In steady-state hematopoiesis, G-CSF (granulocyte-colony stimulating factor) regulates the level of neutrophils in the bone marrow and blood. In this study, we have exploited the availability of G-CSFdeficient mice to evaluate the role of G-CSF in steady-state granulopoiesis and the release of granulocytes from marrow into circulation. The thymidine analogue bromodeoxyuridine (BrdU) was used to label dividing bone marrow cells, allowing us to follow the release of granulocytes into circulation. Interestingly, the labeling index and the amount of BrdU incorporated by blast cells in bone marrow was greater in G-CSF-deficient mice than in wild-type mice. In blood, 2 different populations of BrdU-positive granulocytes, BrdU bright and BrdU dim , could be detected. The kinetics of release of the BrdU bright granulocytes from bone marrow into blood was similar in wild-type and G-CSF-deficient mice; however, BrdU dim granulocytes peaked earlier in G-CSF-deficient mice. Our findings suggest that the mean transit time of granulocytes through the postmitotic pool is similar in G-CSF-deficient and control mice, although the transit time through the mitotic pool is reduced in G-CSF-deficient mice. Moreover, the reduced numbers of granulocytes that characterize G-CSF-deficient mice is primarily due to increased apoptosis in cells within the granulocytic lineage. Collectively, our data suggest that at steady state, G-CSF is critical for the survival of granulocytic cells; however, it is dispensable for trafficking of granulocytes from bone marrow into circulation. (Blood. 2002;100:854-861)

Sex in birds is chomosomally based (ZZ male, ZW female), but the mechanism underlying sex determination remains unknown. An unresolved question is whether Z gene dosage plays a role in avian sex determination. DMRT1 is an avian Z-linked gene that shows higher expression in male gonads during embryogenesis and has been proposed as a putative testis-determining gene in birds. The Z-linkage of this gene makes it an ideal candidate for testing the question of gene dosage in avian testis determination. A higher level of DMRT1 expression in male (ZZ) versus female (ZW) embryonic gonads may reflect the presence of two Z-linked copies in the male, or it may be due to specific and active upregulation of DMRT1 during testis formation. A functional interventionist strategy was used to distinguish between these two possibilities. DMRT1 expression was analyzed in chicken embryos during experimentally induced female-to-male sex reversal, using the aromatase enzyme inhibitor fadrozole. DMRT1 expression was analyzed by whole mount in situ hybridization and reverse transcription polymerase chain reaction (for mRNA) and indirect immunofluorescence (for protein). Female-to-male sex-reversed embryos (genetically ZW) showed elevated levels of DMRT1 expression similar to those of normal males (with two copies of the Z chromosome). Elevated levels of DMRT1 are therefore associated with testis development, both in normal males (ZZ) and in sex-reversed females (ZW). SOX9 expression was also activated during female-to-male sex reversal but appeared delayed relative to DMRT1 upregulation. These results show that testis development does not require two Z-linked copies of DMRT1, but it does involve active upregulation of the gene. Higher levels of DMRT1 expression during testis differentiation therefore do not simply reflect a gene dosage difference between the two sexes but imply active involvement in male development.

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