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The gamma common (gamma c) chain is a partner in several interleukin receptor complexes, including the interleukin-2 receptor (IL-2R), IL-4R, and IL-7R. Mutations in the gamma c gene are associated with X-linked severe combined immunodeficiency (SCID). Using reverse transcriptase-PCR, we examined the level of mRNA-encoding gamma c and its partners in mouse pluripotent hematopoietic stem cells (PHSCs), which repopulate both bone marrow and thymus. We also assayed developing lymphocytes to define which, if any, IL-R complexes are expressed at the earliest stage of T and B lymphocyte maturation. RNA extracted from bone marrow-derived PHSCs did not contain detectable levels of mRNA-encoding IL-7R alpha. However, the most primitive (CD4- CD8-) T cells from the thymus and the most primitive (c-kit+ B220+) B cells from bone marrow contained high levels of IL-7R alpha mRNA. There were no detectable differences between PHSCs and primitive or more mature T and B cells for expression of gamma c mRNA. We conclude that the onset of IL-7R formation occurs at the earliest stage of differentiation of T and B lymphocytes. Our findings are consistent with the hypothesis that the absence of an intact IL-7R (IL-7R alpha and gamma c) may be a critical loss that interrupts lymphopoiesis.

Dtk (Tyro 3/Sky/Rse/Brt/Tif) belongs to a recently recognized subfamily of receptor tyrosine kinases that also includes Ufo (Axl/Ark) and Mer (Eyk). Ligands for Dtk and Ufo have been identified as protein S and the related molecule Gas6, respectively. This study examined expression of Dtk during ontogeny of the hematopoietic system and compared the pattern of expression with that of Ufo. Both receptors were abundantly expressed in differentiating embryonic stem cells, yolk sac blood islands, para-aortic splanchnopleural mesoderm, fractionated AA4+ fetal liver cells, and fetal thymus from day 14 until birth. Although Ufo was expressed at moderate levels in adult bone marrow, expression of Dtk in this tissue was barely detectable. In adult bone marrow subpopulations fractionated using counterflow centrifugal elutriation, immunomagnetic bead selection for lineage-depletion and FACS sorting for c-kit expression, very low levels of Dtk and/or Ufo were detected in some cell fractions. These results suggest that Dtk and Ufo are likely to be involved in the regulation of hematopoiesis, particularly during the embryonic stages of blood cell development.

We have studied several features of pluripotent hematopoietic stem cells (PHSC) and day-12 spleen colony-forming units (CFU-S12) in murine bone marrow. C57BL/6J marrow cell suspensions were separated by elutriation and fractions were obtained at flow rates (FR) of 25 ml/min, 29/30 ml/min, 35 ml/min, and with the rotor off. All four fractions contained PHSC that could repopulate W/Wv mice, but significant numbers of CFU-S12 were found only in the three higher FR fractions. Cells in the FR29/30 fraction were shown to have almost three-fold more repopulating activity than fresh marrow in a competitive repopulation assay. The PHSC in fractions separated by elutriation were enriched by depleting cells expressing specific lineage markers with monoclonal antibodies and magnetic immunobeads. As few as 10(4) lineage negative (lin-) cells from FR35 or 10(5) lin--cells from FR25 conferred long-term multilineage repopulation in W/Wv mice, as demonstrated by Southern blot analysis of DNA from recipient thymus and bone marrow. We conclude that PHSC are heterogeneous for cell size and density and that the highest concentration of PHSC resides in the subset of intermediate density present in the FR29/30 fraction.

The rate of CFUE production in adult rats varies considerably according to whether the animals are rendered anemic by short or long term treatment with phenylhydrazine. The bone marrow responds during 2 months of phenylhydrazine injections with a progressive increase in CFUE, the spleen shows a rapid and larger rise after 3 daily injections followed by a decline to near zero, control numbers, after prolonged administration of the drug, and the liver never develops erythropoietic activity. Endogenous colonies, that is, colonies that grow in plasma clots without added erythropoietin, are most numerous in bone marrow after phenylhydrazine treatment, are always few in the spleen and are never present in cultures of liver cells. In both marrow and spleen, there is a direct correlation between the rate of erythropoiesis and 2'5'-A polymerase activity. These findings suggest that production of the enzyme may be an early event in erythroid cell differentiation.

Several 2'-5' oligoadenylate (2-5A) core molecules were tested for their inhibitory effect on erythroid colony-forming units (CFU-E). These compounds were introduced into the CFU-E microenvironment by vascular perfusion of isolated rat hind limbs. The marrow microvasculature did not appear to be damaged by these procedures when examined by electron microscopy. After a brief in situ exposure to 2-5A core, the marrow cells were grown in methylcellulose medium with added erythropoietin (Ep). Maximum percent inhibition of CFU-E was obtained with 2-5A trimer core. The tetramer core was less effective, and no inhibition of colony formation was seen in cultures of marrow perfused with 2-5A dimer core. From this it was concluded that the inhibitory effect of 2-5A trimer core was highly specific. Furthermore, this effect was of long duration, since when 2-5A trimer core was given just prior to Ep the CFU-E were blocked in their differentiative response to Ep for 48-72 h.

In response to prolonged, intermittent exposure to hypoxia, the spleens of adult BALB/c mice displayed an initial increase and subsequent decrease in erythropoietic activity. The enzyme 2,5-adenylate synthetase was assayed during this period, and a direct relationship was found between the rate of red cell production and enzyme activity; that is, 2,5-adenylate synthetase activity was maximum in the spleen cell populations that contained the largest number of nucleated erythroid cells and minimum in those populations that contained the fewest nucleated erythroid cells. In contrast to this finding, synthetase activity was inversely related to the number of lymphocytes present in these spleen cell populations. On the basis of these observations, it appears that 2,5-adenylate synthetase is present in nucleated erythroid cells. If active in late erythroblasts, 2,5-adenylate synthetase may function as an inhibitor of DNA synthesis and/or hemoglobin synthesis.

Fetal erythropoiesis was studied in human livers at 10 to 12 weeks of gestation. The most primitive blood cells were often observed in large indentations of the surface of hepatocytes and the plasma membranes of the two cell types were adherent at sites of attachment. Erythroid cell maturation occurred predominantly in the lumen of the sinusoids. Cell suspensions obtained from fetal livers were centrifuged, frozen at -196 degrees C, thawed and studied by electron microscopy. The primitive cells were morphologically altered by these procedures. Changes included damage to mitochondria and cell membranes and vacuole formation. Erythroblasts, by comparison, were virtually intact and even displayed some indications of reestablished functions within 10 minutes after thawing.