Cross section through bone marrow - negative control

A cross section through a femur from a control, non-transgenic mouse. Compare this background fluorescence to the green fluorescence expressed by B lineage progenitors expressing rag2-GFP in slide 4.

B cell development in situ

A cross section through a murine femur reveals B lineage progenitors nestled within the bone cavity.

Lymphocytes within the bone marrow (upper right) are outlined in faint green; greenish line at lower left is edge of bone.

Tracing rag2-GFP expression in T cells

Green fluorescence marks T cell progenitors undergoing V(D)J recombination to assemble a T cell receptor. Slide 6 shows this same section under transmitted light. Note that virtually all T cells express rag2.

Compare to rag2 expression in bone marrow B cells, slide 4.

Tracing rag2-GFP expression in B cells

Dim green fluorescence marks B lineage progenitors (middle and upper right) undergoing V(D)J recombination to make antibodies. Slide 2 show this B cell section under transmitted light, and slide 1 shows a comparable image from a control mouse that does not express rag2-GFP.

Note that rag2+ B lymphocytes in bone marrow are rare as compared to rag2+ T cells in the thymus (slide 3). This is because B cell precursors are not the only hematopoietic subset in the bone marrow while T cell precursors form the bulk of thymus cells.

developing B lymphocytes

B lineage progenitors (small, round) receive critical developmental signals from bone marrow stromal cells (larger, adherent).

thymus cross section

The thymus is packed full of developing T lineage progenitors. Visualize which cells are in the process of making a T cell receptor by seeing this same section with fluorescent imaging in slide 3.

The immune system is characterized by the production of an extraordinarily diverse array of antigen receptors. This diversity is generated through V(D)J rearrangement, a mechanism in which double-strand breaks are intentionally introduced into the DNA, and variable region gene segments at antigen receptor loci are permanently deleted. Tight regulation of V(D)J rearrangement is essential as blocks in recombination lead to severe immune deficiencies while recombination-dependent translocations can cause fatal B and T cell leukemias. Our work aims to characterize the mechanisms by which V(D)J recombination is controlled during normal hematopoietic development, and how mistakes in V(D)J recombinase activity can contribute to cancers of hematopoietic origin.

rag1 and rag2 initiate the process of V(D)J recombination by introducing DNA nicks at immunoglobulin (Ig) and T cell receptor (TCR) loci. Because rag1/2 expression is absolutely essential for production of a diverse antigen receptor repertoire, it is important to understand the mechanisms that regulate transcription of these genes. Currently, it remains unknown how rag gene activity and ultimately, V(D)J recombinase activity, is regulated in B lineage progenitors in vivo. This is a key issue because not only is rag expression already established in the earliest B cells, but defects in the bone marrow from aged individuals correlate with decreases in both recombinase activity and antibody diversity.

The capacity for self-renewal is a key property that enables bone marrow hematopoietic stem cells (HSCs) to replenish the immune system throughout childhood as well as adult life. To date, few transcription factors have been identified that are definitively required for the progression of stem cells to the earliest lymphoid lineage progenitors. It is of significant interest to understand the mechanisms that regulate stem cell renewal for two reasons. First, premature depletion of normal, healthy HSCs leads to loss of the hematopoietic system and hence, death. Second, HSCs, like cancer stem cells, exhibit the key property of self-renewal. Cancer stem cells are a distinct subset of cells that continue to seed new tumors and thus, are responsible for the maintenance of at least some neoplasms. Our recent work shows that E47, a transcription factor mechanistically linked to B and T lineage cancers, is critical regulator of stem cell integrity. We have recently identified several targets of E47 in HSCs, and are examining their specific contribution to HSC self-renewal, proliferation, and differentiation.

B cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of functionally immature B cells which are arrested in the G0 phase of the cell cycle and seem to escape programmed cell death. Leukemic cells from most of B-CLL patients express rearranged immunoglobulin (Ig) VH gene with somatic point-mutations. DNA rearrangements and specific cell-surface markers are regulated by several specific transcription factors including E2A. We are studying the role of the E2A transcription factor in the altered proliferation and survival of B-CLL cells using a targeted siRNA knockdown approach.