Induction of Transient Cytopenia To Analyze Hematopoiesis in Mutant Mice.

Abstract The mouse plays an indispensable role in developing our current understanding of mammalian hematopoiesis. Most hematopoietic phenotyping assays in the mouse are non-viable techniques designed to evaluate homeostatic populations, enumerate progenitor populations, and perform functional analysis. The worldwide effort to generate mouse models of human disease and functionally annotate the mammalian genome using mouse mutagenesis (including dominant ENU screens) requires the development of robust standardized viable phenotyping tools. We have developed a phenotyping assay that induces transient cytopenias using various pharmacological agents (5-fluorouracil, phenylhydrazine, and hydroxyurea), the responses to which are monitored by tracing changes in peripheral blood levels of red blood cells, white blood cells and platelets. We have performed detailed analysis of lineage recovery kinetics, developing lineage recovery curves for various strains of mice for both males and females, which allowed us to identify appropriate testing days to identify phenodeviants. We have compared the recovery data with conventional progenitor assays and analyzed a cohort of well-studied naturally occurring and targeted hematopoietic mutants using the transient anemia assays that has yielded novel phenotypes of hemizygous mutant animals. For example, erythropoietin receptor null embryos die of severe anemia at mid-gestation; however, no defect in erythropoiesis has been reported in EpoR +/− mice. We have found that 5-fluorouracil and phenylhydrazine elicit delayed RBC recovery in EpoR +/− mice, demonstrating a critical dose-dependent role for the erythropoietin receptor in stress erythropoiesis. In addition, Stat5 has been shown to play an important role in erythropoiesis and in the regulation of steady state hematopoiesis. We have found that Stat5a/b+/− mice treated with 5-fluorouracil show altered recovery kinetics in RBC, WBC and platelets. Finally, we have adapted the transient cytopenia assay to develop a sensitized dominant ENU screen, enabling us to identify hematopoietic mutants that do not present abnormal blood cell counts in a homeostatic state. Thus, these standardized cytopenia response assays function as surrogate viable assays to analyze progenitor populations.