Supplementary Material for: Colony-Stimulating Factor-1-Responsive Macrophage Precursors Reside in the Amphibian (Xenopus laevis) Bone Marrow rather than the Hematopoietic Subcapsular Liver

Macrophage precursors originate from and undergo lineage commitment within designated sites of hematopoiesis, such as the mammalian bone marrow. These cells subsequently differentiate in response to stimulation with macrophage colony-stimulating factor-1 (CSF-1). The amphibian bone marrow, unlike that of mammals, has been overlooked as a source of leukocyte precursors in favor of the liver subcapsular region, where hematopoiesis occurs in anurans. Here we report that the bone marrow rather than the liver periphery provides macrophage progenitors to the amphibian Xenopus laevis. We identified the amphibian CSF-1, examined its gene expression in developing and virally infected X. lae vis and produced it in recombinant form (rXlCSF-1). This rXlCSF-1 did not bind or elicit proliferation/differentiation of subcapsular liver cells. Surprisingly, a subpopulation of bone marrow cells engaged this growth factor and formed rXlCSF-1 concentration-dependent colonies in semisolid medium. Furthermore, rXlCSF-1-treated bone marrow (but not liver) cultures comprised of cells with characteristic macrophage morphology and high gene expression of the macrophage marker CSF-1 receptor. Together, our findings indicate that in contrast to all other vertebrates studied to date, committed Xenopus macrophage precursor populations are not present at the central site of hematopoiesis, but reside in the bone marrow.