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Apoptosis and hematopoiesis in murine fetal liver
H Yu, B Bauer, GK Lipke, RL Phillips and G Van Zant
Department of Cell Biology and Anatomy, Texas Tech University Health
Sciences Center, Lubbock 79430.
The fetal mouse liver (FL) is an organ of intense, but transient,
hematopoietic activity during mid-gestation, with erythropoiesis being
predominant during days 11 through 16. It therefore seemed reasonable to
expect that hematopoietic cytokines, such as erythropoietin (epo),
interleukin-3 (IL-3), and stem cell factor (SCF), may play important roles
in maintaining a homeostatic balance of erythropoiesis and apoptosis in
liver during ontogeny. First, we determined the effects of these growth
factors on hematopoiesis by measuring colony formation and hemoglobin
synthesis of cultured FLs. Secondly, we determined the protection from
apoptosis afforded by these cytokines, using electrophoretic analysis of
DNA and by flow cytometry of FL cells deprived in culture of epo, IL-3, and
SCF. Erythropoietin was necessary and alone sufficient for hemoglobin
synthesis in colony-forming units- erythroid colonies, but IL-3 was a
required cofactor to obtain maximal development of burst-forming
units-erythroid colonies. SCF alone caused little colony formation in
methylcellulose cultures of FLs, but when combined with epo and IL-3, it
had dramatic effects both on the number of colonies and their size.
Secondly, indices of apoptosis were determined by measuring DNA
fragmentation caused by endogenous nuclease activity in apoptotic cells.
Liver cells from cultures without cytokines showed the extensive
degradation of DNA to low molecular weight nucleosomal oligomers, which is
characteristic of apoptosis. Protection from apoptosis afforded by epo
directly corresponded to the level of erythropoiesis in FLs of different
gestational age. Erythropoietin was by far the most critical cytokine in
sparing FL cells from apoptosis. Analyses of agarose gels showed that SCF
and IL-3 alone had no apparent effect in reducing the amount of DNA in
fragments, and when combined with epo they had no more protective effect
than that provided by epo alone. However, using the more sensitive flow
cytometric determination of cells with subdiploid amounts of DNA, SCF, and
IL-3 alone had measurable protective effects that were less than those
caused by epo. Thus, we show that normal, untransformed cells of the
developing hematopoietic system not only require cytokines for
proliferation and differentiation, but they have an initial and absolute
requirement of them for protection from apoptosis.
Volume 81,
Issue 2,
pp. 373-384,
01/15/1993
Copyright © 1993 by The American Society of Hematology
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