Senthil Raja Jayapal1, Heather Yin-Kuan Ang3, Chelsia Qiuxia Wang1,4, Xavier Bisteau1, Matias J. Caldez1,2, Gan Xiao Xuan1, Weimiao Yu1, Vinay Tergaonkar1, Motomi Osato4, Bing Lim3, and Philipp Kaldis1.2.*
1 Institute of Molecular and Cell Biology (IMCB), A*STAR (Agency for Science, Technology and Research), 61 Biopolis Drive, Proteos #3-09, Singapore 138673, Republic of Singapore
2 National University of Singapore (NUS), Department of Biochemistry, Singapore 117597
3 Genome Institute of Singapore, 60 Biopolis Street, Genome, Singapore 138672
4 Cancer Science Institute of Singapore, National University of Singapore, 14 Medical Drive, Singapore 117599
*Corresponding author: Philipp Kaldis PhD, Phone +65 6586 9854, Fax +65 6779 1117, Email: email@example.com
Published online in Cell Cycle on 22 September 2016.
Cyclin A2 is an essential gene for development and in hematopoietic stem cells and therefore its functions in definitive erythropoiesis have not been investigated. We have ablated cyclin A2 in committed erythroid progenitors in vivo using erythropoietin receptor promoter-driven Cre, which revealed its critical role in regulating erythrocyte morphology and numbers. Erythroid-specific cyclin A2 knockout mice are viable but displayed increased mean erythrocyte volume and reduced erythrocyte counts, as well as increased frequency of erythrocytes containing Howell-Jolly bodies. Erythroblasts lacking cyclin A2 displayed defective enucleation, resulting in reduced production of enucleated erythrocytes and increased frequencies of erythrocytes containing nuclear remnants. Deletion of the Cdk inhibitor p27Kip1 but not Cdk2, ameliorated the erythroid defects resulting from deficiency of cyclin A2, confirming the critical role of cyclin A2/Cdk activity in erythroid development. Loss of cyclin A2 in bone marrow cells in semisolid culture prevented the formation of BFU-E but not CFU-E colonies, uncovering its essential role in BFU-E function. Our data unveils the critical functions of cyclin A2 in regulating mammalian erythropoiesis.
Figure 1. Ablation of cyclin A2 in erythroid cells in vivo using erythropoietin receptor promoter-driven Cre leads to defective erythropoiesis. (A) Complete blood count data from cyclin A2fl/fl ErGFPcre mice (A2 KO, red dots, n=11) and littermate controls (Control, blue dots, n=9) at 3 months of age. (B-C) May-Grünwald Giemsa stained blood smears. Scale bar=10μm. Arrows indicate erythrocytes with Howell-Jolly bodies. (D) 3D reconstruction of erythrocyte volume using z-stacks from confocal microscopy of blood smears stained with fluorescence-labeled PKH26. Representative wild-type erythrocytes (left panel, blue, inset density diagram in the middle); A2 KO (middle panel, red, inset density diagram in the middle [yellow plane indicates the cut of the inset]; and merge (right panel, inset representing 3D-view); indicating the difference in volume between wild-type and A2 KO erythrocytes. (E-F) Representative flow cytometry analysis plots of peripheral blood from age-matched control (top) and A2 KO (bottom)
mice stained with antibodies against TER119 and CD71, and Hoechst33342 (n=3). The percentage of TER119+ erythrocytes with nuclear remnants (Howell-Jolly bodies, HJ) is indicated in panel E. CD71 versus Hoechst plots for TER119+-gated peripheral blood cells with the frequencies of CD71+Hoechst- reticulocytes (RET) and CD71-Hoechst+ erythrocytes with Howell-Jolly bodies (HJ) are shown in panel F. (G) Bar graphs representing flow cytometry quantification of the percentage of erythrocytes containing Howell-Jolly bodies (HJ) in peripheral blood gated as shown in panel E, and the percentage of reticulocytes (RET) in TER119+-gated peripheral blood cells gated as shown in panel F (n=3). (H) The mean forward scatter values of TER119+Hoechst-CD71- erythrocytes (RBC, red bars), TER119+Hoechst-CD71+ reticulocytes (RET, green bars), and TER119+Hoechst+CD71- Howell-Jolly bodies (HJ, blue bar) in the peripheral blood, gated as shown in panel F (n=3). The forward scatter histogram overlays are shown in Figure S1C. Error bars represent standard deviation. Two-tailed t-test results are indicated by asterisks. *, p<0.05; **, p<0.01; ***, p<0.001.
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