G6b-B Directs Megakaryocyte Transcriptional Program Controlling Differentiation and Bone Marrow Homeostasis

Megakaryocytes (MKs) are large polyploid cells that reside mainly in the bone marrow, where they differentiate from hematopoietic stem cells. Their primary function is to generate new platelets; yet MKs are also important for immunity and maintaining bone marrow homeostasis. Recent findings suggest that transcriptionally distinct MK subtypes are responsible for specific functions. However, the molecular mechanisms of MK subset differentiation are still unclear. While Thrombopoietin (TPO) receptor signaling is well recognized as a critical pathway regulating MK differentiation, the pathways involved in regulating MK maturation are the subject of ongoing research. Various transcription factors, including GATA1, RUNX1, and FLI1, are crucial for MK development, but the exact mechanisms governing the expression and repression of these transcription factors during MK differentiation are not entirely understood.

Another protein indispensable for normal platelet production is G6b-B. This immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptor, uniquely expressed in the MK lineage, is of significant interest because both patients and mice lacking functional G6b-B develop severe macrothrombocytopenia and myelofibrosis.

We previously identified G6b-B as a key player in MK maturation (Becker, Nagy et al. Blood Adv. 2022) and are currently investigating the underlying molecular processes.

To identify the role of G6b-B in regulating the MK transcriptional program, we analyzed native bone marrow cells of Mpig6b^mut mice lacking G6b-B expression and compared them to wild-type mice using magnetic cell separation and single-cell RNA sequencing in combination with flow cytometry, immunoblotting and immunofluorescence microscopy.

Sequencing of about 30,000 cells allowed for high-resolution analysis of the bone marrow environment, especially MKs and their heterogeneity. Our data revealed that the loss of G6b-B leads to aberrant MK subset distribution, including the expansion of hematopoietic stem and progenitor cells (HSPCs), along with a decrease in the relative proportion of platelet-generating mature MKs. Remarkably, gene expression analysis within MK subsets revealed a plethora of differentially expressed genes (DEGs) in Mpig6b^mut cells. In mature MKs, the expression of several well-known transcripts for this lineage, including Vwf, Thbs1, Itga2, and Nbeal2, as well as cytoskeletal regulators such as Flna and Actn1, was reduced, while multiple fibrosis-promoting genes were upregulated. The loss of G6b-B resulted in upregulation of more than 400 transcripts in HSPCs and more than 2000 in MK progenitors. Gene ontology (GO) analysis of these subsets concluded a significant enrichment of GO terms related to regulation of transcription and DNA synthesis, with approximately 10% of the upregulated genes being transcription factors, including Nfkb1, Fli1 and Runx1. Furthermore, GO analysis revealed that a multitude of upregulated genes in mutant HSPCs and MK progenitors are associated with lymphocyte differentiation. Flow cytometry supported these findings, showing a significant increase in CD45+ cells in Mpig6b^mut bone marrow and more than double the number of lymphocytes and granulocytes in peripheral blood.

In conclusion, G6b-B plays a critical role in MK differentiation. Loss of functional G6b-B results in an abnormal distribution of MK subtypes, leukocytosis, and myelofibrosis, as well as an increased expression of transcription factors in MK progenitors. The vast transcriptional differences already observed in Mpig6b^mut HSPCs and MK progenitors indicate an earlier role in differentiation than previously anticipated. Our data supports a novel working hypothesis that G6b-B is essential for suppressing alternative lineage gene expression programs, ensuring proper MK development, platelet production, and bone marrow homeostasis. Understanding this process bears the potential to reveal new molecular targets for modulation of megakaryocyte differentiation in pathologic conditions.

No relevant conflicts of interest to declare.