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.

Public Health Relevance

This project addresses issues relating to the development and growth control of B cell precursors, important issues for human health, since chronic lymphocytic leukemia may arise during aging from natural autoreactive B cells generated during this process and the failure of normal precursor growth regulation can result in the most prevalent childhood leukemia, pre-B acute lymphocytic leukemia. Thus information gained during the course of this work will increase our understanding of the origins and growth of distinctive B cells, potentially aiding in the design of rational therapies for leukemias of childrn and the aged.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Cellular and Molecular Immunology - B Study Section (CMIB)
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Ferguson, Stacy E
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Research Institute of Fox Chase Cancer Center
United States
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