Mpl expression on megakaryocytes and platelets is dispensable for thrombopoiesis but essential to prevent myeloproliferation Ashley P. Nga,b,1, Maria Kauppia,b, Donald Metcalfa,b,1, Craig D. Hylanda, Emma C. Josefssona,b, Marion Leboisa, Jian-Guo Zhanga,b, Tracey M. Baldwinc, Ladina Di Ragoa, Douglas J. Hiltonb,c, and Warren S. Alexandera,b Divisions of aCancer and Haematology, and cMolecular Medicine, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia; and bDepartment of Medical Biology, The University of Melbourne, Parkville, VIC 3010, Australia Contributed by Donald Metcalf, March 10, 2014 (sent for review February 4, 2014) Thrombopoietin (TPO) acting via its receptor, the cellular homologue Mpl mass, circulating TPO concentration, and the degree of of the myeloproliferative leukemia virus oncogene (Mpl), is the stimulation of megakaryopoiesis may not always hold. major cytokine regulator of platelet number. To precisely define the Whereas expression of Mpl on megakaryocytes and platelets role of specific hematopoietic cells in TPO-dependent hematopoiesis, contributes to regulation of available TPO, the role of direct we generated mice that express the Mpl receptor normally on stem/ TPO stimulation of megakaryocytes for effective platelet pro- progenitor cells but lack expression on megakaryocytes and plate- duction is unclear. Administration of TPO in vivo or stimulation PF4cre/PF4cre PF4cre/PF4cre lets (Mpl ). Mpl mice displayed profound mega- of bone marrow in vitro elevates megakaryocyte numbers and karyocytosis and thrombocytosis with a remarkable expansion of increases mean DNA ploidy (10, 11), and the thrombocyto- − − megakaryocyte-committed and multipotential progenitor cells, the penia in TPO / mice is accompanied by reduced megakaryocyte latter displaying biological responses and a gene expression signa- ploidy (12). However, although exposure of megakaryocytes to ture indicative of chronic TPO overstimulation as the underlying TPO stimulates intracellular signaling (13), in vitro studies sug- causative mechanism, despite a normal circulating TPO level. Thus, gesting direct action of TPO on megakaryocytes to increase TPO signaling in megakaryocytes is dispensable for platelet produc- DNA ploidy, promote cytoplasmic maturation, or to stimulate CELL BIOLOGY tion; its key role in control of platelet number is via generation and proplatelet production (14–16) are balanced by reports that TPO stimulation of the bipotential megakaryocyte precursors. Neverthe- is dispensable for these megakaryocyte functions (15, 17–19). less, Mpl expression on megakaryocytes and platelets is essential to To comprehensively define Mpl-expressing stem and pro- prevent megakaryocytosis and myeloproliferation by restricting the genitor cell populations in vivo and resolve the specific require- amount of TPO available to stimulate the production of megakar- ments for Mpl expression in megakaryocytes and platelets for yocytes from the progenitor cell pool. platelet production and feedback control of TPO levels, we gen- erated a mouse strain in which green fluorescent protein (GFP) is bone marrow | essential thrombocythemia expressed from the Mpl locus and, when crossed to Platelet Factor 4(PF4)cre transgenic mice (20), specifically lacks Mpl expression hrombopoietin (TPO) is the principal hematopoietic cytokine in megakaryocytes and platelets. Tthat regulates platelet production at steady state and is re- quired for rapid responses to platelet loss. TPO acts by binding Results to a specific cell surface receptor, the cellular homologue of the A targeting vector was constructed for generation, via homolo- myeloproliferative leukemia virus oncogene (Mpl), leading to gous recombination in embryonic stem cells, of a modified Mpl receptor dimerization, activation of intracellular signal trans- duction pathways, and responses of target cells. Mice lacking TPO Significance or Mpl are severely thrombocytopenic and deficient in mega- karyocytes and their progenitor cells, a phenotype consistent with Blood platelets, the small circulating cells that coordinate he- a role for TPO in maintaining appropriate megakaryocyte num- mostasis, are produced by specialized bone marrow cells called bers in vivo. In addition to its role in megakaryopoiesis, TPO is megakaryocytes. The cytokine thrombopoietin (TPO) is a key also an indispensible regulator of hematopoietic stem cells (HSC), regulator of platelet production acting via its specific cell re- essential for maintenance of quiescence and self-renewal (1). ceptor, Mpl. Via genetic modification of the Mpl allele in mice, TPO is produced primarily in the liver (2) and upon binding to we precisely define the bone marrow cells that express Mpl the Mpl receptor on target cells, is internalized and degraded. and, by genetically removing Mpl from megakaryocytes and The prevailing model posits that circulating TPO concentration platelets, we show TPO signaling via Mpl is not required in is inversely proportional to the “Mpl mass” contributed by the megakaryocytes for their expansion, maturation, or platelet total number of megakaryocytes and platelets. In normal indi- production. Rather, Mpl expression on megakaryocytes is es- viduals, this model describes an effective feedback system to sential for regulating TPO availability in the bone marrow mi- regulate TPO-driven megakaryocyte and platelet production croenvironment to prevent myeloproliferation, a model we according to need. The reciprocal relationship between platelet suggest is important for human disease. number and circulating TPO level is clearly evident in bone marrow transplant patients (1), and the key role of the TPO Author contributions: A.P.N., M.K., D.M., C.D.H., E.C.J., M.L., J.-G.Z., T.M.B., L.D.R., D.J.H., − − and W.S.A. designed research, performed research, analyzed data, and wrote the paper. receptor is illustrated by the elevated circulating TPO in Mpl / mice (3) and the modest elevation of platelet counts in trans- The authors declare no conflict of interest. Data deposition: The sequences reported in this paper have been deposited in Array genic mice expressing low levels of Mpl (4, 5). However, the Express database, www.ebi.ac.uk/arrayexpress (accession no. E-MTAB-2389). relationship between circulating TPO concentration and pe- 1To whom correspondence may be addressed. E-mail: [email protected] or metcalf@wehi. ripheral platelet counts is not always conserved in pathological edu.au. – states of thrombocytosis and thrombocytopenia (6 9), suggesting This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. that a simple relationship among megakaryocyte and platelet 1073/pnas.1404354111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1404354111 PNAS Early Edition | 1of6 Downloaded by guest on September 23, 2021 allele (Mpl fl) designed to retain Mpl expression and provide mice to produce progeny in which the intracellular domain of a green fluorescent protein (GFP) reporter for transcriptional Mpl and the IRES-GFP cassette had been specifically deleted in activity of the Mpl locus. The inclusion of loxP sites allowed for megakaryocytes and platelets (MplPF4cre/PF4cre). Western blot cre recombinase-mediated deletion of the GFP reporter as well analysis of extracts from megakaryocytes and platelets demon- as exons 11 and 12, resulting in a null allele (Fig. 1A and SI strated efficient ablation of Mpl expression in MplPF4cre/PF4cre Appendix, SI Materials and Methods). PF4-cre transgenic mice mice (Fig. 1B). This finding was confirmed by analysis of Mpl- (20) have been reported to allow specific and efficient deletion GFP reporter expression: although megakaryocytes generated of conditional alleles in megakaryocytes and platelets. To verify in culture from Mplfl/fl mice expressed GFP, GFP expression was the activity and cell-type specificity of PF4-cre mice, particularly lost in MplPF4cre/PF4cre mice, consistent with recombination and in view of a recent report of activity in HSCs (21), we crossed deletion of the targeted Mplfl intracellular domain and IRES- PF4-cre mice to mice in which enhanced yellow fluorescent protein GFP cassette in these cells (SI Appendix, Fig. S3A and Fig. 1A) (EYFP) is expressed in a cre-dependent manner (Rosa26EYFP; and, consequently, absence of GFP in platelets (SI Appendix, Fig. ref. 22). Whereas, as expected, EYFP was not expressed in the S3B). As anticipated, no activity of the Mpl locus was evident in absence of cre recombinase, in Rosa26EYFPPF4Cre mice, EYFP was lymphocytes, granulocytes, or erythroid cells (SI Appendix, Fig. expressed in megakaryocytes and platelets, consistent with previous S3B). In LSK cells, MplPF4cre/PF4cre mice expressed Mpl at reports (20) and not in erythroid, lymphoid, or myeloid cells equivalent levels to Mplfl/fl controls, both via flow cytometry with (SI Appendix, Fig. S1A). Megakaryocyte formation progresses anti-Mpl antibody and via the Mpl-GFP reporter (Fig. 1 C and D from a series of TPO-responsive bipotential erythroid-megakaryo- and SI Appendix, Fig. S3 C and D). As expected, Mpl expression cyte progenitors (23). EYFP was not expressed in lineage-negative was not detected in pregranulocyte-macrophage (preGM) or + + Sca-1 Kit (LSK) bone marrow cells, the population containing granulocyte-macrophage (GMP) progenitor cells, nor in CFU-E. stem and primitive multipotential progenitor cells, nor in any pro- In Mplfl/fl control megakaryocyte progenitor cells, Mpl expression + genitor cell fraction tested,