PROJECT SUMMARY: Hematopoietic stem cells (HSCs) are at the top of a hierarchy of differentiating progenitor cells that can reconstitute the entire blood and immune system. Proper regulation of HSC differentiation is critical during homeostasis and response to stress or infection. Dysregulation of HSCs naturally occurs during aging, and aberrant differentiation can cause blood cancers and other diseases. Decades of research have been devoted to understanding and defining the hierarchy of HSC differentiation. However, the limitations of current biological tools and animal models has left key questions unanswered. Foremost, we have not been able to track individual HSCs and multipotent progenitors (MPPs), directly and continuously, in their endogenous microenvironment. Previously, HSCs and MPPs in mouse models could only be identified by dissociation of bone marrow, labeling with antibodies to detect markers of differentiated lineages (Lin) and at least four other markers; for example, c-Kit (aka Kit or CD117), Sca-1 (aka Ly6a), CD48, CD150 (aka Slamf1). More recently, single color transgenic HSC reporter lines have labeled a proportion of HSCs that could be detected by intravital imaging, but without additional markers for MPPs, the dynamics of HSC fate decisions are lost. We hypothesize that newly discovered combinations of cell type-specific markers will allow live tracking of HSC/MPP behaviors and key lineage decisions. The convergence of multiple cutting-edge technologies makes it possible to address this hypothesis by generating a multi-color, multi-loci mouse reporter system in the following aims. Aim 1) Generate an HSC/MPP reporter mouse by targeting knock-in 2A fusion reporters to four loci. We will use CRISPR/Cas9-dependent homology directed repair (HDR) in mouse zygotes, with repair template delivered by adeno-associated virus (AAV). Each locus will retain functional proteins that cleave to express distinct fluorescent proteins (e.g., BFP, GFP, tdTomato, iRFP), representing a positive or negative marker for HSCs and/or MPPs. Aim 2) Characterize the HSC/MPP reporter line using complementary assays to assess faithful labeling of HSCs and MPPs. After knock-in validation, we will interbreed the lines to generate four color transgenics. Labeled HSCs and MPPs will be quantified by flow cytometry and functionally validated by limiting dilution transplantation. Bones will be imaged ex vivo to confirm that spatial maps of endogenously labeled HSCs and MPPs are consistent with published immunolabeling data. Outstanding questions about the migration of HSCs and MPPs will be answered by intravital microscopy. Our research proposal fits this funding opportunity because it represents a significant “improvement of animal models for stem cell-based regenerative medicine.” It addresses the research interests of multiple NIH Institutes and Centers because the models generated would apply to the basic biology of the blood system (NHLBI), immune cells (NIAI...