MYB IS Required for B-Selection During Thymopoises 713 views

c‐Myb is a transcription factor required during hematopoiesis and is crucial for multiple stages of thymopoiesis. Understanding c‐Myb function during hematopoiesis has been greatly impeded due to the lack of a tractable genetic system. We have used the Cre/loxP approach to conditional mutagenesis to study c‐Myb function during thymopoiesis. Thymocyte specific inactivation of the Myb locus demonstrated a block in DN3 to DN4 differentiation concomitant with impaired Vβ to DJβ recombination of the Tcrb locus suggesting that the inability to generate a TCRβ protein blocked DN3 differentiation. However, some DN3 thymocytes expressed a TCRβ protein but very few thymocytes transitioned to the DN4 stage suggesting that c‐Myb is required for DN3 to DN4 differentiation. To determine if c‐Myb plays additional role(s) during transition from the DN3 to DN4 stage of thymocyte differentiation, we bred mice with either Mybff cwlckCre Rag2‐/‐ or Mybff cwlckCre Rag2‐/‐ Tcrb‐Tg genotypes. c‐Myb does not appear to be required for maintenance of the DN3E compartment in Rag2‐deficient thymocytes, and defective V(D)J recombination in the Mybf/f C mice is not simply a consequence of reduced survival and the inability to complete V(D)J recombination. Moreover, the presence of a Tcrb transgene (Tg) was not able to rescue DN3 to DN4 differentiation in the absence of c‐Myb, demonstrating for the first time that c‐Myb is required for normal β‐selection independent of the impairment in V(D)J recombination. We present evidence that c‐Myb deficient thymocytes can initiate some events associated with pre‐TCR signaling and upregulate surface markers indicative of β‐selection such as CD5, CD2 and CD27. In addition, Myb‐ and Rag2‐/‐ doubly deficient mice are able to respond to pre‐TCR stimulation by anti‐CD3ε treatment to down‐regulate CD25 and up‐regulate CD8. However, survival of pre‐TCR+ DN3 thymocytes is impaired in the absence of c‐Myb which is not rescued by expression of a Bcl‐2‐transgene or loss of p53. Moreover, in DN4 thymocytes both survival and proliferation is impaired. The inability of DN4 thymocytes to proliferate could be due to the massive amount of cell death that was occurring; however, pre‐TCR+ DN and ISP thymocytes fail to receive signals to enter the cell cycle and do not express cyclin D3. Despite recent studies that demonstrated that mature B lymphocytes can proliferate without c‐Myb, we demonstrate that c‐Myb is required for proliferation during β‐selection of DN4 and ISP thymocytes. We performed gene expression microarrays on several DN subsets from Mybf/f cwlckCre and control mice to gain insight into how c‐Myb regulates proliferation and survival during β‐selection. The microarrays suggest changes in expression of genes involved in interaction with the thymic microenvironment which could cause the impaired proliferation and survival in c‐Myb‐deficient thymocytes. Moreover, we provide preliminary data that suggests that c‐Myb may be involved in migration to CXCL12 and in interaction with extracellular matrix protein laminin. Thus, we provide evidence that c‐Myb plays multiple roles during differentiation of DN3 thymocytes independent of V(D)J recombination that is most likely due to the altered expression of multiple transcriptional targets of c‐Myb. Note: Abstract extracted from PDF text

PHD (Doctor of Philosophy)

English

All rights reserved (no additional license for public reuse)

2009-08-01