N=6 animals per group. that JAK/STAT pathway activation in megakaryocytes induces myeloproliferation and is necessary for MPN maintenance allele burden, and support the development of MPN therapies aimed at targeting megakaryocytes. in the hematopoietic compartment is sufficient to cause MPN in Telaprevir (VX-950) mouse models (5C8). These observations, along with gene expression studies in primary patient samples (9), establish aberrant JAK/STAT signaling as a central molecular hallmark of MPN pathogenesis. Clonality studies using MPN patient samples have traced driver mutations, including can be detected across the hematopoietic ontogeny (11,12). Telaprevir (VX-950) This implies that can have differential effects on discrete cell types, such as progenitor cells versus mature lineage-committed cells (13). However, most studies performed to date have investigated the effects of when expressed throughout hematopoiesis. We initiated this study to explore the contributions of mutant megakaryocytes to MPN pathogenesis. In many ET/PMF patients, manifests primarily in the megakaryocyte (Mk) lineage, with less significant Telaprevir (VX-950) involvement of erythroid and myeloid cells as determined by mutant allele burden profiling (14) of specific hematopoietic subsets. Furthermore, aberrant megakaryopoiesis is a pathological hallmark of MPN, regardless of clinical subtype (15). These abnormal megakaryocytes are known to secrete increased levels of pro-inflammatory cytokines and other factors (such as TGF-), which presumably contribute to various MPN-related pathologies, such as bone marrow fibrosis (16,17). Megakaryocytes are also regulators of HSC quiescence (18,19), and we previously revealed that (20). These observations raise the possibility that wild type) cells. Indeed, earlier work has revealed that knock-in mouse where mutant is expressed from the endogenous murine locus, with transgenic mice to produce a model wherein is expressed specifically in megakaryocyte lineage-committed cells (13,23). While steady-state megakaryopoiesis Telaprevir (VX-950) was significantly elevated as expected, we unexpectedly observed that knock-in animals (a gift of Dr. Ann Mullally (13)) were bred with transgenic mice (a gift of Dr. Radek Skoda(23)) to induce expression in megakaryocyte lineage-committed cells. (Stock# 008610) and (Stock# 003556) transgenic mice were purchased from Jackson Laboratory (Bar Harbor, ME) and bred with mice to induce pan-hematopoietic expression. For megakaryocyte depletion studies, mice (which harbor a conditional Cre-inducible diphtheria toxin receptor (DTR) (24)) were obtained from Jackson Laboratory (Stock# 007900) and crossed to switch reporter mice (25) were obtained from Jackson Laboratory (Stock# 007676) and bred to mice to confirm lineage-specific Cre expression. Animal studies were approved by the Telaprevir (VX-950) IACUC of both Northwestern University and Memorial Sloan Kettering Cancer Center. Antibodies and reagents Antibodies used to characterize mouse cell surface makers by flow cytometry include: mouse PE/Cy7-CD41 (Cat# 25C0411-80) and APC-Gr1 (Cat# 17C5931-82) were purchased from eBioscience. Mouse PE-CD42b antibody (Cat# M040C3) was purchased from Emfret Analytics (Wurzburg, Germany). Antibodies for mouse APC-Ter119 (Cat# 116211), PE/Cy7-CD71 (Cat# 113811), FITC-Mac1 (Cat# 101205), APC/Cy7-cKit (Cat# 105825), FITC-Sca1 (Cat# 108105), PerCP/Cy5.5-FcR (Cat# 101323), PE-CD150 (Cat# 115903), APC-CD105 (Cat# 120413) and PE-CD34 (Cat# 128609) were purchased from BioLegend (San Diego, CA). PE-Phospho-Stat5 (Cat# 5387) antibody was purchased from Cell Signaling (Danvers, MA). Rabbit polyclonal Von Willebrand Factor (VWF) antibody (Cat# A008229C5) used for immunohistochemistry was purchased from Agilent Technologies (Santa Clara, CA). Recombinant mouse stem cell factor (mSCF, Cat# 250C03), recombinant human EPO (hEPO, Cat# 100C64), recombinant mouse IL-3 (mIL-3, Cat# 213C13), recombinant human TPO (hTPO, Cat# 300C18), recombinant mouse Cxcl1 (mCxcl1, Cat# 250C01), and recombinant mouse Cxcl2 (mCxcl2, Cat# 250C15) were purchased from Peprotech (Rocky Hill, NJ). Flow cytometry Bone marrow SMAD2 and spleen cells were harvested from mice. Surface marker staining for mouse CD41, CD42b, Ter119, CD71, Gr1, and Mac1 was performed by incubating cells in antibodies diluted in PBS + 0.5% BSA for 30 minutes. To characterize the myelo-erythroid stem/progenitor cell compartment, bone marrow cells were stained as follows: cells were first stained using.