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Letters to the Editor
S5A, B). Perhaps Wip1 is involved in the endothelial to hematopoietic transition and hematopoietic cell expan- sion by regulating Runx1 and Gata2. To test which step in hematopoiesis Wip1 deletion was affected, we sorted EC (CD31+CD41–CD45–Ter119–). The percentages of EC were indistinguishable between WT and Wip1-/- AGM (Online Supplementary Figure S5C). Three-day co-cultures of OP9 and EC showed similar numbers of hematopoietic clusters, related to the earlier stage of endothelial to hematopoietic transition in Wip1-/- AGM (Online Supplementary Figure S5D, E). However, after co-culture for 8 days, fewer CD45+ cells were found derived from Wip1-/- EC co-cultures (Online Supplementary Figure S5F), indicating that Wip1 mainly affects HPC maturation/expansion.
As previously reported, Wip1 influences the cell prolif- eration/cell cycle status of bone marrow HSC.11,12,15 Flow cytometric assays showed no differences in cell cycle sta- tus of total cells between E11.5 Wip1-/- and WT AGM. Conversely, significantly higher percentages of EC in G1 phase were observed after Wip1 deletion (33.1±1.4% vs. 27.3±2.5%), with no differences in proliferation. Furthermore, a lower percentage of pre-HSC I was in the G0 phase (25.5±3.4% vs. 37.2±4.5%), however, the per- centages of pre-HSC II in G1 phase were significantly higher in the Wip1-/- AGM region (40.0±3.1% vs. 31.26±2.6%) (Figure 3P). S/G2/M phases and prolifera- tion status were not changed by Wip1 deficiency (Figure 3P, Online Supplementary Figure S6A-C). The apoptosis assay did not show any differences in the EC, pre-HSC I and II in Wip1-/- AGM (Online Supplementary Figure S6D- 6F), suggesting influences of Wip1 on the cell cycle.
In summary, our study identifies a regulatory role for Wip1 in hematopoietic development during the mid-ges- tation stage. Transplantation assays after co-culture revealed that Wip1 regulates pre-HSC maturation in embryonic hematopoiesis, likely mediated by altered cell cycle status, laying a theoretical foundation for HSC regeneration in vitro.
Wenyan He,1 Xiaobo Wang,2 Yanli Ni,2 Zongcheng Li,2 Wei Liu,2 Zhilin Chang,2 Haowen Li,1 Zhenyu Ju3
and Zhuan Li4
1China National Clinical Research Center for Neurological Diseases, Beijing Tiantan Hospital, Capital Medical University, Beijing; 2Laboratory of Oncology, Fifth Medical Center, General Hospital of PLA, Beijing; 3Key Laboratory of Regenerative Medicine of Ministry
of Education, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Institute of Aging and Regenerative Medicine, Jinan University, Guangzhou and 4Department of Developmental Biology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China
Correspondence: ZHUAN LI - zhuanli2018@smu.edu.cn ZHENYU JU - zhenyuju@163.com doi:10.3324/haematol.2019.235481
Disclosures: no conflicts of interests to disclose.
Contributions: ZL, ZJ and WH designed research; WH performed research; XW did some transplantation experiments; YN gave support on FACS; ZL supported bioinformatics analysis, WL, LC, HL pre- pared embryos; ZL and WH analyzed data; ZL, ZJ and WH wrote the paper.
Acknowledgments: we are grateful to Dr. L.A. Donehower from Baylor College of Medicine for the Wip1 knockout mice. We thank Dr. B. Liu and Dr. Y. Lan for useful discussions.
Funding: this work was supported by grants from the National Natural Science Foundation of China (81570095, 81870087, 91749203, 81525010), the National Key Research and Development Program (2019YFA0801802, SQ2019YFA0111100, 2016YFA0100602, 2017YFA0103302) and the Program for Guangdong Introducing Innovative and Entrepreneurial Teams (2017ZT07S347) and the Innovative Team Program of Guangzhou Regenerative Medicine and Health Guangdong Laboratory (2018GZR110103002).
References
1. Muller AM, Medvinsky A, Strouboulis J, Grosveld F, Dzierzak E. Development of hematopoietic stem cell activity in the mouse embryo. Immunity. 1994;1(4):291-301.
2. MedvinskyA,DzierzakE.Definitivehematopoiesisisautonomous- ly initiated by the AGM region. Cell. 1996;86(6):897-906.
3. Li Z, Lan Y, He W, Chen D, et al. Mouse embryonic head as a site for hematopoietic stem cell development. Cell Stem Cell. 2012; 11(5):663-675.
4. LiZ,VinkCS,MarianiSA,DzierzakE.Subregionallocalizationand characterization of Ly6aGFP-expressing hematopoietic cells in the mouse embryonic head. Dev Biol. 2016 ;416(1):34-41.
5. OttersbachK,DzierzakE.Themurineplacentacontainshematopoi- etic stem cells within the vascular labyrinth region. Dev Cell. 2005; 8(3):377-387.
6. Mikkola HK, Gekas C, Orkin SH, Dieterlen-Lievre F. Placenta as a site for hematopoietic stem cell development. Exp Hematol. 2005; 33(9):1048-1054.
7. Dzierzak E, Bigas A. Blood development: hematopoietic stem cell dependence and independence. Cell Stem Cell. 2018;22(5):639-651.
8. Chen MJ, Yokomizo T, Zeigler BM, Dzierzak E, Speck NA. Runx1 is required for the endothelial to haematopoietic cell transition but not thereafter. Nature. 2009;457(7231):887-891.
9. Bertrand JY, Chi NC, Santoso B, Teng S, Stainier DY, Traver D. Haematopoietic stem cells derive directly from aortic endothelium during development. Nature. 2010;464(7285):108-111.
10. Boisset JC, van Cappellen W, Andrieu-Soler C, Galjart N, Dzierzak E, Robin C. In vivo imaging of haematopoietic cells emerging from the mouse aortic endothelium. Nature. 2010;464(7285):116-120.
11. ChenZ,YiW,MoritaY,etal.Wip1deficiencyimpairshaematopoi- etic stem cell function via p53 and mTORC1 pathways. Nature Commun. 2015;6:6808.
12. Schito ML, Demidov ON, Saito S, Ashwell JD, Appella E. Wip1 phosphatase-deficient mice exhibit defective T cell maturation due to sustained p53 activation. J Immunol. 2006;176(8):4818-4825.
13. ZhouF,LiX,WangW,etal.Tracinghaematopoieticstemcellforma- tion at single-cell resolution. Nature. 2016;533(7604):487-492.
14. Yokomizo T, Dzierzak E. Three-dimensional cartography of hematopoietic clusters in the vasculature of whole mouse embryos. Development. 2010;137(21):3651-3661.
15. Uyanik B, Grigorash BB, Goloudina AR, Demidov ON. DNA dam- age-induced phosphatase Wip1 in regulation of hematopoiesis, immune system and inflammation. Cell Death Discov. 2017; 3:17018.
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