Multiphoton In Vivo Microscopy of Embryonic Thrombopoiesis Reveals the Generation of Platelets through Budding

Huan Liu; Hellen Ishikawa-Ankerhold; Julia Winterhalter; Michael Lorenz; Mykhailo Vladymyrov; Steffen Massberg; Christian Schulz; Mathias Orban

doi:10.3390/cells12192411

Multiphoton In Vivo Microscopy of Embryonic Thrombopoiesis Reveals the Generation of Platelets through Budding

Cells. 2023 Oct 6;12(19):2411. doi: 10.3390/cells12192411.

Authors

Huan Liu¹, Hellen Ishikawa-Ankerhold¹, Julia Winterhalter¹, Michael Lorenz¹, Mykhailo Vladymyrov^{2

3

4}, Steffen Massberg^{1

5}, Christian Schulz^{1

5}, Mathias Orban^{1

5}

Affiliations

¹ Department of Internal Medicine I, Ludwig Maximilians University, 81377 Munich, Germany.
² Laboratory for High Energy Physics (LHEP), Albert Einstein Center for Fundamental Physics, University of Bern, 3012 Bern, Switzerland.
³ Theodor Kocher Institute, University of Bern, 3012 Bern, Switzerland.
⁴ Data Science Lab, Mathematical Institute, University of Bern, 3012 Bern, Switzerland.
⁵ DZHK (German Centre for Cardiovascular Research), Partner Site Munich Heart Alliance, 80802 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 (Pf4^CreRosa26^mTmG). 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.

Keywords: c-Myb; fetal liver; megakaryocyte; multiphoton intravital microscopy; thrombopoiesis; yolk sac.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Blood Platelets
Megakaryocytes
Mice
Microscopy*
Platelet Count
Thrombopoiesis*

Grants and funding

This research was funded by the SFB 914 project Z01 (to H.I. and S.M.) and SFB 914 project A10 (C.S.), SFB 1123 projects A07 (C.S.) and B06 (S.M.), the DZHK (German Centre for Cardiovascular Research), and the BMBF (German Ministry of Education and Research) grants #81Z0600201 (S.M.), as well as the ERC AdvGrant “Immunothrombosis” #833440 (S.M.).