Genes and signals controlling mammalian hematopoiesis.

我们的研究主要集中在三个方面。第一个涉及T细胞抗原受体(TCR)信号的特征，特别是单个TCR信号转导亚单位和信号转导模体在T细胞发育中的作用。其次，我们扩大了我们的研究范围，包括分析TCR下游的信号转导分子或抑制TCR信号的分子。这些研究的目的是了解这些分子如何参与TCR介导的信号转导，并确定它们及其调控的信号通路在T细胞成熟和T细胞激活中所起的作用。最后，该实验室启动了一个新的研究领域，研究控制造血干细胞生成和维持的基因。 T细胞抗原受体(TCR)信号在胸腺细胞发育中的作用 信号转导序列(称为免疫受体酪氨酸激活基序；ITAM)包含在多聚体TCR复合体的四个不同的亚基中(Zeta，CD3-Gamma，-Delta，-epsilon)。ITAM中的二酪氨酸残基在TCR参与时被磷酸化，功能是将信号分子，如蛋白酪氨酸激酶，招募到TCR复合体，从而启动T细胞激活级联反应。为了确定TCR信号转导亚单位在发育过程中是否发挥着不同或相似的功能，我们以前通过基因打靶的方法产生了Zeta缺陷和CD3-epsilon缺陷小鼠，用编码野生型或信号缺陷(ITAM-突变)形式的Zeta和CD3-epsilon的转基因重组了这些小鼠，并表征了这些变化对TCR信号转导的发育和功能影响。这些研究的结果表明，TCR-ITAM在功能上是等同的，但协同作用放大TCR信号。TCR信号扩增被发现对胸腺细胞选择至关重要，在胸腺中，潜在有用的未成熟T细胞被指示存活并进一步分化-(正选择)，以及可能导致自身免疫性疾病的潜在的自身反应细胞被删除(负选择)。因此，TCR的多亚基结构可能已经进化，使复杂的生物体能够发展出广泛的、自我限制的、自我耐受的T细胞库。在目前的研究中，我们正在使用条件基因表达系统来分析TCR信号在特定发育阶段的重要性。此外，我们正在使用微阵列和消减克隆来识别与T细胞信号和T细胞发育有关的基因。 TCR下游的信号分子或对TCR信号进行“微调”的分子。 我们对TCR-ITAM突变小鼠的研究结果表明，其他信号分子可以补偿TCR信号强度的降低。最初基于流式细胞仪的候选补偿分子的搜索使我们找到了CD5，一种与TCR相关的跨膜蛋白，它抑制TCR信号转导。重要的是，我们发现CD5表面的表达受TCR信号强度的调节，并且与TCR信号强度平行。因此，CD5不是简单地作为辅助受体发挥作用，而是在胸腺细胞选择过程中微调TCR信号，因为它的表面表达水平取决于TCR信号的强度。这样微调TCR信号响应的一个明显的好处是能够产生具有最大可能多样性的T细胞谱系，因为它将允许更大范围的TCR通过正选择的信号窗口。由于对CD5如何调节TCR信号知之甚少，我们发起了一个项目，从遗传和生化两个方面表征CD5的功能。这些实验的结果表明了CD5介导的TCR信号抑制的机制，我们目前正在进行实验测试。我们还鉴定了一种新的T-谱系限制性假定接头蛋白，暂时命名为TLAP。在此期间进行的生化研究表明，TLAP在TCR信号通路中发挥作用。已经产生了TLAP-/-小鼠，它们的表型揭示了该蛋白在胸腺细胞发育中的重要作用。目前和计划中的实验旨在阐明TLAP在T细胞信号和发育中的功能。 控制造血干细胞规范和维持的基因。 造血系统由一组功能不同的细胞组成，这些细胞起源于一个共同的造血干细胞(HSC)，能够长期自我更新和多谱系分化。自我更新确保造血干细胞池在一生中持续存在，而分化则导致包括淋巴细胞、髓系细胞、红细胞和血小板在内的所有循环血细胞的持续生成。我们最近启动了旨在识别对HSC生成和维持至关重要的基因的实验。我们的初步研究集中在LIM结构域结合蛋白-1(LDB1)在造血中的作用。这些实验结果揭示了LDB1在调节造血干细胞自我更新/分化细胞命运决定中的关键作用，并进一步表明LDB1核转录复合体可能控制谱系特异性干细胞的维持。

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.