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Updated: Jul 13, 2025
In Vivo Two-photon Imaging of Megakaryocytes and Proplatelets in the Mouse Skull Bone Marrow
Published on: July 28, 2021
Multiphoton In Vivo Microscopy of Embryonic Thrombopoiesis Reveals the Generation of Platelets through Budding
Huan Liu1, Hellen Ishikawa-Ankerhold1, Julia Winterhalter1
1Department of Internal Medicine I, Ludwig Maximilians University, 81377 Munich, Germany.
Abstract:
Platelets are generated by specialized cells called megakaryocytes (MKs). However, MK's origin and platelet release mode have remained incompletely understood. Here, we established direct visualization of embryonic thrombopoiesis in vivo by combining multiphoton intravital microscopy (MP-IVM) with a fluorescence switch reporter mouse model under control of the platelet factor 4 promoter (Pf4CreRosa26mTmG). Using this microscopy tool, we discovered that fetal liver MKs provide higher thrombopoietic activity than yolk sac MKs. Mechanistically, fetal platelets were released from MKs either by membrane buds or the formation of proplatelets, with the former constituting the key process. In E14.5 c-Myb-deficient embryos that lack definitive hematopoiesis, MK and platelet numbers were similar to wild-type embryos, indicating the independence of embryonic thrombopoiesis from definitive hematopoiesis at this stage of development. In summary, our novel MP-IVM protocol allows the characterization of thrombopoiesis with high spatio-temporal resolution in the mouse embryo and has identified membrane budding as the main mechanism of fetal platelet production.
Insights
This study visualizes embryonic platelet production in mice. Membrane budding, not proplatelets, is the primary method fetal megakaryocytes use to generate platelets.
Area of Science:
- Hematology
- Developmental Biology
- Cell Biology
Background:
- Megakaryocytes (MKs) produce platelets, but their origin and platelet release mechanisms are not fully understood.
- Embryonic thrombopoiesis, the process of platelet formation during embryonic development, requires further investigation.
Purpose of the Study:
- To visualize and characterize embryonic thrombopoiesis in vivo with high spatio-temporal resolution.
- To elucidate the primary mechanism of fetal platelet production by megakaryocytes.
Main Methods:
- Developed a novel multiphoton intravital microscopy (MP-IVM) protocol.
- Utilized a fluorescence switch reporter mouse model (Pf4Cre;Rosa26mTmG) for direct visualization.
- Analyzed megakaryocyte and platelet production in wild-type and c-Myb-deficient embryos.
Main Results:
- Fetal liver megakaryocytes exhibit higher thrombopoietic activity than yolk sac megakaryocytes.
- Identified membrane budding as the predominant mechanism for fetal platelet release from megakaryocytes.
- Embryonic thrombopoiesis at E14.5 is independent of definitive hematopoiesis, as shown in c-Myb-deficient embryos.
Conclusions:
- The novel MP-IVM protocol enables detailed characterization of embryonic thrombopoiesis.
- Membrane budding is the principal mechanism of fetal platelet production.
- Embryonic megakaryocyte origin and platelet release modes are distinct from previously assumed pathways.
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