Arhgef2 regulates HSC function through RhoA activation and its loss disrupts HSPC mitotic spindle orientation. (A) Arhgef2^−/− or wild-type (WT) Lin^– cells supplemented with CD45.1 bone marrow support cells were transplanted into recipient mice for 13 weeks (n = 3 recipients per group). BrdU was administered 72 hours before the end point, and cell cycle distribution of wild-type or Arhgef2^−/− Lin^–CD150⁺CD48^– cells were examined by flow cytometry. (B) Quantification of 4N/4N⁺ cell frequency by 7-AAD staining. (C) Expression of TNF-RIP–related genes evaluated by qPCR in wild-type or Arhgef2^−/− Lin^– graft cells. (D) Experimental schematic of rescue competitive transplants. (E) Proportion of blue fluorescent protein positive (BFP⁺) transduced Arhgef2^−/− cells after 12 weeks (left), HSC content (middle), and myeloid content as measured by Gr-1 (right) of the grafts. Empty vector (EV), n = 5 recipients; ARHGEF2, n = 3 recipients; RhoA Q63L, n = 4 recipients. (F) Multinuclearity (4N and above) assessed by live cell fluorescence microscopy of dividing wild-type (51 events) or Arhgef2^−/− (50 events) HSPCs. Differences between groups for multinuclearity enumeration were not significant by Fisher’s exact test (2-tailed). (G) Representative z-stack stitched images of LSK HSPCs retrovirally transduced with both H2B-EGFP and mCherry-α-tubulin imaged under live cell fluorescence microscopy to capture telophase events; observed division axes (yellow) as measured in reference to the horizontal axis (red). (H) Quantification of cytokinesis events indicate that Arhgef2^−/− LSK HSPCs exhibit a significantly increased frequency of random divisional orientations whereas wild-type HSPCs preferentially divide parallel to an underlying retronectin substrate (55 cells for wild-type background and 56 cells for Arhgef2^−/− background). Error bars represent SEM. *P < .05; **P < .01; ***P < .001. FACS, fluorescence-activated cell sorter.