Cross-Talk between c-Myb and GATA-1 Regulates the Entry into Megakaryocyte Differentiation.

Abstract

Megakaryocytic differentiation and platelet formation are regulated by the combined function of multiple transcription factors. Previously, we have shown that knockdown (KD) mice expressing low levels of c-Myb have an increased number of bone marrow megakaryocytes and a corresponding thrombocytosis. In contrast, mice engineered to express low levels of GATA-1 in the megakaryocytic lineage exhibit aberrant megakaryocytopoiesis with hyperproliferation of progenitors and defective terminal differentiation leading to thrombocytopenia. Here, making use of mice expressing low levels of both c-Myb and GATA-1 (referred to as “double-KD”) and comparison with the single-KD counterparts and wild type animals, we describe how c-Myb and GATA-1 act together to control megakaryocytopoiesis. Few double-KD mice are born live, most likely as a result of hypocellularity in the hematopoietic organs, and platelet numbers are low, indicating that they are not capable of overcoming the thrombocytopenia characteristic of the GATA-1 KD mice. Proliferation assays performed using E14 fetal liver cells, growing in liquid cultures containing thrombopoietin to induce megakaryocytic differentiation, revealed that double-KD cells lacked the hyperproliferative capacity of GATA-1 KD cells. A similar conclusion was reached using colony assays in semi-solid media capable of supporting multilineage differentiation. The colony assays also revealed that double-KD hematopoietic progenitors could only give rise to megakaryocyte and erythroid lineage cells, unlike the single-KD fetal liver cells, which additionally yielded both macrophage and neutrophil-containing colonies. The reduced hyperproliferative capacity of megakaryocytic progenitors in the double-KD compared to the GATA-1 KD suggests that c-Myb and GATA-1 have opposing effects on megakaryocytic progenitors, the reduced level of c-Myb overcoming the block to differentiation imposed by low expression of GATA-1. However, the persistent thrombocytopenia observed in double-KD mice suggests that the low level of GATA-1 prevents terminal differentiation even when commitment has been enhanced by the reduction in c-Myb. To better define the consequences of this functional interaction between c-Myb and GATA-1, we compared the phenotype of the megakaryocytic cells deriving from single- and double-KD progenitors. We observed that the c-Kit+CD41+ megakaryocytic progenitor population present in GATA-1 KD fetal liver is replaced in the double-KD by a more differentiated c-Kit-CD41+ population, similar to that seen in wild type and c-Myb-KD fetal liver. However, the levels of CD41 on double-KD cells are low, suggesting that megakaryocytic terminal differentiation is affected. This was confirmed by staining for acetylcholinesterase, which revealed an abundance of morphologically aberrant megakaryocytes that are incapable of forming proplatelets, as has been described for the GATA-1 KD megakaryocytes. Based on these observations, we conclude that c-Myb and GATA-1 act in concert to achieve correct megakaryocytic differentiation. GATA-1 regulates both the proliferation of megakaryocytic progenitors and their terminal differentiation towards platelet-producing megakaryocytes. c-Myb also acts at the level of the progenitor by influencing its commitment to differentiation, but in contrast to GATA-1 it does not have any effect on the process of terminal differentiation.