Transcriptional regulation of B lymphocyte commitment.

The transcription factor Pax5 is essential for commitment to the B lineage as the development of these cells is arrested at an early stage in the bone marrow of Pax5 deficient mice. Pax5 deficient pro-B cells display remarkable plasticity and are able to differentiate into other cell lineages both in vitro and in vivo. Several Pax5 target genes have been previously reported but none are able to explain the developmental block observed at the early to late pro-B cell stage. To determine the exact mechanisms by which Pax5 controls B cell development, I have undertaken a cDNA microarray screen with a custom generated B cell-specific cDNA library. By identifying genes that are differentially expressed between Pax5 deficient and wild type pro-B cells, I have identified a number of potential Pax5 target genes. The microarray screen identified lymphoid-restricted membrane protein (Lrmp or Jaw1) as a novel Pax5 activated transcript. Using RT-PCR and a Pax5-estrogen receptor inducible system, I confirmed that Jaw1 is a direct Pax5 target gene whose expression is confined, in resting cells, to the earliest stages of B and T cell development. The biological relevance of Jaw1 for cell fate specification has been tested by transducing bone marrow progenitor cells with murine retroviral vectors and reconstituting haemopoiesis in vivo. I have reported that over-expression of Jaw1 in these cells results in a decrease in the development of B and NK lymphocytes and a marked increase in the development of myeloid cells in the bone marrow of reconstituted mice. This result suggests that Jaw1 may play an important role in the early development of lymphocytes in the bone marrow. Whilst initial analysis of Jaw1 deficient mice has revealed no overt defects in lymphopoiesis, I postulate that Jaw1 is involved in IP3-induced calcium signaling downstream of the pre-BCR and BCR. This hypothesis has resulted from analysis of the function of IRAG, the only known Jaw1 homologue combined with data that Jaw1 is expressed in early B cells in the BM as well as in germinal centers in the spleen. Pax5 mutant mice usually die before weaning and the cause of death is currently unknown, suggesting that Pax5 is expressed in a previously unreported tissue. To investigate this hypothesis I produced a monoclonal antibody to Pax5 and screened for novel expression domains during embryonic development and also in neonate mice. These studies did not detect any new Pax5 expression domains, but did reveal that this antibody cross-reacts with Pax2 and/or Pax8 in the developing kidney and brain. Biochemical analysis of the serum from Pax5 deficient mice also did not reveal the likely cause of death in these animals, beyond the general signs of dehydration and starvation that are likely to be secondary to the underlying defect