Cdc42 Is Critical for Multi-Lineage Blood Cell Development from Hematopoietic Stem and Progenitor Cells.

Abstract Cdc42 is a member of the Rho GTPase family regulating multiple functions in eukaryotic cells. However, its involvement in blood cell development is not well studied. In our recent work using an inducible, conditional gene targeted mouse model (Mx-Cre;Cdc42loxP/loxP), we have shown that Cdc42 is important for hematopoietic stem cell (HSC) homing, lodging and engraftment, as well as maintenance of long-term HSC quiescence in the hematopoietic microenvironment. To define the contribution of Cdc42 to multi-lineage differentiation of HSCs, we have examined this mouse model for lymphoid, erythroid and myeloid lineage development. Firstly, although Cdc42 gene disruption in HSCs does not alter the overall bone marrow (BM) cellularity, it affects the frequency of multipotent progenitor cells (Table). The number of common lymphoid progenitors (CLPs) remain unchanged, but the number of phenotypic common myeloid progenitors (CMP) decreases, the number of phenotypic granulocytic myeloid progenitors (GMP) increases, and the myeloerythroid progenitors (MEP) decreases, in the Cdc42−/− mice. Secondly, despite the normal CLP content in the BM, the cellularity of Cdc42−/− thymus and T-lymphoid populations in peripheral blood is markedly reduced (Table). The T-cell developmental defect is associated with a thymus-homing defect of the Cdc42−/− BM cells. Thirdly, the Cdc42−/− mice show severe anemia and splenomegaly that are associated with dramatically decreased BFU-E and CFU-E activities of the BM cells (Table). FACS analysis showed an accumulation of early (CD71hiTER119−/low) erythroid cells and a reduction of the more mature TER119hi populations of erythroid progenitors in the Cdc42−/− bone marrow (Table), suggesting that Cdc42-deficiency causes a block in erythroid differentiation. Finally, Cdc42-deficiency in the HSCs led to a myeloproliferative disorder (MPD) phenotype, characterized by significantly increased monocytes, granulocytes and myeloid progenitors in the peripheral blood (Table), massive deposit of Gr-1+/Mac-1+ myeloid cells in the BM, and infiltration by the myeloid cells into distal organs such as spleen, liver and lung. These effects are a consequence of increased cell cycle progression and cell expansion accompanying the markedly increased mobilization of the short-term HSCs and myeloid progenitors from the BM to the peripheral organs found in the Cdc42−/− mice. Taken together, our results show that in addition to an essential role in maintaining HSC quiescence in the bone marrow niche, the Rho GTPase Cdc42 is critically involved in regulating multi-lineage differentiation of HSCs, including lymphopoiesis, erythropoiesis and myelopoiesis, through distinct mechanisms. WT Cdc42−/− BM cellularity (X106) 113.0±15.2 116.8±26.4 CLP (X106) 0.0223±0.0139 0.0390±0.0277 CMP (X106) 0.228±0.093 0.077±0.029* GMP (X106) 1.47±0.33 2.70±1.07* MEP (X106) 0.16±0.058 0.051±0.031* Thymus weight (X10−3g/g body weight) 2.37±0.78 0.49±0.36*** CD4+CD8− (/ul PB) 614.3±340.7 282.2±179.1* CD4−CD8+ (/ul PB) 446.1±332.7 181.2±56.7* BFU-E (/105 BM cells) 36±8.9 14.4±3.7 CFU-E (/105 BM cells) 87.2±12.4 26.4±5.7* CD71+Ter119− cells in BM (Stage I) 1.3%±0.06% 3.4%±0.38%*** CD71−Ter119+ cells in BM (StageII-IV) 39.7%±10.8% 7.8%±1.3%** Gr1/Mac1+ cells in BM 47.8%±27.2% 83.3%±8.9%** *p