IHEM   20887
INSTITUTO DE HISTOLOGIA Y EMBRIOLOGIA DE MENDOZA DR. MARIO H. BURGOS
Unidad Ejecutora - UE
artículos
Título:
Both quiescent and proliferating cells circulate in the blood of the invasive apple snail Pomacea canaliculata
Autor/es:
CONGET, PAULETTE; RODRÍGUEZ, CRISTIAN; VEGA, ISRAEL A; SIMON, VALESKA
Revista:
Fish and Shellfish Immunology
Editorial:
Academic Press Inc.
Referencias:
Año: 2020 vol. 107 p. 95 - 103
ISSN:
1095-9947
Resumen:
Gastropod hematopoiesis occurs at specialized tissues in some species, but the evidence also suggests that hemocytegeneration is maybe widespread in the connective tissues or the blood system in others. In Ampullariidae(Caenogastropoda), both the kidney and the lung contain putative hematopoietic cells, which react to immunechallenges. In the current study, we wanted to explore if hematopoiesis occurs in the blood of Pomacea canaliculata.Thus, we obtained circulating hemocytes from donor animals and tested their ability to proliferate in theblood of conspecific recipients. We tracked cell proliferation by labeling the donors´ hemocytes with the fluorescentcell proliferation marker carboxyfluorescein diacetate succinimidyl ester (CFSE). Transferred CFSE-labeledhemocytes survived and proliferated into the recipients? circulation for at least 17 days. We also determined thecell cycle status of circulating hemocytes by using the propidium iodide (PI) and acridine orange (AO) stainingmethods. Flow cytometry analyses showed that most PI-stained hemocytes were in the G1 phase (~96%), whilea lower proportion of cells were through the G2/S-M transition (~4%). When we instead used AO-staining, wefurther distinguished a subpopulation of cells (~5%) of low size, complexity-granularity, and RNA content. Weregarded this subpopulation as quiescent cells. In separate experimental sets, we complemented these findings byassessing in circulating hemocytes two evolutionary conserved features of quiescent, undifferentiated cells. First,we used JC-1 staining to determine the mitochondrial membrane potential (Ψm) of circulating hemocytes, whichis expected to be low in quiescent cells. Most hemocytes (~87%) showed high aggregation of JC-1, which indicatesa high Ψm. Besides that, a small hemocyte subpopulation (~11%) showed low aggregation of the dye, thusindicating a low Ψm. It is known that the transition from a quiescent to a proliferating state associates with anincrease of the Ψm. The specificity of these changes was here controlled by membrane depolarization with the Ψmdisruptor CCCP. Second, we stained hemocytes with Hoechst33342 dye to determine the efflux activity of ABCtransporters, which participate in the multixenobiotic resistance system characteristic of undifferentiated cells.Most hemocytes (>99%) showed a low dye-efflux activity, but a small proportion of cells (0.06?0.12%) showeda high dye-efflux activity, which was significantly inhibited by 100 and 500 μM verapamil, and thus is indicativeof an undifferentiated subpopulation of circulating hemocytes. Taken together, our results suggest that, amongcirculating hemocytes, there are cells with the ability to proliferate or to stay in a quiescent state and behave asprogenitor cells later, either in the circulation or the hematopoietic tissues/organs.