Regulation of the Rho GTPase, RhoA, Activity Is Critical for Erythropoiesis during Embryo Development.

Hematopoiesis is critical during fetal development for the survival and growth of the embryo and is regulated by growth factors, chemokines and adhesion interactions necessary for stem cell trafficking, proliferation, differentiation and survival. The fetal liver (FL) is the major hematopoietic organ at midgestation where erythroid cells in particular undergo dramatic expansion before birth. However, the molecular regulation of hematopoiesis during embryogenesis is still poorly defined. Rho GTPases, Rac1, Rac2, CDC42 and Rho, are best known for their role in cell migration via actin rearrangement. Rho GTPases have also been implicated in cell proliferation and differentiation in non-hematopoietic cells. Using genetic approaches, we have implicated Rac1 and Rac2 in adult HSC regulating migration and proliferation and demonstrated crosstalk between Rac and other members of the Rho GTPase family (Yang, PNAS 2001; Gu, Science 2003), suggesting a role for other RhoGTPase in hematopoietic cell functions. P190Rho GTPase Activating Protein (GAP) which regulates RhoA activity has previously been implicated in IGF-1 signaling regulating cell size during fetal development (Sordella, Dev Cell, 2002) and adipogenesis-myogenesis cell fate decision (Sordella, Cell, 2003). However, there have been no reports on its role on hematopoiesis. Here, we show that p190RhoGAP is also a regulator of hematopoiesis during fetal development. FL p190RhoGAP^−/− showed 2-fold increase in RhoA activity, indicating that p190RhoGAP regulates RhoA activity in hematopoietic cells. At 14.5 day post coitum (dpc) and most prominently at 15.5 dpc, p190RhoGAP^−/− embryos appear paler than WT embryos. FL of 14.5 dpc p190RhoGAP^−/− embryos appear to be smaller than WT embryos. Hemoglobin concentration of blood from 14.5 dpc p190RhoGAP^−/− animals was 45% lower than WT (p< 0.01). The cellular content of p190RhoGAP^−/− FL was also reduced compared to WT (5.5±2.7x10⁶ vs 13.3±4.2x10⁶, p<0.05). In contrast, the size of other organs of p190RhoGAP^−/− mice was similar to WT mice, suggesting a hematopoietic specific phenotype at this stage of development. P190RhoGAP^−/− mice showed deficiency in erythroid colony forming-unit (CFU) erythroid (CFU-E, 285±84 vs 469±42 per 5x10⁴ FL cells, p<0.05) with normal frequency of burst forming-unit (BFU) E and CFU-granulo-monocytic (GM) (80±6 vs 79±7 per 5x10⁴ FL cells). Overall, total myeloid colonies and CFU-E per p190RhoGAP^−/− FL were lower than WT (10±3x10³ vs 24±3x10³, p<0.05; 40±5x10³ vs 130±3x10³, p<0.05) suggesting a defect in fetal hematopoiesis with a specific defect in erythropoiesis. To further examine the mechanism of anemia in 190RhoGAP^−/− mice, 14.5 dpc FL cells were cultured in the presence of EPO, SCF, IGF-1 and on fibronectin-coated plate, predominant regulators of erythropoiesis. Erythroid differentiation was assessed by expression of erythroid markers, CD71 and Ter119, at 5 days of culture. P190RhoGAP^−/− cells showed reduction in percentage of mature erythroid cells compared to WT cells (CD71⁺Ter119⁺: 15±6 vs 25±6.8; CD71⁻Ter119⁺: 6±1.2 vs 13±1.6, p<0.05), while increase in immature erythroid cells (CD71⁺Ter119⁻: 71.5±5 vs 53±5). Overall, this study reveals a previously unrecognized function for the family of GTPases in hematopoiesis and suggests that RhoA signaling may be critical for erythropoiesis during embryo development by regulating terminal erythroid differentiation.