Recombinant goldfish thrombopoietin up-regulates expression of genes involved in thrombocyte development and synergizes with kit ligand A to promote progenitor cell proliferation and colony formation

• tpo was highly expressed in heart, followed by spleen, liver, brain, intestine, and kidney tissues.
• Recombinant goldfish TPO up-regulated expression of genes involved in thrombocyte development.
• rgTPO synergized with recombinant goldfish kit ligand A (rgKITLA) to promote progenitor cell proliferation and thrombocytic colony-formation.

Thrombopoietin (TPO) is the principal regulator of thrombopoiesis and promotes the proliferation, differentiation and maturation of megakaryocytic progenitor cells in mammals. In this study we report on the molecular and functional characterization of goldfish TPO. Quantitative expression analysis of goldfish tpo revealed the highest mRNA levels in heart, followed by spleen, liver, brain, intestine and kidney tissues. Significant decrease of tpo and c-mpl expressions in goldfish primary kidney macrophage (PKM) cultures, as progenitor to macrophage development progressed, indicates that TPO is not involved in monopoiesis. Recombinant goldfish TPO (rgTPO) alone did not induce significant proliferation of progenitor cells, but TPO in cooperation with recombinant goldfish kit ligand A (rgKITLA) supported proliferation of progenitor cells in a dose-dependent manner. In response to rgTPO or a combination of rgTPO and rgKITLA, the mRNA levels of thrombopoietic markers cd41 and c-mpl as well as thrombo/erythropoietic transcription factors gata1 and lmo2 in sorted progenitor cells were up-regulated, while the mRNA levels of granulopoietic markers (cebpα and gcsfr) and the lymphoid transcription factor gata3 were down-regulated. Furthermore, rgTPO and rgKITLA synergistically stimulated thrombocytic colony-formation. Our results demonstrate that goldfish TPO has similar functions to mammalian TPO as a regulator of thrombopoiesis, and suggests a highly conserved molecular mechanism of thrombocyte development throughout evolution of vertebrates.