RhoA GTPase is known to regulate cell adhesion, actomyosin cytoskeleton, and cytokinesis. Specifically, it contributes to cytokinesis in sea urchin embryos, Xenopus and HeLa cells by associating with the microtubule ends at the abscission area marking the position for the daughter cell separation and by activating myosin through phosphorylation of the myosin regulatory light chain (MRLC) (Piekny et al, Trends in Cell Biology 2005). Erythroblast enucleation, the process through which orthochromatic erythroblasts expel their nucleus during the final stage of mammalian erythropoiesis, is a multi-step process resembling asymmetric cytokinesis (reviewed by Li, Developmental Cell 2013). It requires establishment of cell polarity through microtubule function, followed by formation of a contractile actomyosin ring between reticulocyte and pyrenocyte (Konstantinidis et al, Blood 2012).
To define the mechanistic contribution of RhoA signaling in terminal erythroid maturation, we generated mice with erythroid specific RhoA deletion (EpoR-GFPcre-driven). Erythroid-specific deletion of RhoA, confirmed in the CD71-positive cells by PCR and immunoblotting, caused severe anemia, fatal in utero by E15.5. RhoA-deficient peripheral primitive blood cells were large with significant poikilocytosis and anisocytosis, frequently binuclear or multinuclear with polyploidy of the genetic material as demonstrated by flow cytometry with propidium iodide, and with incomplete clearance of the Golgi network and mitochondria. RhoAΔ/Δ fetal livers had progressively decreased cellularity. The erythroblast populations as determined by CD71-Ter119-FSC flow cytometry as well as by multispectral high-speed cell imaging in flow, demonstrated progressive decline and significantly decreased reticulocyte production (n=3, p<0.05), compatible with a fatal intrauterine anemia. RhoAΔ/Δ definitive erythroblasts also exhibited increased frequency of polyploidy. These polyploid cells were Ki67-negative by immunohistochemistry, indicating that they were not actively mitotic. Apoptosis was not significantly increased in the RhoA-deficient erythroblasts as determined by Annexin-V positivity. However, the late RhoAΔ/Δ erythroblasts demonstrated a significant increase in necrosis as determined by 7AAD and Annexin-V double-positivity (n=3, p<0.05). Increased phosphorylation of p53 was evident by immunoblotting, likely functioning as a polyploidy checkpoint after failure of abscission due to RhoA deficiency. Further evidence that RhoA is significant for microtubule organization in erythroblasts was attained by immuno-localization of RhoA at the microtubule ends and by decreased nuclear polarization in EpoR-Cre RhoAΔ/Δ orthochromatic erythroblasts, as shown by decreased delta centroid Ter119-Draq5 (the distance between the centers of the erythroblast and the nucleus) in multispectral high-speed cell imaging in flow. Phosphorylated myosin regulatory light chain (pMRLC) was found significantly decreased by immunoblotting of RhoAΔ/Δ fetal liver erythroblasts, indicating that RhoA controls phosphorylation of MRLC in erythropoiesis. Increased PAK phosphorylation was noted pointing towards compensatory upregulation of Rac GTPases in the RhoA-deficient cells, which was confirmed by quantitative RT-PCR. We further examined the erythroblastic islands in RhoAΔ/Δfetal liver by transmission electron microscopy and found that RhoA-deficient erythroblasts, frequently dysplastic and binucleated, were loosely associated with the central macrophage and there was a paucity of intracellular vacuoles in comparison to WT erythroblasts, indicating possible defects in vesicular transport, which has also been shown to play a role in erythroblast enucleation (Keerthivasan et al, Blood 2010).
Thus, RhoA GTPase regulates erythroblast differentiation and enucleation by dynamic coordination of the microtubule-actomyosin machineries to achieve successful abscission. In addition, RhoA may also regulate erythroblast adhesive interactions with the central macrophage in erythroblastic islands and intracellular vesicular transport. Our results reveal novel molecular components and cellular processes in erythropoiesis that may be important for improving the efficiency of red blood cell production in vitro.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.