement our cellular tagging approaches, we will develop imaging platforms to elucidate the entire progeny sets of labeled clones and their tissue distribution. We will also develop novel computational methods to track clones across development and to generate a digitized map that recapitulates the cellular dynamics that underlie the adult body plan. Clonal data sets will be utilized to develop probabilistic models to infer how proliferation and differentiation dynamics give rise to observed lineage restriction. Collectively, this massive technological achievement will yield templates of organ morphogenesis that we will make facile for any user to access, visualize, and manipulate. These body plan reconstructions can be employed as a baseline for interpreting cell lineage decisions in response to tissue damage, genetic manipulation, or malignancy.
Knowledge regarding the cellular paths that form each organ system has become increasingly important to human health. In this proposal, we will utilize the unique strengths of the zebrafish embryo, notably its translucency, external and rapid development, genetic amenability, and suitability to lineage tracing techniques, to follow the fate of every cell in the early embryo by direct imaging approaches. These studies will provide an unprecedented visualization of organ development, and they will generate lineage maps of unprecedented resolution. Our work will illuminate fundamental mechanisms that construct, enlarge, adapt, and repair tissues, inspiring potential methods for regenerative medicine.
