Genes and signals controlling mammalian hematopoiesis.

我们的研究集中在三个方面。第一部分涉及T细胞抗原受体（TCR）信号的作用表征，特别是单个TCR信号转导亚基和信号转导基序在T细胞发育中的作用。其次，我们扩展了我们的研究，包括分析在TCR下游起作用或抑制TCR信号传导的信号转导分子。这些研究的目的是了解这些分子如何参与TCR介导的信号传导，并确定它们及其调节的信号通路在T细胞成熟和T细胞活化中起什么作用。最后，本实验室开启了研究控制造血干细胞产生和维持的基因的新领域。

Our studies are focused in three areas. The first involves characterization of the role of T cell antigen receptor (TCR) signals, and in particular, individual TCR signal transducing subunits and signal transducing motifs in T cell development. Second, we have extended our studies to include analysis of signal transducing molecules that function downstream of the TCR or that inhibit TCR signaling. The aim of these studies is to understand how these molecules participate in TCR mediated signaling and to determine what roles they and the signaling pathways they regulate play in T cell maturation and T cell activation. Finally, the lab has initiated a new area of investigation of the genes controlling the generation and maintenance of Hematopoietic Stem Cells. Role of T cell antigen receptor (TCR) signaling in thymocyte development. Signal transduction sequences (termed Immunoreceptor Tyrosine-based Activation Motifs; ITAMs) are contained within four distinct subunits of the multimeric TCR complex (zeta, CD3-gamma, -delta, -epsilon). Di-tyrosine residues within ITAMs are phosphorylated upon TCR engagement and function to recruit signaling molecules, such as protein tyrosine kinases, to the TCR complex, thereby initiating the T cell activation cascade. To determine if TCR signal transducing subunits perform distinct or analogous functions in development, we previously generated zeta deficient and CD3-epsilon deficient mice by gene targeting, genetically reconstituted these mice with transgenes encoding wild-type or signaling-deficient (ITAM-mutant) forms of zeta and CD3-epsilon, and characterized the developmental and functional consequences of these alterations on TCR signaling. The results of these studies demonstrated that TCR-ITAMs are functionally equivalent but act in concert to amplify TCR signals. TCR signal amplification was found to be critical for thymocyte selection, the process by which potentially useful immature T cells are instructed to survive and differentiate further-(positive selection), and potentially auto-reactive cells that may cause auto-immune disease are deleted in the thymus (negative selection). Thus, the multi-subunit structure of the TCR may have evolved to enable complex organisms to develop a broad, self-restricted yet auto-tolerant T cell repertoire. In current studies we are using conditional gene expression systems to analyze the importance of TCR signaling at specific stages of development. In addition, we are using microarray and subtractive cloning to identify genes involved in T cell signaling and T cell development. Signaling molecules that function downstream of the TCR or that function to "fine-tune" the TCR signal. Our results with TCR-ITAM mutant mice suggested that other signaling molecules can compensate for the reduction in TCR signal strength. An initial FACS-based search for candidate compensatory molecules led us to CD5, a TCR associated trans-membrane protein that inhibits TCR signaling. Importantly, we found that CD5 surface expression is regulated by and parallels TCR signal intensity. Thus, rather than simply functioning as a co-receptor, CD5 acts to fine-tune TCR signals during thymocyte selection since its level of surface expression depends upon the intensity of TCR signaling. An obvious benefit of such fine-tuning of the TCR signaling response would be to enable the generation of a T cell repertoire with the maximum possible diversity since it would allow a broader range of TCRs to pass through the signaling window of positive selection. Since little was known about how CD5 regulates TCR signaling, we initiated a project to characterize CD5 function, both genetically and biochemically. The results of these experiments suggest a mechanism for CD5 mediated TCR signal inhibition that we are currently testing experimentally. We have also identified a novel T-lineage restricted putative adaptor protein, provisionally named TLAP. Biochemical studies conducted in the interim indicate that TLAP functions in the TCR signaling pathway. TLAP-/- mice have been generated and their phenotype reveals an important role for this protein in thymocyte development. Current and projected experiments are directed at elucidating the function of TLAP in T cell signaling and development. Genes controlling Hematopoietic Stem cell specification and maintenance. The hematopoietic system is composed of a functionally diverse group of cells that originate from a common hematopoietic stem cell (HSC) capable of long-term self-renewal and multi-lineage differentiation. Self-renewal ensures that a pool of HSCs persists throughout life, whereas differentiation leads to the continuous generation of all circulating blood cells including lymphocytes, myeloid cells, erythrocytes and platelets. We have recently initiated experiments aimed at identifying genes important for HSC generation and maintenance. Our initial studies focused on the role of LIM domain binding protein-1 (Ldb1) in hematopoiesis. The results of these experiments revealed a critical function for Ldb1 in regulating the self-renewal/differentiation cell fate decision in hematopoietic stem cells and suggest further that Ldb1 nucleated transcription complexes may control maintenance of lineage specific stem cells.