Tropomodulin3-Null Mice Are Embryonic Lethal with Anemia and Impaired Definitive Erythropoiesis in the Fetal Liver

Abstract 1036 Tropomodulin1 binds tropomyosin and caps the pointed ends of the short actin filaments in the spectrin-actin network of red blood cells (RBCs). Tmod1-null mice display a mild compensated, sphero-elliptocytic anemia with fragile RBCs, due to mis-regulation of actin filament lengths and a disrupted membrane skeleton lattice (Moyer et al. 2010. Blood 116:2590–2599). The mild phenotype may be explained by the appearance of Tmod3, which is not found in wild-type RBCs but is in Tmod1-null RBCs, albeit at only 1/5th of normal Tmod1 levels. Tmod3 is ubiquitously expressed, including in erythroblasts, and thus the presence of Tmod3 in Tmod1-null RBCs is likely due to aberrant persistence of Tmod3 during terminal differentiation and maturation. To investigate the role of Tmod3 in RBCs, we created a Tmod3 knockout mouse from ES cells (#RRF004, BayGenomics) in which intron 1 of the Tmod3 locus was disrupted by retroviral-mediated insertion of a gene-trap vector. The insertion creates a novel fusion transcript joining sequences from exons 59 to the insertion site to the beta-galactosidase marker. Tmod3+/− mice are viable and fertile, but Tmod3−/− animals are embryonic lethal, with 94% of nulls dying between E13.5 – E15.5 (175 total embryos, E12.5-E18.5). Tmod3−/− embryos are pale and anemic starting at E13.5, with much smaller livers. Tmod3 mRNA and protein are reduced in Tmod3+/− and absent in Tmod3−/−, demonstrating that this is a true null. Tmod1 mRNA and protein are also reduced, consistent with anemia, fewer erythroblasts and RBCs, indicating that Tmod1 expression does not compensate for absence of Tmod3. The peripheral blood of wild-type E13.5-E15.5 embryos contains abundant large primitive and smaller definitive RBCs, but few or no definitive enucleated RBCs are observed in Tmod3-null embryos. Fetal livers from Tmod3−/− embryos were about ½ the size of wild-type livers, and histology showed altered cellularity. However, the proliferative potential of erythroid progenitors appears not to be impaired based on similar numbers of BFU-E and CFU-E colonies from Tmod3+/+ and Tmod3−/− fetal livers. Cytospins of dissociated fetal liver cells from E13.5-E14.5 embryos reveal the presence of erythroblast/macrophage islands in both genotypes, but with some macrophages containing abundant ingested cellular debris in the Tmod3−/− cytospins. High magnification images show increased erythroblast blebbing in absence of Tmod3, suggesting a weaker cortex, and/or apoptosis of erythroblasts. Flow cytometry of E14.5 fetal liver erythroblasts labeled with Ter119 and CD71 indicate that the late stage R3 population is reduced by about 40–50% in absence of Tmod3, while R1-R2 populations are somewhat increased (possibly as a compensatory response to fetal anemia). In addition, Annexin V staining shows a 2-fold increase in apoptotic cells in the fetal liver, most of which are in the R1 population. Measurement of enucleation frequency in R populations shows a marked reduction of enucleated cells as the erythroblasts mature through the R3-R5 populations. To explore the role of macrophages in the Tmod3−/− phenotype, we performed immunofluorescence staining and confocal microscopy of fetal liver cryosections stained with F4/80 (macrophages), Ter119 (erythroblasts) and Hoechst (nuclei). Macrophages displayed strikingly less dendritic morphologies in the Tmod3−/−, with macrophages sometimes containing Ter119-positive nucleated erythroblasts. Our data show that definitive erythropoiesis is impaired during terminal differentiation in absence of Tmod3, and we speculate that this is due to defective erythroblast-macrophage interactions during terminal differentiation. Experiments are in progress to determine whether Tmod3 function is required in erythroblasts or macrophages, and to identify the molecular pathway in which Tmod3 functions in erythropoiesis. Supported by NIH/NHLBI grant R01HL083464 to V.M.F. Disclosures: No relevant conflicts of interest to declare.