Positional Cloning of the Mouse Scat Mutation Reveals a Critical Role for RASA3, a Ras GTPase Activating Protein, in Vertebrate Erythropoiesis.

Abstract 776

The spontaneous recessive mutation scat (severe combined anemia and thrombocytopenia) arose on the inbred BALB/cBy (BALB) mouse strain. The phenotype of scat is cyclic. All homozygotes are severely anemic and thrombocytopenic at birth. Leukocytes are also significantly depleted (Table). Approximately 13% die during this first “crisis” episode that lasts, on average, until the 9^th postnatal day. Remarkably, a spontaneous remission ensues in those surviving the neonatal crisis period wherein all peripheral blood values revert to normal. A second crisis follows, and 94% of the mice die by 30 days of age. The recessive ENU (N-ethyl-N-nitrosourea)-induced mutation, hlb381, on the C57BL/6J (B6) background, is characterized by severe thrombocytopenia and leukopenia with mild anemia. Unlike scat, hlb381 is not cyclical. The phenotype is present at birth and persists throughout life. Despite the phenotypic differences, scat and hlb381 interact genetically; double heterozygotes show non-cyclical severe thrombocytopenia and leukopenia, and mild anemia (Table). This interaction implies that the scat and hlb381 genetic defects affect the same gene or distinct genes within the same pathway. Both scat and hlb381 mapped to overlapping intervals on mouse chromosome 8. Sequence analysis of genes within the interval identified Rasa3 (GAPIII, GAP1^IP4BP) as a strong candidate gene for both scat and hlb381. In scat, Rasa3 carries a missense mutation near the N-terminus (G125V, exon 5) and, in hlb381, a missense mutation near the C-terminus (H794L, exon 23). RASA3 is a GTPase activating protein (GAP) that negatively regulates p21 Ras function by accelerating GTP hydrolysis and converting Ras to the inactive GDP bound form. Analysis in Panther and SIFT predicts that both residues are highly conserved and substitutions are likely to be deleterious. Rasa3 is widely expressed throughout embryonic and fetal development in mice, and is ubiquitously expressed in zebrafish 24 hours post fertilization (hpf). RASA3 protein is detected in erythroid tissues and platelets in the adult mouse. Analysis of scat spleen and bone marrow erythrocyte populations by FACS (dual staining for Ter119 and CD71 followed by forward scatter of the Ter119 high population) reveals a severe block in erythropoiesis during crisis periods. The proerythroblast, EryA (basophilic erythroblasts), and EryB (late basophilic and polychromatophilic erythroblasts) populations are significantly increased in frequency vs. wild type, and the EryC (orthochromatophilic erythroblasts and reticulocytes) population is markedly decreased. Annexin V staining revealed no significant differences in any of these populations. Notably, a similar delay in erythroid maturation, albeit much milder, is also seen in hlb381. In pull-down assays using the Ras-binding domain of Raf1 to affinity purify active GTP-bound Ras followed by detection by western blotting with pan-Ras antibody, active GTP-bound Ras is deficient in scat crisis red cells but recovers during remission. Finally, injection of two independent splice-blocking morpholinos designed to disrupt exon 5 and induce disruption of rasa3 mRNA processing resulted in a major decrease in the number of hemoglobinized cells when stained with o-dianisidine at 48 and 72 hpf in zebrafish. Over 90% of morphants showed no hemoglobinized cells at all, or vastly reduced numbers (<20) of hemoglobinized cells. We conclude that RASA3 plays a critical role in vertebrate erythropoiesis. Differences in the scat and hlb381 phenotypes likely result from allele-specific interactions mediated by the different genetic backgrounds (B6 vs. BALB) or domain-specific functions of the RASA3 protein.