Developmentally regulated expression of a chicken muscle-specific gene in stably transfected rat myogenic cells.

To test the evolutionary conservation of DNA sequences specifying the developmentally regulated expression of the skeletal muscle actin gene, a recombinant plasmid containing the chicken skeletal muscle actin gene was introduced into rat myogenic cells. In a significant number of isolated clones, the accumulation of chicken actin mRNA increased greatly during differentiation. To test the expression in myogenic cells of a gene that is normally expressed during terminal differentiation of another tissue, rat myogenic cells were transfected with a mouse/human beta-globin chimeric gene. A decrease by a factor of 2-3 in the amount of globin mRNA during differentiation was observed in most clones in which the gene was expressed. The results indicate the conservation of the muscle-specific regulatory DNA sequences for more than 300 Myr.     

Tumor suppressor gene Rb is required for self-renewal of spermatogonial stem cells in mice

The retinoblastoma tumor suppressor gene Rb is essential for maintaining the quiescence and for regulating the differentiation of somatic stem cells. Inactivation of Rb in somatic stem cells typically leads to their overexpansion, often followed by increased apoptosis, defective terminal differentiation, and tumor formation. However, Rb’s roles in germ-line stem cells have not been explored. We conditionally disrupted the Rb gene in mouse germ cells in vivo and discovered unanticipated consequences for GFRa1-protein-expressing A_single (GFRa1⁺ A_s) spermatogonia, the major source of male germ-line stem cells. Rb-deficient GFRa1⁺ A_s spermatogonia were present at normal density in testes 5 d after birth, but they lacked the capacity for self-renewal, resulting in germ cell depletion by 2 mo of age. Rb deficiency did not affect the proliferative activity of GFRa1⁺ A_s spermatogonia, but their progeny were exclusively transit-amplifying progenitor spermatogonia and did not include GFRa1⁺ A_s spermatogonia. In addition, Rb deficiency caused prolonged proliferation of progenitor spermatogonia, transiently enlarging this population. Despite these defects, Rb deficiency did not block terminal differentiation into functional sperm; offspring were readily obtained from young males whose germ cell pool was not yet depleted. We conclude that Rb is required for self-renewal of germ-line stem cells, but contrary to its critical roles in somatic stem cells, it is dispensable for their proliferative activity and terminal differentiation. Thus, this study identifies an unexpected function for Rb in maintaining the stem cell pool in the male germ line.     

Activation of the Jak3 pathway is associated with granulocytic differentiation of myeloid precursor cells

Jak3, a member of the Janus kinase family of cytoplasmic tyrosine kinases, is expressed at low levels in immature hematopoietic cells and its expression is dramatically up-regulated during the terminal differentiation of these cells. To better understand the role of Jak3 in myeloid cell development, we have investigated the role of Jak3 in myeloid cell differentiation using the 32Dcl3 cell system. Our studies show that Jak3 is a primary response gene for granulocyte colony-stimulating factor (G-CSF) and the accumulation of tyrosine phosphorylated Jak3 correlated with cell growth inhibition and terminal granulocytic differentiation in response to G-CSF. Ectopic overexpression of Jak3 in 32Dcl3 cells resulted in an acceleration of the G-CSF-induced differentiation program that was preceded by G(1) cell cycle arrest, which was associated with the up-regulation of the cyclin-dependent kinase inhibitor p27(Kip1) and down-regulation of Cdk2, Cdk4, Cdk6, and Cyclin E. In addition, ectopic overexpression of Jak3 appears to result in the inactivation of PKB/Akt and Stat3-mediated proliferative pathways in the presence of G-CSF. Similarly, overexpression of Jak3 in primary bone marrow cells resulted in an acceleration of granulocytic differentiation in the presence of granulocyte-macrophage colony-stimulating factor, which was associated with their growth arrest in the G(1) phase of the cell cycle. Taken together, these results indicate that Jak3-mediated signals play an important role in myeloid cell differentiation.     

Accumulation of a differentiation regulator specifies transit amplifying division number in an adult stem cell lineage

A key feature of many adult stem cell lineages is that stem cell daughters destined for differentiation undergo several transit amplifying (TA) divisions before initiating terminal differentiation, allowing few and infrequently dividing stem cells to produce many differentiated progeny. Although the number of progenitor divisions profoundly affects tissue (re)generation, and failure to control these divisions may contribute to cancer, the mechanisms that limit TA proliferation are not well understood. Here, we use a model stem cell lineage, the Drosophila male germ line, to investigate the mechanism that counts the number of TA divisions. The Drosophila Bag of Marbles (Bam) protein is required for male germ cells to cease spermatogonial TA divisions and initiate spermatocyte differentiation [McKearin DM, et al. (1990) Genes Dev 4:2242–2251]. Contrary to models involving dilution of a differentiation repressor, our results suggest that the switch from proliferation to terminal differentiation is triggered by accumulation of Bam protein to a critical threshold in TA cells and that the number of TA divisions is set by the timing of Bam accumulation with respect to the rate of cell cycle progression.     

胚胎干细胞标志物Oct-4在胰腺癌的表达

目的分析胚胎干细胞标志物Oct-4在胰腺癌组织和胰腺癌细胞株中的表达状况。方法应用RT-PCR检测Oct-4mRNA在25例胰腺癌组织以及胰腺癌细胞株SW1990、PC3、JF305和BxPC3中的表达;应用Westernblot法检测胰腺癌组织及胰腺癌细胞株中Oct-4蛋白表达;应用异硫氰荧光素(FITC)标记的流式细胞仪分选法检测Oct-4在胰腺癌细胞株中的表达率。结果4株胰腺癌细胞株均表达Oct-4mRNA和Oct-4蛋白。88%(22/25)胰腺癌组织高表达Oct-4mRNA和Oct-4蛋白,其表达率与胰腺癌的分化程度无关。Oct-4在SW1990的表达率为50.21%、BxPC3为68.53%、PC3为66.27%、JF305为67.61%。结论Oct-4异常表达可能与胰腺癌始发有关,胰腺癌可能来源于胰腺干细胞。     

C/EBPalpha bypasses granulocyte colony-stimulating factor signals to rapidly induce PU.1 gene expression, stimulate granulocytic differentiation, and limit proliferation in 32D cl3 myeloblasts.

Within hematopoiesis, C/EBPalpha is expressed only in myeloid cells, and PU.1 is expressed mainly in myeloid and B-lymphoid cells. C/EBPalpha-deficient mice lack the neutrophil lineage and retain monocytes, whereas PU.1-deficient mice lack monocytes and have severely reduced neutrophils. We expressed a C/EBPalpha-estrogen receptor ligand-binding domain fusion protein, C/EBPalphaWT-ER, in 32D cl3 myeloblasts. 32D cl3 cells proliferate in interleukin-3 (IL-3) and differentiate to neutrophils in granulocyte colony-stimulating factor (G-CSF). In the presence of estradiol, C/EBPalphaWT-ER induced morphologic differentiation and the expression of the myeloperoxidase, lactoferrin, and G-CSF receptor mRNAs. C/EBPalphaWT-ER also induced a G1/S cell cycle block, with induction of p27 and Rb hypophosphorylation. bcr-ablp210 prevented 32D cl3 cell differentiation. Activation of C/EBPalpha-ER in 32D-bcr-ablp210 or Ba/F3 B-lymphoid cells induced cell cycle arrest independent of terminal differentiation. C/EBPalphaWT-ER induced endogenous PU.1 mRNA within 8 hours in both 32D cl3 and Ba/F3 cells, even in the presence of cycloheximide, indicating that C/EBPalpha directly activates the PU.1 gene. However, activation of a PU.1-ER fusion protein in 32D cl3 cells induced myeloperoxidase (MPO) RNA but not terminal differentiation. Thus, C/EBPalpha acts downstream of G-CSF and upstream of PU.1, p27, and potentially other factors to induce myeloblasts to undergo granulocytic differentiation and cell cycle arrest.     

The role of the testicular factor INSL3 in establishing the gonadal position

INSL3, also designated Leydig insulin-like (Ley I-L) or relaxin-like factor (RLF), belongs to the insulin-like hormone superfamily. It is expressed in pre- and postnatal Leydig cells of the testis and in postnatal theca cells of the ovary. This sexual dimorphic pattern of INSL3 expression during development led us to suggest that the INSL3 factor could play an essential role in sexual differentiation, gonadal function and germ cell development. Key insights into the role of INSL3 came from analyses of INSL3 knockout mice. These mice showed impaired development of the gubernaculum ligament, a structure that is believed to mediate transabdominal descent of the testis during male embryogenesis. In double mutant XY-mice lacking INSL3 and a functional androgen receptor, it was demonstrated that both are essential for establishment of the sexual dimorphic position of the gonads through regulation of gubernaculum development and regression of the cranial suspensory ligament (CSL) during fetal life. Defects in this developmental process can cause cryptorchidism in the male, which is a most common disorder of sexual differentiation in human.