Abstract
Abstract 4577
In mammalians, primitive hematopoiesis occurs in yolk sac at early embryonic, and alters to a liver and spleen turning to definitive hematopoiesis at fetal stage. And then, finally, bone marrow becomes a main hematopoietic organ in adult. In contrast, adipocytes and fat fill the bone marrow in African clawed frog Xenopus laevis, limiting the space available for generating red blood cells. This prompted us to look at blood formation in other organs including liver, lung, kidney, and spleen. From previous reports of morphological observations and results of in vitro colony-forming units assay, it is considered that many of erythroid cells exist in the liver, and thrombocytic and leukocytic cells mainly exist in the spleen or the bone marrow. This indicates that blood cells of frogs are produced in different organ attending to blood cell kinds and it is seemed that frogs are favorable to investigate microenvironments proper to each blood cell kinds. Here, we used a progenitor assay based on label retention of thymidine analog 5-bromo-2′-deoxyuridine (BrdU) in frogs with phenylhydrazine (PHZ)-induced acute hemolytic anemia and those with phlebotomy-caused anemia. We gave sub-lethal, intraperitoneal doses of PHZ (25 mg/Kg) to adult frogs and injected BrdU (170 mg/Kg) after seven days to trace proliferating cells in each organ. One week later, we found significant increases in the number of BrdU-positive cells in liver, lung, kidney and spleen compared to controls. To role out the possibility that BrdU was incorporated in non-hematopoietic cells of tissues, especially damaged by PHZ administration, we also examined phlebotomy-caused anemic model. Though the number of BrdU-positive cells are less than PHZ administrated frog, two days of phlebotomy similarly increased the number of BrdU-positive cells in those tissues. These results indicated that, in anemic condition, proliferating cells were resided in liver, lung, spleen and kidney. We also localized slow cycling, immature cells. Twenty-five days after BrdU injection, few labeled cells remained in the tissues, so we re-injected PHZ to induce acute hemolytic anemia again. Eight days later, BrdU-positive cells reappeared but only in liver, suggesting that progenitor cells divided during the second PHZ-induced anemia. In control frogs that did not receive a second PHZ injection, BrdU-positive cells were still present in liver after seventy days. In adult liver, histochemistry revealed BrdU-positive cells with the morphological appearance of hematopoietic cells. Immunostaining with polyclonal anti-Xenopus laevis erythropoietin receptor (xlEPOR) antibody further suggested the presence of erythropoiesis. This observation led us to examine where erythroid progenitor cells locate in the liver and what kinds of cells exist around them to support erythropoiesis. The localization of hepatocytes, kupffer cells and vascular endothelial cells were visualized, by detecting with in situ hybridization for albumin, use of carmine phagocytosis and lectin labeling, respectively. Furthermore transmission electron microscopy confirmed the ultrastructure of these cells. Based on morphological analysis, we showed the localization of erythroid cells in the liver spatially and its time-dependent change through becoming anemia. In the mammalian liver, erythropoiesis occurs during fetal development or as extramedullary hematopoiesis. Our findings may offer a new model for analyzing red blood cell formation in the hepatic microenvironment in response to anemia.
Disclosures:
No relevant conflicts of interest to declare.
Characteristic Distribution of Hematopoietic Cells in Bone Marrow of Xenopus Laevis
