Immature erythroid cells with novel morphology and cytoskeletal organization in adultXenopus

Abstract

Nucleated erythrocytes of non-mammalian vertebrates are a useful model system for studying the correlation between changes in cell shape and cytoskeletal organization during cellular morphogenesis. They are believed to transform from spheres to flattened discs to ellipsoids. Our previous work on developing erythroblasts suggested that pointed cells containing incomplete, pointed marginal bands (MBs) of microtubules might be intermediate stages in the larval axolotl. To test whether the occurrence of such pointed cells was characteristic of amphibian erythrogenesis, we have utilized phenylhydrazine (PHZ)-induced anemia in adultXenopus. In this system, circulating erythrocytes are destroyed and replaced by erythroblasts that differentiate in the blood, making them experimentally accessible. Thus, we followed the time-course of morphological and cytoskeletal changes in the new erythroid population during recovery. During days ∼ 7–9 post-PHZ, pointed cells did indeed begin to appear, as did spherical and discoidal cells. The percentage of pointed cells peaked at days ∼ 11–13 in different animals, subsequently declining as the percentage of elliptical cells increased. Since degenerating “old” erythrocytes were still present when pointed cells appeared, we tested directly whether pointed ones were “old” or “new” cells. Blood was removed via the dorsal tarsus vein, and the erythrocytes washed, fluorescently tagged, and re-injected. In different animals, ∼ 2–8% of circulating erythrocytes were labeled. Subsequent to induction of anemia in these frogs, time-course sampling showed that no pointed cells were labeled, identifying them as “new” cells. Use of propidium iodide revealed large nuclei and cytoplasmic staining indicative of immaturity, and video-enhanced phase contrast and anti-tubulin immunofluorescence showed that the pointed cells contained pointed MBs. The results show that pointed cells, containing incomplete, pointed MBs are a consistent feature of amphibian erythrogenesis. These cells may represent intermediate stages in the formation of elliptical erythrocytes.