Induction of Transient Cytopenia to Detect Hematopoietic Defects in the Mouse.

In an attempt to generate new mouse models of human hematopoietic diseases and increase our knowledge of the genetic networks that orchestrate hematopoiesis, we are developing a sensitized screen that will more effectively identify hematopoietic in heterozygous mutants. The chemical mutagen ethylnitrososurea (ENU) is a highly efficient mutagen in the mouse and other model organisms, generating primarily point mutations. As a part of the Centre for Modeling Human Disease (CMHD), we have used simple clinical assays such as blood morphology and automated blood cell counts to test saphenous vein collected peripheral blood from approximately 6500 offspring of ENU mutagenized mice to identify clinically relevant dominantly inherited hematopoietic disease models. Phenodeviant G1 animals are bred to wild type mice to generate G2 mice, and G2 strains demonstrating high penetrance undergo a genome scan. This strategy has yielded 14 heritable dominant mutants to date. However, many genes and pathways will not express phenotypes in a dominant manner due to the homeostatic balance maintained by complex genetic networks, possessing excess capacity or redundancy, a problem exacerbated by using a viable peripheral blood assay. For example, only one of the 14 heritable hematopoietic mutants demonstrates anemia. Because environmental sensitization can exacerbate latent phenotypes, such as the effect of a high salt diet in an individual with prehypertension, sensitized screens should identify subtle and additional phenotypes of dominant mutations. Thus, we are developing a sensitized screen to uncover subtle dominant phenotypes by inducing transient cytopenia and following the kinetics and magnitude of the recovery. We are using a variety of drugs (5-fluorouracil, phenylhydrazine, and hydroxyurea) to induce cytopenia and recruit hematopoietic stem cells and progenitors to repopulate the blood with differentiated cells. We hypothesize that animals with defects in blood development will demonstrate altered recovery kinetics. This strategy will allow us to determine if there is a defect in blood cell progenitors, while keeping the animals viable for further screening and breeding. We are testing these drugs on known dominant and recessive mutants to validate the assay, determine the best dosages, and compare the effects of each drug. However, we have already begun to use 5-fluoruracil on a cohort of G1 animals and have identified two G1s that display latent recovery in at least 1 blood lineage. The two G1s are confirmed heritable and are currently undergoing a genome scan to map position the mutation. We believe this strategy will further our understanding of the molecular mechanisms that regulate hematopoietic development, as well as generate new mouse models of human hematopoietic diseases and identify the underlying molecular defects of diseases whose etiological origins are currently unknown.