Conditional Knockout of Sox4 Markedly Affects Early B Lymphopoiesis in Adult Mice

Abstract 913

The development of mature B cells from self-renewing pluripotent hematopoietic stem cells (HSCs) involves a complex process that is regulated in a hierarchical order by various proteins, particularly transcription factors. Sox4 is an SRY-related HMG-box-containing transcription factor and is known to be implicated in B cell development. However, Sox4's role in B cell development has not been systematically investigated. In this project, we used conditional knockout mouse strains to study the effect of Sox4 deletion on B lymphopoiesis in adult mice.

To determine when in the B cell developmental process Sox4 is required, we crossed Sox4-floxed mice with 5 Cre-recombinase-expressing mouse strains that were expected to result in deletion of the floxed Sox4 gene at different developmental stages, as follows: Vav-Cre (HSC stage in early embryos), Mx1-Cre (HSC stage in adults, induced by pIpC injection), MB1-Cre (early progenitor B cell stage), CD19-Cre (pro-B cell stage), and CD21-Cre (mature B cell stage). We demonstrated that deletion of Sox4 caused arrest of B lymphopoiesis at the transition from pre-pro-B cell stage (fraction A) to pro-B cell stage (fraction B); fraction B and later-stage B cells were nearly absent. There was a significant reduction in the number of hematopoietic stem (LSK) cells upon Sox4 deletion, but the number of common lymphocyte progenitors (CLPs) was not significantly changed and that of fraction A cells was only slightly reduced, suggesting that reduction in HSCs was not directly responsible for the B cell deficiency. Deletion of Sox4 in the early embryonic stage (Vav-Cre) and in adults (Mx1-Cre) resulted in similar phenotypes with regard to B lymphopoiesis, except that the number of peritoneal B1 cells was reduced more significantly in Vav-Cre than in Mx1-Cre mice. MB1-Cre yielded results similar to those of Vav-Cre, but B lymphopoiesis arrest was not as dramatic. Sox4^f/f CD19-Cre and Sox4^f/f CD21-Cre mice produced no significant difference in B cell phenotype compared with Sox4^f/+ CD19-Cre and Sox4^f/+ CD21-Cre mice, respectively. These data suggest that Sox4 is required for early B cell development at the transition from pre-pro-B cells to pro-B cells and is not required for mature B cell development.

To further understand the mechanism by which Sox4 deletion induced pro-B cell deficiency, we performed semi-quantitative RT-PCR on residual pro-B cells and showed that Sox4 deletion did not change the mRNA expression of the well-known genes involved in early B cell development (EBF1, E2A, Rag1, CXCR4, IL7R, EZH2, PAX5, FoxP1, STAT5), indicating that Sox4 might be functioning in early B lymphopoiesis through a novel mechanism. We next studied pro-B cell viability in Sox4^f/+ and Sox4^f/f Vav-Cre mice. We found that, when Sox4 was depleted, bone marrow pro-B cells, but not pre-pro-B cells, underwent remarkable apoptosis, as shown by increased annexin V staining. In accordance, the level of the antiapoptotic protein Bcl2 in Sox4-deleted pro-B cells was much lower than that in control pro-B cells. Introduction of Bcl2 transgenic gene into the Sox4 conditional knockout mice decreased pro-B cell apoptosis and partially rescued the Sox4-deficient B cell development. We also observed striking molecular communication between Sox4 and the c-Kit-mediated cell survival signaling pathway; after intraperitoneal injection of the c-Kit inhibitor imatinib, the apoptotic rate was much higher in Sox4^f/+Vav-Cre mice than in Sox4^+/+ mice, suggesting that reduction of Sox4 level sensitized the cells to the inhibitor.

Our data suggest that Sox4 deletion markedly decreases pro-B cell viability by modulating Bcl2 and that the c-Kit signaling pathway and Sox4 function is interlinked. We are currently investigating Sox4 transcription program and the mechanism of Sox4-induced Bcl2 protein reduction in progenitor B cells.