application) Hematopoiesis is a cascade of events leading from a pluripotent stem cell to terminally differentiated blood cells. Defects in this signaling cascade can results in dyserythropoiesis and reveal important molecular events and underlying processes in erythrocyte differentiation. Dyserythropoiesis is observed in a variety of congenital and acquired anemias indicative of bone marrow stress, such as congenital dyserythropoietic anemias (CDA), myelodysplastic syndromes, leukemia megaloblastic anemia, HIV infection, recovery from bone marrow transplantation, and hemolytic anemia. A full understanding of this marrow stress response awaits identification of genes involved in this cascade of events. To achieve this understanding, we propose to take an approach using zebrafish (Danio rerio) as a genetic model system. The combined genetic and embryological advantages of zebrafish make it an ideal model system to study the developmental events of hematopoiesis. Nearly 45 zebrafish mutations, representing about 25 genes, defective in hematopoiesis have been identified. One of these zebrafish blood mutations called retsina is an autosomal recessive disorder which results in a profound anemia at 3 days post-fertilization. Using molecular markers and histologic features, we showed that myeloid and lymphoid lineages are unaffected by this mutation. Retsina mutants that survive to adulthood show complete maturation arrest at the polychromatophilic erythroblast stage with a large percentage of cells have bilobed nuclei. The striking histologic feature of retsina mutant erythroblast are most reminiscent of smears from human patients with dyserythropoietic anemias, particularly HEMPAS (Hereditary Erythroblastic Multinuclearity with Positive Acidified Serum, CDA type 2).
The specific aim of this proposal is to clone the retsina gene by a positional cloning strategy in zebrafish. I have mapped the retsina locus with a closely linked marker (0.7 cM based on 10/1380 meiotic recombinants). Candidate genes have been isolated and excluded for retsina using linkage analysis. Genetically linked YAC, BAC and PAC clones have been isolated, and a chromosome walk has been undertaken. After the isolation of the retsina gene in zebrafish, its human homolog will be cloned and examined in human patients with CDA and other dyserythropoietic anemias. The isolation of the human gene will be facilitated by the extensive synteny within this region of the zebrafish and human genome. The cloning of the retsina gene would further our understanding of molecular events in both normal erythrocyte differentiation and dyserythropoietic processes in CDA, myelodysplasia.
