Mechanisms of embryonic thrombopoiesis

Liu, Huan (2021): Mechanisms of embryonic thrombopoiesis. Dissertation, LMU München: Medizinische Fakultät

Abstract

Platelets are generated by specialized cells called megakaryocytes (MKs) throughout embryonic development and adulthood. In adult bone marrow, MKs form and release proplatelets into circulation which mature into functional peripheral platelets. However, platelet generation in embryos is not fully understood. This is due to limitations of existing mouse models, challenges in handling fragile embryonic structures and lack of suitable imaging methods. Our goal was to assess the physiological generation of platelets in embryos. We used flow cytometry to globally assess the different components of embryonic megakaryopoiesis and thrombopoiesis. We quantified platelets in embryonic blood and MKs in the fetal liver from wild type mice and mouse models of absent definitive hematopoiesis with c-Myb deficient embryos, and deficient megakaryocyte differentiation and thrombocytopenia with NFE2 deficient embryos. We found a dramatic increase of platelet count from E13.5 to E14.5, which could be the result of an observed strong increase in absolute MK number and further maturation with increasing fetal liver MK ploidy. Embryos with absence of c-Myb showed normal peripheral platelet count and MK numbers in fetal liver, although MKs had an immature phenotype. This finding suggests that megakaryopoiesis and thrombopoiesis in the fetal liver could be of dual original from yolk sac progenitors and later definitive hematopoietic precursors. Moreover, the master regulator of MK differentiation, NFE2, appeared to have also a role in embryonic thrombopoiesis. NFE2 deficient embryos presented with thrombocytopenia and increased numbers of apparently less mature MKs. Second, we developed a highly advanced protocol for direct visualization of embryonic thrombopoiesis in vivo over time by adapting multiphoton intravital microscopy (MP-IVM) in the fluorescent thrombopoietic reporter mouse model Rosa26 mTmG x Pf4 Cre. We imaged directly platelet generation from MKs in the yolk sac and fetal liver identifying various types of proplatelets at different stages of embryonic development. We found that fetal liver MKs had higher thrombopoietic activity than yolk sac MKs. Embryonic platelets were released from MKs either by proplatelet or by membrane bud formation. Membrane bud formation seemed to be the predominant form of platelet generation. Our modified embryonic flow cytometry protocol and our novel three-dimensional MP-IVM protocol can be applied for studies of thrombopoiesis, hematopoiesis and other dynamic developmental processes. Comprehending the underlying mechanism by which embryonic platelets are generated could be critical to develop novel therapeutic strategies for congenital defects of hematopoiesis and thrombopoiesis such as neonatal thrombocytopenia. Our work could help to foster embryonic disease modeling beyond the hematopoietic system.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: megakaryocyte, platelet, yolk sac, fetal liver
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften ; 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• Fakultäten: Medizinische Fakultät
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 9. Dezember 2021
• 1. Bericht­erstatter:in: Massberg, Steffen