Heterogenous Nuclear Ribonucleoprotein L (hnRNPL) Is Required for the Functional Integrity of Hematopoietic Stem Cells.

Abstract 1486

Poster Board I-509

Hematopoietic differentiation has to be tightly regulated since uncontrolled or exaggerated development of blood cells may lead to the development of leukemia or autoimmune diseases. Many mechanisms exist to control hematopoiesis on a molecular level, including the regulation of transcription, which has been intensely studied. However, new evidence suggests the process of alternative splicing to be an important regulator of the maturation and activation of blood- and immune effector cells. One of the factors that has been identified as a potential regulator of the immune response and controls alternative splicing is “heterogenous nuclear ribonucleoprotein L” (hnRNP L). This factor affects among others the alternative splicing of the CD45 gene, which encodes the major tyrosine phosphatase expressed on all hematopoietic cells. To investigate the biological role of hnRNP L as a regulator of alternative splicing in hematopoiesis, we have generated conditional hnRNP L knockout (KO) mice carrying floxed alleles that can be deleted by co expression of Cre recombinase. Both the inducible MxCre transgene or Vav-Cre transgene, which is active in all hematopoietic cells were introduced into hnRNP L^fl/fl mice. We found that the conditional deletion of hnRNP L by the Vav Cre transgene led to early mortality before birth (at stage E17.5) and flow cytometric analysis of fetal liver of hnRNP L^fl/fl, Vav-Cre mice or bone marrow from pIpC induced hnRNP L^fl/fl Mx-Cre mice showed a deficit in erythrocyte maturation. In addition, fetal thymi from hnRNP L^fl/fl X Vav-Cre mice were severely reduced in cellularity and showed disturbed T cell maturation. Moreover, the deletion of hnRNP L results in reduced numbers of Lin⁻Sca1⁺ckit⁺ (LSK) cells, common lymphoid progenitors (CLPs), common myeloid progenitors (CMPs), granulocyte-monocyte progenitors (GMPs) and megakaryocyte-erythrocyte progenitors (MEPs). Strikingly, while most of the progenitors and the short-term hematopoietic stem cells (HSCs) were affected by the deletion of hnRNP L, the population of long term HSCs was not reduced. We found a high percentage of Annexin V positive cells in the LSK population suggesting that the loss of progenitors and short term HSCs in hnRNP L deficient mice is due to an accelerated cell death. To test whether stem cells lacking hnRNP L were still functional, we sorted Lin⁻Sca1⁺ckit⁺ (LSK) cells and cultured them on either methylcellulose or the feeder cell lines OP9 and OP9-DL1. The co-culture with OP9 or OP9-DL1 cells demonstrated that hnRNP L^−/− LSK cells had lost their potential to differentiate into B and T lymphocytes. Similarly, hnRNP L deficient LSK cells were unable to give rise to lymphoid, myeloid or erythroid colonies on methylcellulose. This suggests that hnRNP L is required to maintain not only the numbers of hematopoietic stem cells, but also their ability for multilineage differentiation. We conclude that the regulation of alternative splicing is an essential component of the regulatory network required to maintain hematopoietic differentiation and the functional integrity of hematopoietic stem cells.