Mechanisms regulating the B lymphopoiesis fetal/adult switch

B 淋巴细胞生成胎儿/成人转换的调节机制

• 批准号: 8964046
• 负责人: RICHARD R HARDY
• 金额: $ 22.31万
• 依托单位: RESEARCH INST OF FOX CHASE CAN CTR
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-15 至 2019-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8964046
• 关键词: Acute; Adaptor Signaling Protein; Address; Adult; Aging; Alleles; Animals; Autoantibodies; B Cell Proliferation; B-Cell Acute Lymphoblastic Leukemia; B-Cell Development; B-Lymphocyte Subsets; B-Lymphocytes; BLNK gene; Biological Models; Bone Marrow; Cell Cycle; Cells; Childhood Leukemia; Chronic; Chronic Lymphocytic Leukemia; Complex; Data; Development; Engineering; Engraftment; Estrogen Analogues; Failure; Fetal Development; Fetal Liver; Fetus; Flow Cytometry; Gene Expression; Generations; Genetic Engineering; Greater sac of peritoneum; Growth; Growth and Development function; Health; Hematopoiesis; Human; In Vitro; Knock-out; Knockout Mice; Life; Lymphopoiesis; Maintenance; Mediating; Messenger RNA; MicroRNAs; Molecular; Monitor; Mus; Outcome; Pathway interactions; Phenotype; Phosphorylation; Play; Porifera; Process; Regulation; Reporting; Role; Scaffolding Protein; Signal Transduction; Signaling Molecule; Spleen; Staging; Stem Cell Factor; System; Tamoxifen; Testing; To specify; Transgenic Mice; Transgenic Model; Work; aged; autoreactive B cell; base; design; embryonic stem cell; experience; fetal; fetus cell; in vivo; insight; knock-down; leukemia; mutant; novel; progenitor; programs; public health relevance; reconstitution; response; retroviral transduction; scaffold; signal processing; small hairpin RNA; transcription factor; zinc finger nuclease

 DESCRIPTION (provided by applicant): Fetal hematopoiesis produces a type of B lymphocyte, termed B1a, which is not generated by adult bone marrow progenitors. Fetal hematopoiesis, and the development of B1a B cells, is dependent upon the action of the stem cell factor, Lin28b; however, the basis by which Lin28b directs fetal hematopoiesis remains poorly understood. In this application, we seek to understand how Lin28b directs fetal B lymphopoiesis and whether it is responsible for the change in proliferative response to Pre-BCR expression during fetal and adult development. We will also determine the mechanism for this difference. We have already made two critical insights that will serve as the basis for this proposal: 1) The transcription factor Arid3A (Bright) is capable of recapitulating many aspects of fetal development when ectopically expressed in adult progenitors, which do not express Lin28b, indicating that Arid3A is a critical effector through which of Lin28b promotes fetal hematopoiesis; and 2) The signaling adaptor protein, BCAP, is dispensable for adult B lymphopoiesis, but is essential for fetal development of B1 B cells, indicating that it plays a critical role in supporting the signaling processes required to specify fetal B cells. We intend to gain insight into the molecular processes that underlie fetal hematopoiesis, with a particular emphasis on elucidating the roles of Arid3A and BCAP. In Aim 1, we will determine how the Lin28b/Let-7 axis regulates Arid3A and orchestrates the developmental outcomes uniquely associated with fetal B cell development, such as the altered linkage of Pre-BCR signaling to proliferation. In doing so, we will employ shRNA knockdown, miRNA sponges, a conditional Arid3a knockout mouse, and novel Lin28b and Let-7 transgenic mice. In Aim 2, we will use zinc finger nuclease mediated engineering to generate an inducible allele of BLNK (SLP-65), a key scaffold protein required for Pre-BCR signaling. By appending an ERT2 module to BLNK it will be sequestered away from the Pre-BCR, thereby negating signaling, unless B cell precursors are treated with the estrogen analog tamoxifen, which will release BLNK to assemble with the Pre- BCR, restoring signaling. This will enable us to acutely control the initiation of Pre-BCR signaling, which under normal circumstances is cell-autonomous and chronic. We will use this novel system to characterize phosphorylation in fetal and adult Pre-B cells by PhosFlow analysis. Finally, in Aim 3, we will determine how Pre-BCR signaling differs in fetal versus adult Pre-B cells and how this is perturbed by Btk- and BCAP- deficiency. Pre-BCR signaling will be assessed in fetal cells lacking these key signaling molecules and then fetal BCAP-deficient progenitors will be reconstituted with BCAP mutants to assess which domains and signaling cascades are critical to support fetal lymphopoiesis. In summary, these studies will determine the key regulators and signals that distinguish fetal and adult Pre-BCR responses. Our work combines state-of-the-art flow cytometry with powerful in vitro and in vivo analysis and sophisticated manipulation of gene expression to dissect the fetal/adult switch in B lymphopoiesis and alterations in Pre-BCR signaling.

 描述(申请人提供)：胎儿造血产生一种类型的B淋巴细胞，称为B1a，不是由成人骨髓祖细胞产生的。胎儿的造血和B1a B细胞的发育依赖于干细胞因子Lin28b的作用；然而，Lin28b指导胎儿造血的基础仍然知之甚少。在这一应用中，我们试图了解Lin28b是如何引导胎儿B淋巴细胞生成的，以及它是否与胎儿和成人发育过程中对前BCR表达的增殖反应的变化有关。我们还将确定这种差异的机制。我们已经得到了两个关键的见解，将作为这一提议的基础：1)转录因子Arid3A(Bright)在不表达Lin28b的成年祖细胞中异位表达时，能够概括胎儿发育的许多方面，这表明Arid3A是一个关键的效应器，Lin28b通过它促进胎儿造血；2)信号适配器蛋白BCAP对成人B淋巴细胞的生成是必不可少的，但对B1 B细胞的胎儿发育是必不可少的，这表明它在支持指定胎儿B细胞所需的信号过程中发挥关键作用。我们打算 深入了解胎儿造血的分子过程，特别强调阐明Arid3A和BCAP的作用。在目标1中，我们将确定Lin28b/let-7轴如何调节Arid3A并协调与胎儿B细胞发育独特相关的发育结果，例如BCR前信号与增殖的变化联系。在此过程中，我们将使用shRNA敲除、miRNA海绵、条件Arid3a基因敲除小鼠，以及新型Lin28b和let-7转基因小鼠。在目标2中，我们将利用锌指核酸酶介导的工程技术来产生BLNK(SLP-65)的诱导等位基因，SLP-65是BCR前信号转导所需的关键支架蛋白。通过将ERT2模块附加到BLNK上，它将与前BCR隔离，从而否定信号，除非B细胞前体用雌激素类似物他莫昔芬处理，它将释放BLNK与前BCR组装，恢复信号。这将使我们能够准确地控制BCR前信号的启动，在正常情况下，BCR前信号是细胞自主的和慢性的。我们将使用这个新的系统通过PhosFlow分析来表征胎儿和成人前B细胞的磷酸化。最后，在目标3中，我们将确定Pre-BCR信号在胎儿和成人Pre-B细胞中的差异，以及BTK和BCAP缺乏如何干扰这一差异。在缺乏这些关键信号分子的胎儿细胞中，将评估Pre-BCR信号，然后将BCAP缺失的祖细胞与BCAP突变体重组，以评估哪些结构域和信号级联对支持胎儿淋巴生成至关重要。总之，这些研究将确定区分胎儿和成人BCR前反应的关键调节因子和信号。我们的工作将最先进的流式细胞术与强大的体外和体内分析以及对基因表达的复杂操作相结合，剖析了B淋巴细胞生成和BCR前信号变化中胎儿/成人的开关。