RNA-binding motif protein 15 (knockout (family a group of proteins with homology to Tyrphostin AG 183 the split ends (spen) protein. in a knockout (is a proto-oncogene that has been studied extensively in many tissue types. Nonetheless the role of in the SAP155 regulation of adult mouse HSCs has been described only recently due to limitations in the analysis of mice lacking the gene because of early embryonic lethality; however using an inducible Cre-LoxP system was conditionally deleted in the adult hematopoietic system and new unexpected roles for gene were discovered roles involving more than just the enhancement of hematopoietic progenitor cell proliferation.13 14 In these studies Wilson and coworkers found that the increased long-term (LT) HSCs in c-Myc-deficient BM were caused not by alterations in HSC proliferation or survival but rather by an accumulation of LT-HSCs associated with a differentiation block caused by increased HSC-niche adhesion.13 14 In this study we report that Rbm15 has an important role in regulating HSCs and megakaryocyte development which may Tyrphostin AG 183 Tyrphostin AG 183 occur partly through its regulation of expression. expression is down-regulated in and expression suggesting a possible functional interplay between Rbm15 and c-Myc in the regulation of both HSC and megakaryocyte development. Methods Mice To generate mice we constructed a targeting vector in which the entire exon 1 was flanked by 2 sites (supplemental Figure 1 available on the website; see the Supplemental Materials link at the top of the online article). The construct was introduced by homologous recombination into 129SvJ embryonic stem cells and the targeted embryonic stem cells used to produce mouse chimeras. The Tyrphostin AG 183 mice were subsequently backcrossed and are maintained on a pure C57BL/6 background. To delete conditionally in the hematopoietic system mice were crossed with transgenic mice (The Jackson Laboratory). By proper mating we were able to obtain (or transgene both mice and their test was used to assess statistical significance. Results expression in hematopoietic cells of adult mice To determine the expression pattern of in hematopoietic cells we isolated different stages and lineages of mouse BM cells based on their surface marker expression. This was accomplished by FACS and analysis of levels was done by semiquantitative reverse transcription-PCR. was found to be expressed in ST-HSC granulocyte/monocyte progenitor (GMP) and megakaryocytic/erythroid progenitor (MEP) stages as well as mature B cells and all stages of T-cell maturation (supplemental Figure 1A). These murine expression data closely parallel the expression pattern of human conditionally in the hematopoietic system mice were crossed with transgenic animals. ((= .010 n = 18 mice per group; Figure 1A-B). Previous studies have demonstrated that LT-HSCs and ST-HSCs can be distinguished based on expression; LT-HSCs are Flk2 negative (LSK/Flk2?) whereas ST-HSCs are Flk2 positive (LSK/Flk2+).16 Therefore we examined Flk2 expression in the LSK cell population in < .001 n = 18 per group; Figure 1A C). The absolute number of LT-HSCs was also increased significantly in < .001 n = 17 per group; Figure 1D). By contrast although the percentage of ST-HSCs was decreased in the LSK population it was comparable between expression during this process (supplemental Figure 1A). Figure 1 Increased percentages and absolute numbers of LSK cells and LT-HSCs in by retroviral transduction decreased N-cadherin expression (1.8-fold decrease) in WT LSK cells (Figure 3B) suggesting that the gene may regulate expression of the adhesion molecule and in turn can alter HSC-niche interactions. The increase in major adhesion molecules in was discovered due to its involvement in the AMkL fusion gene = .003 n = 7 per group; in the spleen: = .015 n = 7 per group. Histopathologic examination of the spleens and BM from each group of mice revealed a statistically significant increase in the numbers of morphologically identified megakaryocytes present in the spleens (average of 2.8-fold higher in KO vs WT spleens = .005) but not the marrows from Rbm15-KO animals (supplemental Figure 4A-B). This increase of megakaryocyte numbers was also confirmed by acetylcholinesterase (AchE) staining in the spleens of the KO animals (Figure 4Aiii). To further explore the role of Rbm15 in Tyrphostin AG 183 megakaryocyte development we quantitated megakaryocytic progenitors by.