Deficiency of Rac1 and Rac2 GTPases Perturbs Erythroid Proliferation and Differentiation but Not Enucleation.
Authors: Kalfa Theodosia A. , Pushkaran Suvarnamala , Cancelas Jose A. , Jansen Michael , Johnson James F. , Hebel Kristen , Williams David A. , Zheng Yi 
Published: April 2020

Abstract
The small Rho GTPases Rac1 and Rac2 have overlapping as well as distinct roles in actin organization, cell survival, and proliferation in various hematopoietic cell lineages. However their role in erythropoiesis has not yet been fully elucidated. Using conditional gene-targeted mice we demonstrated that deficiency of Rac1 and Rac2 GTPases causes a significant phenotype in erythroid lineage. The mice develop anemia that is both hemolytic (abnormal structure of the erythrocyte cytoskeleton and decreased deformability; Kalfa et al. Blood 2006) and dyserythropoietic in nature. Cre-recombinase-induced deletion of Rac1 genomic sequence was accomplished as previously described (Gu et al. Science, 2003) on a Rac2-null genetic background. Colony assays revealed that although BFU-E frequency was similar, Rac1−/ −;Rac2−/ − BFU-E colonies had a strikingly different morphology appearing as round, small, dense colonies with solid edges, likely a manifestation of migration defects associated with Rac GTPase deficiency. CFU-E formation from hematopoietic stem/progenitors (HSC/Ps) derived from bone marrow (BM) of Rac1−/ −;Rac2−/ − mice was decreased more than 50% in comparison to WT (p=0.01). On the other hand, Rac1−/ −;Rac2−/ − mice developed marked splenomegaly (2-fold enlargement) and low density spleen cells demonstrated a 2-fold increase in CFU-E frequency in comparison to WT (p=0.008). To further assess erythroblast differentiation, BM and spleen cells were immunostained with fluorescent label-conjugated anti-CD71 and anti-Ter119, as previously described (Socolovski et al. Blood, 2001). Flow cytometry analysis revealed that the BM content of proerythroblasts and basophilic erythroblasts was significantly decreased (&gt;5-fold) in Rac1−/ −;Rac2−/ − vs. WT mice. In contrast, the same erythroblast populations were 4-fold increased in the spleens of Rac1−/ −;Rac2−/ − animals. However, the terminal differentiation to orthochromatic erythroblasts was comparable. No survival difference was found between WT and Rac1−/ −;Rac2−/ − erythroid precursors by flow cytometry with annexin-V, indicating that apoptosis was not contributing to the changes in erythropoiesis in Rac-deficient mice. Differentiation of Rac1−/ −;Rac2−/ − HSC/Ps to proerythroblasts and basophilic erythroblasts was delayed significantly at the early stages in ex vivo erythropoiesis culture (Giarratana et al. Nat Biotechnol, 2005) in the presence of SCF and erythropoietin. Later in the culture the cytokine-independent terminal differentiation to orthochromatic erythroblasts was similar between WT and Rac1−/ −;Rac2−/ − mice. The phosphorylation of AKT in WT and Rac1−/ −;Rac2−/ − erythroid precursors revealed by immunoblotting was similar, but the phosphorylation of extracellular signal-regulated kinase (ERK) (p42/p44) in Rac1−/ −;Rac2−/ − erythroid precursors was significantly decreased. The enucleation process was evaluated quantitatively, in ex vivo erythropoiesis cultures, by flow cytometry, using SYTO16, a cell-permeable DNA-staining dye. The frequency of enucleated red cells (SYTO16-negative, Ter119-positive population) was similar in the WT and Rac1−/ −;Rac2−/ − erythroid cultures. These data suggest that Rac1 and Rac2 deficiency does not affect enucleation but causes a significant decrease of early erythroid precursor populations in the bone marrow.