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The proximal promoter of lck directs gene expression exclusively in T cells. To investigate the developmental regulation of the lck proximal promoter activity and its relationship to T cell lineage commitment, a green fluorescence protein (GFP) transgenic (Tg) mouse in which the GFP expression is under the control of the proximal promoter of lck was created. In the adult GFP-Tg mice, >90% of CD4⁺CD8⁺ and CD4⁺CD8^- thymocytes, and the majority of CD4^-CD8⁺ and CD4^-CD8^- [double-negative (DN)] thymocytes were highly positive for GFP. Slightly lower but substantial levels of expression of GFP was also observed in mature splenic T cells. No GFP⁺ cells was detected in non-T lineage subsets, including mature and immature B cells, CD5⁺ B cells, and NK cells, indicating a preserved tissue specificity of the promoter. The earliest GFP⁺ cells detected were found in the CD44⁺CD25^- DN thymocyte subpopulation. The developmental potential of GFP^- and GFP⁺ cells in the CD44⁺CD25^- DN fraction was examined using in vitro culture systems. The generation of substantial numbers of αβ and γδ T cells as well as NK cells was demonstrated from both GFP^- and GFP⁺ cells. However, no development of B cells or dendritic cells was detected from GFP⁺ CD44⁺CD25^- DN thymocytes. These results suggest that the progenitors expressing lck proximal promoter activity in the CD44⁺CD25^- DN thymocyte subset have lost most of the progenitor potential for the B and dendritic cell lineage. Thus, progression of T cell lineage restriction in the earliest thymic population can be visualized by lck proximal promoter activity, suggesting a potential role of Lck in the T cell lineage commitment.

The proximal promoter of lck directs gene expression exclusively in T cells. To investigate the developmental regulation of the lck proximal promoter activity and its relationship to T cell lineage commitment, a green fluorescence protein (GFP) transgenic (Tg) mouse in which the GFP expression is under the control of the proximal promoter of lck was created. In the adult GFP-Tg mice, >90% of CD4(+)CD8(+) and CD4(+)CD8(-) thymocytes, and the majority of CD4(-)CD8(+) and CD4(-)CD8(-) [double-negative (DN)] thymocytes were highly positive for GFP. Slightly lower but substantial levels of expression of GFP was also observed in mature splenic T cells. No GFP(+) cells was detected in non-T lineage subsets, including mature and immature B cells, CD5(+) B cells, and NK cells, indicating a preserved tissue specificity of the promoter. The earliest GFP(+) cells detected were found in the CD44(+)CD25(-) DN thymocyte subpopulation. The developmental potential of GFP(-) and GFP(+) cells in the CD44(+)CD25(-) DN fraction was examined using in vitro culture systems. The generation of substantial numbers of alphabeta and gammadelta T cells as well as NK cells was demonstrated from both GFP(-) and GFP(+) cells. However, no development of B cells or dendritic cells was detected from GFP(+) CD44(+)CD25(-) DN thymocytes. These results suggest that the progenitors expressing lck proximal promoter activity in the CD44(+)CD25(-) DN thymocyte subset have lost most of the progenitor potential for the B and dendritic cell lineage. Thus, progression of T cell lineage restriction in the earliest thymic population can be visualized by lck proximal promoter activity, suggesting a potential role of Lck in the T cell lineage commitment.

Inactivation of the klotho gene in mice results in multiple disorders that resemble human aging after 3 weeks of age. Because hematopoiesis, especially B lymphopoiesis, is affected in humans and mice by aging, we analyzed the hematopoietic state in homozygous klotho (kl/kl) mice. The kl/kl mice showed thymic atrophy and a reduced number of splenocytes. These mice had almost the normal number of myeloid cells, erythroid cells, IL-3-responsive myeloid precursors and colony forming units in spleen (CFU-S) in bone marrow (BM), but had a substantially decreased number of B cells in BM and peripheral blood as compared with wild-type mice. IL-7-responsive B cell precursors and all of the maturation stages of B cells in BM were also reduced. However, the function of hematopoietic stem cells including their capacity of B lymphopoiesis in vivo and in vitro was normal. Early B cell development was also normal in neonates and young kl/kl mice until 2 weeks old without aging phenotypes. RT-PCR analysis revealed that the level of IL-7 gene expression was significantly reduced in freshly isolated kl/kl BM cells. However, injection of IL-7 in kl/kl mice could not rescue the B lymphopenia. These findings indicate that Klotho protein may regulate B lymphopoiesis via its influence on the hematopoietic microenvironment.

We have studied the contribution of c-Fos/activator protein-1 (AP-1) to antigen-specific T cell response with reference to T cell anergy by increasing c-Fos/AP-1 in vivo and in vitro. First, after injection of a high dose of staphylococcus enterotoxin B (SEB), clonal deletion of SEB-reactive V_β8⁺ CD4 T cells occurred both in control B6 and H2-c-fos transgenic (fos) mice, whereas proliferation of T cells against SEB was profoundly depressed in B6 but not in fos mice. Second, the keyhole limpet hemocyanin-specific CD4 T_h1 cell clone produced decreasing amounts of IL-2 in response to increasing amounts of concanavalin A (Con A) in vitro, whereas the decrease was less significant in the T_h1 clones stably transfected with c-fos gene. Electrophoretic mobility shift assay with nuclear protein from the transformants showed that overexpression of the c-fos gene compensated the amounts of AP-1 in the nuclei of Con A-treated T_h1 clones. Thus, increased c-Fos/AP-1 confers resistance against anergy induction on antigen-specific T cells.

The murine Ncx (Enx, Hox11L1) gene is specifically expressed in a neuronal subset of neural crest-derived tissues. In attempts to elucidate the regulatory DNA element of the tissue-specific expression, we sequenced the 5'-flanking region of the Ncx gene. The transcriptional initiation site was determined at 297 nucleotides (-297) upstream from the ATG start codon (+1). A retinoic acid response element was located on the region between -1163 and -1150. Transient transfection assays with the 5'-flanking sequences fused to the luciferase gene showed that the region between -1387 and -1368 was crucial for the tissue-specific enhancer activity. Furthermore, nuclear proteins extracted from neural crest-derived cells such as murine and human neuroblastoma cells bind to the DNA region between -1387 and -1368. This DNA element was also conserved in the 5'-flanking region of the human NCX gene. Our observations strongly suggest that the DNA element (between -1387 and -1368) contributes to tissue-specific expression of the Ncx gene in murine and human species.