Mary Barbara

Representation is faithfully preserved in global cDNA amplified exponentially from sub-picogram quantities of mRNA

Analysis of transcript representation on gene microarrays requires microgram amounts of total RNA or DNA. Without amplification, such amounts are obtainable only from millions of cells. However, it may be desirable to determine transcript representation in few or even single cells in aspiration biopsies, rare population subsets isolated by cell sorting or laser capture, or micromanipulated single cells. Nucleic-acid amplification methods could be used in these cases, but it is difficult to amplify different transcripts in a sample without distorting quantitative relationships between them. Linear isothermal RNA amplification has been used to amplify as little as 10 ng of total cellular RNA, corresponding to the amount obtainable from thousands of cells, while still preserving the original abundance relationships. However, the available procedures require multiple steps, are labor intensive and time consuming, and have not been shown to preserve abundance information from smaller starting amounts. Exponential amplification, on the other hand, is a relatively simple technology, but is generally considered to bias abundance relationships unacceptably. These constraints have placed beyond current reach the secure and routine application of microarray analysis to single or small numbers of cells. Here we describe results obtained with a rapid and highly optimized global reverse transcription–PCR (RT-PCR) procedure. Contrary to prevalent expectations, the exponential approach preserves abundance relationships through amplification as high as 3 × 1011-fold. Further, it reduces by a million-fold the input amount of RNA needed for microarray analysis, and yields reproducible results from the picogram range of total RNA obtainable from single cells.

Transcriptome Analysis of the Normal Human Mammary Cell Commitment and Differentiation Process

Mature mammary epithelial cells are generated from undifferentiated precursors through a hierarchical process, but the molecular mechanisms involved, particularly in the human mammary gland, are poorly understood. To address this issue, we isolated highly purified subpopulations of primitive bipotent and committed luminal progenitor cells as well as mature luminal and myoepithelial cells from normal human mammary tissue and compared their transcriptomes obtained using three different methods. Elements unique to each subset of mammary cells were identified, and changes that accompany their differentiation in vivo were shown to be recapitulated in vitro. These include a stage-specific change in NOTCH pathway gene expression during the commitment of bipotent progenitors to the luminal lineage. Functional studies further showed NOTCH3 signaling to be critical for this differentiation event to occur in vitro. Taken together, these findings provide an initial foundation for future delineation of mechanisms that perturb primitive human mammary cell growth and differentiation.

Essential role for Ptpn11 in survival of hematopoietic stem and progenitor cells

Src homology 2 domain-containing phosphatase 2 (Shp2), encoded by Ptpn11, is a member of the nonreceptor protein-tyrosine phosphatase family, and functions in cell survival, proliferation, migration, and differentiation in many tissues. Here we report that loss of Ptpn11 in murine hematopoietic cells leads to bone marrow aplasia and lethality. Mutant mice show rapid loss of hematopoietic stem cells (HSCs) and immature progenitors of all hematopoietic lineages in a gene dosage-dependent and cell-autonomous manner. Ptpn11-deficient HSCs and progenitors undergo apoptosis concomitant with increased Noxa expression. Mutant HSCs/progenitors also show defective Erk and Akt activation in response to stem cell factor and diminished thrombopoietin-evoked Erk activation. Activated Kras alleviates the Ptpn11 requirement for colony formation by progenitors and cytokine/growth factor responsiveness of HSCs, indicating that Ras is functionally downstream of Shp2 in these cells. Thus, Shp2 plays a critical role in controlling the survival and maintenance of HSCs and immature progenitors in vivo.

GATA-3 regulates the self-renewal of long-term hematopoietic stem cells

The transcription factor GATA-3 is expressed and required for differentiation and function throughout the T lymphocyte lineage. Despite evidence it may also be expressed in multipotent hematopoietic stem cells (HSCs), any role for GATA-3 in these cells has remained unclear. Here we found GATA-3 was in the cytoplasm in quiescent long-term stem cells from steady-state bone marrow but relocated to the nucleus when HSCs cycled. Relocation depended on signaling via the mitogen-activated protein kinase p38 and was associated with a diminished capacity for long-term reconstitution after transfer into irradiated mice. Deletion of Gata3 enhanced the repopulating capacity and augmented the self-renewal of long-term HSCs in cell-autonomous fashion without affecting the cell cycle. Our observations position GATA-3 as a regulator of the balance between self-renewal and differentiation in HSCs that acts downstream of the p38 signaling pathway.

cDNAs Generated from Individual Epidermal Cells Reveal that Differential Gene Expression Predicting Subsequent Resistance or Susceptibility to Rust Fungal Infection Occurs Prior to the Fungus Entering the Cell Lumen

As the cowpea rust fungus penetrates the wall of a cowpea epidermal cell, resistant and susceptible plants exhibit different ultrastructural and cytochemical changes within the epidermal protoplast. To examine plant gene expression at this stage of infection, cytoplasm was extracted from individual inoculated or uninoculated epidermal cells before the fungal penetration peg reached the cell lumen. Initial differential colony hybridization screening of an expressed sequence tag library constructed from globally amplified cDNAs generated from the inoculated resistant cells resulted in 80 clones (out of 835) with a differential hybridization pattern. Further slot-blot screening and screening of the amplified cDNAs generated from inoculated or uninoculated, resistant or susceptible cells revealed 28 separate genes, mostly with no matching sequences in the databases, that were up-regulated in response to the growth of the fungus through the wall of resistant or susceptible cells. Five genes, including those coding for beta- and alpha-tubulin, were found to be down-regulated specifically in inoculated, susceptible cells, and five were specifically up-regulated in inoculated, resistant cells, including a PR-10 homolog and a phenylalanine ammonia-lyase gene. Probing the amplified cDNAs from each cell type for the expression of cell death-related genes revealed that an LLS1 homolog (vuLLS1), cloned from cowpea, was up-regulated by infection in both resistant and susceptible cells and that a homolog of HSR203J was differentially up-regulated in resistant cells. These data show that changes in gene expression predicting the subsequent expression of susceptibility or hypersensitive resistance to fungal infection occur prior to the fungus entering the cell lumen.

OLIG2 (BHLHB1), a bHLH Transcription Factor, Contributes to Leukemogenesis in Concert with LMO1

OLIG2 (originally designated BHLHB1) encodes a transcription factor that contains the basic helix-loop-helix motif. Although expression of OLIG2 is normally restricted to neural tissues, overexpression of OLIG2 has been shown in patients with precursor T-cell lymphoblastic lymphoma/leukemia (pre-T LBL). In the current study, we found that overexpression of OLIG2 was not only found in oligodendroglioma samples and normal neural tissue but also in a wide spectrum of malignant cell lines including leukemia, non-small cell lung carcinoma, melanoma, and breast cancer cell lines. To investigate whether enforced expression of OLIG2 is oncogenic, we generated transgenic mice that overexpressed OLIG2 in the thymus. Ectopic OLIG2 expression in the thymus was only weakly oncogenic as only 2 of 85 mice developed pre-T LBL. However, almost 60% of transgenic mice that overexpressed both OLIG2 and LMO1 developed pre-T LBL with large thymic tumor masses. Gene expression profiling of thymic tumors that developed in OLIG2/LMO1 mice revealed up-regulation of Notch1 as well as Deltex1 (Dtx1) and pre T-cell antigen receptor alpha (Ptcra), two genes that are considered to be downstream of Notch1. Of note, we found mutations in the Notch1 heterodimerization or proline-, glutamic acid-, serine-, and threonine-rich domain in three of six primary thymic tumors. In addition, growth of leukemic cell lines established from OLIG2/LMO1 transgenic mice was suppressed by a gamma-secretase inhibitor, suggesting that Notch1 up-regulation is important for the proliferation of OLIG2-LMO1 leukemic cells.

Committed hemopoietic progenitors, not stem cells, are the principal responders to Hox gene transduction

As hemopoietic stem cells differentiate, their proliferative lifespan shortens by unknown mechanisms. Homeobox cluster (Hox) genes have been implicated by their enhancement of self-renewal when transduced into hemopoietic cells, but gene deletions have been inconclusive because of functional redundancy. Here we enforced HOXB4 expression in purified precursor stages, and compared responses of early stages expressing the endogenous genes with later stages that did not. Contrary to the prevalent view that transduced Hox genes enhance the self-renewal of hemopoietic stem cells, stem cells or their multipotent progeny expressing the endogenous genes showed little response. Instead, immortalization, extensive self-renewal and acquired reconstituting potential occurred in committed erythroid and myeloid progenitors where the endogenous genes were shutting down. The results change our understanding of the stages affected by exogenous HOX proteins and point to shutdown of the endogenous genes as a principal determinant of the shortened clonal lifespans of committed progenitor cells.