Non-coding RNAs have emerged as crucial regulators of gene expression and cell fate decisions. However, their expression patterns and regulatory functions during normal and malignant human hematopoiesis are incompletely understood. Here we present a comprehensive resource defining the non-coding RNA landscape of the human hematopoietic system. Based on highly specific non-coding RNA expression portraits per blood cell population, we identify unique fingerprint non-coding RNAs—such as LINC00173 in granulocytes—and assign these to critical regulatory circuits involved in blood homeostasis. Following the incorporation of acute myeloid leukemia samples into the landscape, we further uncover prognostically relevant non-coding RNA stem cell signatures shared between acute myeloid leukemia blasts and healthy hematopoietic stem cells. Our findings highlight the importance of the non-coding transcriptome in the formation and maintenance of the human blood hierarchy.

The patterning of the mammalian brain is orchestrated by a large battery of regulatory genes. Here we examine the developmental function of the Gsh-2 nonclustered homeobox gene. Whole-mount and serial section in situ hybridizations have been used to better define Gsh-2 expression domains within the developing forebrain, midbrain, and hindbrain. Gsh-2 transcripts are shown to be particularly abundant in the hindbrain and within the developing ganglionic eminences of the forebrain. In addition, mice carrying a targeted mutation of Gsh-2 have been generated and characterized. Homozygous mutants uniformly failed to survive more than 1 day following birth. At the physiologic level the mutants experienced apnea and reduced levels of hemoglobin oxygenation. Histologically, the mutant brains had striking alterations of discrete components. In the forebrain the lateral ganglionic eminence was reduced in size. In the hindbrain, the area postrema, an important cardiorespiratory chemosensory center, was absent. The contiguous nucleus tractus solitarius, involved in integrating sensory input to maintain homeostasis, was also severely malformed in mutants. Immunohistochemistry was used to examine the mutant brains for alterations in the distribution of markers specific for serotonergic and cholinergic neurons. In addition, in situ hybridizations were used to define expression patterns of the Dlx 2 and Nkx 2.1 homeobox genes in Gsh-2 mutant mice. The mutant lateral ganglionic eminences showed an abnormal absence of Dlx 2 expression. These results better define the genetic program of development of the mammalian brain, support neuromeric models of brain development, and further suggest similar patterning function for homeobox genes in phylogenetically diverse organisms.

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Plasminogen activation has been proposed to play a critical role in cancer invasion and metastasis. The effects of complete ablation of plasminogen activation in cancer was studied by inoculation of a metastatic Lewis lung carcinoma expressing high levels of plasminogen activator into plasminogen-deficient (Plg−/−) mice and matched control mice. Primary tumors developed in all mice with no difference in the rate of appearance between Plg−/− and control mice. However, the primary tumors in Plg−/− mice were smaller and less hemorrhagic and displayed reduced skin ulceration. In addition, dissemination of the tumor to regional lymph nodes was delayed in Plg−/− mice. Surprisingly, no quantitative differences were observed in lung metastasis between Plg−/− and control mice. In addition, Plg deficiency was compatible with metastasis of the primary tumor to a variety of other organs. Nevertheless, Plg−/− mice displayed a moderately increased survival after primary tumor resection. These findings suggest that plasmin-mediated proteolysis contributes to the morbidity and mortality of Lewis lung carcinoma in mice, but sufficient proteolytic activity is generated in Plg−/− mice for efficient tumor development and metastasis.

A human megakaryoblastic cell line, designated CHRF-288–11, has been established in vitro through the use of adherent stromal cells in long- term human bone marrow culture. Long-term bone marrow cultures were required for the initial adaptation of the megakaryoblastic cells to culture conditions; however, once adapted, the cells were weaned from the stromal layer until they proliferated in the complete absence of any feeder layers. The seed cells for the establishment of this line were derived from a solid tumor; the cloned cell line derived from this tumor exhibits markers characteristic of megakaryocytes and platelets. Specifically, the cells express platelet peroxidase, platelet factor 4, and platelet Ca+(+)-adenosine triphosphatase (ATPase), glycoprotein IIb- IIIa (CDw41), factor VIII antigen, and the MY7 (CD13) and MY9 (CD33) antigens. The cells do not express the erythroid markers glycophorin A and hemoglobin, the myeloid marker myeloperoxidase, nor markers specific for T and/or B cells. The established cell line produces both basic fibroblast growth factor and transforming growth factor-beta, properties demonstrated previously for the solid tumor. The clonal cell population exhibited a unique, singular karyotype, indicating cellular homogeneity. The cells display a doubling time of approximately 33 hours in either 25% horse or calf serum. Treatment of the cells with 1 X 10(-8) mol/L phorbol 12-myristate 13-acetate (PMA) leads to the induction of multi-nucleation and hyperploidy in the cells, with approximately 35% of the cells exhibiting two or more nuclei per cell, and greater than 80% of the cells enlarging in size. The establishment of this unique cell line under defined culture conditions will be beneficial for the future study of megakaryocytic properties expressed by this cell line.

Plasminogen activation has been proposed to play a critical role in cancer invasion and metastasis. The effects of complete ablation of plasminogen activation in cancer was studied by inoculation of a metastatic Lewis lung carcinoma expressing high levels of plasminogen activator into plasminogen-deficient (Plg−/−) mice and matched control mice. Primary tumors developed in all mice with no difference in the rate of appearance between Plg−/− and control mice. However, the primary tumors in Plg−/− mice were smaller and less hemorrhagic and displayed reduced skin ulceration. In addition, dissemination of the tumor to regional lymph nodes was delayed in Plg−/− mice. Surprisingly, no quantitative differences were observed in lung metastasis between Plg−/− and control mice. In addition, Plg deficiency was compatible with metastasis of the primary tumor to a variety of other organs. Nevertheless, Plg−/− mice displayed a moderately increased survival after primary tumor resection. These findings suggest that plasmin-mediated proteolysis contributes to the morbidity and mortality of Lewis lung carcinoma in mice, but sufficient proteolytic activity is generated in Plg−/− mice for efficient tumor development and metastasis.