The role of Wnt/β-catenin signaling as a mediator of megakaryopoietic cell fate decision
Hematology, Oncology
Final Report Abstract
The haematopoietic system resides in the bone marrow and gives rise to all blood cells originating from haematopoietic stem cells (HSCs). HSCs undergo continuous restrictions and commitments thereby generating megakaryocytic erythroid progenitors (MEPs), which further differentiate into mature megakaryocytes (MKs) and red blood cells (erythrocytes; RBCs). Although many details on how this cell-fate decision is regulated on the molecular level have been elucidated, some steps and details remain to be clarified which are highly relevant for the understanding and treatment of haematopoietic neoplasms. Leukemia can develop into chronic myelogenous leukemia (CML), which show elevated levels of Wnt/β-catenin signalling. Wnt/β-catenin is an evolutionarily highly conserved signalling pathway for embryonic development but participates also in carcinogenesis in adult organisms. To explore the role of Wnt/β-catenin for MK and RBC differentiation, we dominantly activated β-catenin signaling in platelet derived growth factor b (Pdgfb)-expressing cells of the haematopoietic system using a Cre-lox approach (Ctnnb1BM-GOF), given that Pdgfb is known to be expressed in MKs and platelets in the haematopoietic system. This allowed the activation of β-catenin specifically in the MK lineage. Fluorescence-activated cell sorting (FACS) revealed that mainly megakaryocytic progenitors (MKPs) expressed Pdgfb, and Ctnnb1BM-GOF was associated with a rapid lethal phenotype in heterozygous (Ctnnb1BM-GOFwt/fl) and homozygous (Ctnnb1BM- GOFfl/fl) mutants around the 3rd week after the last tamoxifen injection. Furthermore, the mutants developed splenomegaly and patches of hair-loss, which was characterised as hyperkeratosis. Given that Pdgfb is also highly expressed in the vascular endothelium, we compared the Ctnnb1BM-GOF mice with the Cdh5-(PAC)CreERT2 driver line, allowing for endothelial-specific β-catenin activation (Ctnnb1EC-GOF). Interestingly, Ctnnb1EC-GOF did not show any signs of MKP alteration, suggesting that Wnt/β-catenin in the vascular endothelium is not promoting MK differentiation. Bone marrow (BM) transplantation (BMT) of Ctnnb1BM-GOF BM cells into lethally irradiated wildtype recipients of the Ly5.1 mice (BMT-Ctnnb1BM-GOF) confirmed the megakaryocytic phenotype, but mice did not develop a lethal phenotype and splenomegaly after three weeks of tamoxifen induction as observed in the Ctnnb1 BM-GOF condition. However, FACS analysis and immunofluorescent microscopy revealed an increase in megakaryocytes in the BM and the spleen of Ctnnb1BM-GOF mice. Moreover, Ctnnb1BM-GOF animals developed anaemia. Additionally, a CFU-MK assay in vitro with BM cells of Ctnnb1BM-GOF mice supported an increase in MK at the expense of erythroid colonies. At the molecular level we could show that runt related transcription factor 1 (RUNX1) mRNA, which is known to suppress erythropoiesis, was upregulated in the BM of Ctnnb1BM-GOF animals, whereas friend leukaemia integration 1 (FLI1) was in trend increased. In conclusion, β-catenin activation plays a key role for cell-fate decision favouring MK development at the expense of erythroid production.
