Supplementary Material for: Colony-Stimulating Factor-1-Responsive Macrophage Precursors Reside in the Amphibian <b><i>(Xenopus laevis)</i></b> 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 <i>Xenopus laevis</i>. We identified the amphibian CSF-1, examined its gene expression in developing and virally infected <i>X. lae</i><i> vis</i> and produced it in recombinant form (r<i>Xl</i>CSF-1). This r<i>Xl</i>CSF-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 r<i>Xl</i>CSF-1 concentration-dependent colonies in semisolid medium. Furthermore, r<i>Xl</i>CSF-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 <i>Xenopus </i>macrophage precursor populations are not present at the central site of hematopoiesis, but reside in the bone marrow.巨噬细胞前体起源并完成谱系定型于特定的造血位点，例如哺乳动物的骨髓。此类细胞后续会在巨噬细胞集落刺激因子-1（macrophage colony-stimulating factor-1, CSF-1）的刺激下发生分化。与哺乳动物骨髓不同，两栖动物骨髓作为白细胞前体来源的地位长期被忽视，学界此前更关注无尾目动物造血发生的肝脏被膜下区域。本研究证实，在两栖动物非洲爪蟾（Xenopus laevis）中，巨噬细胞祖细胞的来源是骨髓而非肝脏外周区域。我们鉴定了两栖动物的CSF-1，检测了其在发育中以及病毒感染的非洲爪蟾体内的基因表达，并制备了其重组形式（rXlCSF-1）。该重组蛋白rXlCSF-1无法与肝脏被膜下细胞结合，也不能诱导其增殖或分化。令人意外的是，骨髓细胞的一个亚群可结合该生长因子，并在半固体培养基中形成依赖于rXlCSF-1浓度的细胞集落。此外，经rXlCSF-1处理的骨髓（而非肝脏）培养物中，存在具有典型巨噬细胞形态的细胞，且高表达巨噬细胞标志物CSF-1受体的基因。综上，本研究结果表明：与迄今已研究的所有其他脊椎动物不同，定型的爪蟾巨噬细胞前体群体并不存在于主要造血中心，而是定居于骨髓中。