A Platelet Shedding Defect in Mice Lacking the Basic Helix-Loop-Helix Transcription Factor Scl.

Abstract

Transcriptional regulation of megakaryocyte maturation including platelet shedding is poorly characterised. One potential regulator is Scl, a basic helix-loop-helix transcription factor essential for hematopoietic specification in embryonic development. Scl is expressed in megakaryocytes and erythroid cells, forming DNA binding complexes with other transcription factors including GATA-1. To overcome embryonic lethality of Scl knockout mice, we have generated conditional Scl knockout mice to allow deletion of Scl in cell lineages including megakaryocytes. Scl-null adult mice have mild thrombocytopenia (730±90x109/L compared with 1400±220x109/L in control mice) despite normal numbers of megakaryocytes. Megakaryocyte ploidy was increased in the absence of Scl (modal ploidy 32n compared with 16n in control mice) indicating that Scl is not required for megakaryocyte development and endomitosis. Platelet survival studies demonstrated a normal platelet half-life of Scl-null platelets, suggesting that thrombocytopenia was due to impaired platelet production. This defect was more pronounced in the setting of pharmacologic administration of thrombopoietin (Tpo) or 5-fluorouracil, with the platelet count in Scl-null mice remaining static despite appropriate increases in megakaryocyte number and ploidy. Ultrastructural studies of Scl-null megakaryocytes were performed to examine the function of Scl in later stages of megakaryocyte maturation. Transmission electron microscopy demonstrated markedly disordered platelet demarcation membranes and a lack of platelet granules, defects reminiscent of GATA-1-null megakaryocytes. Gene expression analyses of FACS-sorted Scl-null megakaryocytes demonstrated a 5-fold reduction in β-1 tubulin, a proposed target gene of GATA-1 and NF-E2 in megakaryocytes. Consistent with the role of this gene in maintaining platelet discoid shape, Scl-null platelets were abnormally round. The similarity of defects of GATA-1- and Scl-null mice implies that these genes act together to regulate platelet shedding and structure.