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Members of the tristetraprolin (TTP) family of tandem CCCH finger proteins can bind to AU-rich elements in the 3′-untranslated region of mRNAs, leading to their deadenylation and subsequent degradation. In previous work, we disrupted the first exon of one of the four mouse TTP family members, Zfp36l2 (Tis11d, Brf2, Erf2), resulting in the production of decreased levels of a truncated protein lacking the first 29 amino acids. These mice exhibited complete female infertility, with embryos not progressing past the two-cell stage. In order to establish a true null phenotype for this gene, we have generated mice completely lacking the second exon, encoding the RNA-binding tandem zinc finger domain, resulting in a true knockout (KO) with complete lack of mRNA and protein. Surprisingly, these mice exhibited a completely unexpected phenotype involving the development of the hematopoietic system; they appeared otherwise anatomically normal. Homozygous Zfp36l2 KO mice on a mixed C57Bl/6 – 129SvEv background were born with normal Mendelian frequency but generally died about two weeks after birth, apparently from intestinal or other hemorrhage. Analysis of peripheral blood from KO mice at two weeks of age showed significant decreases in red cells (2.3 fold), white cells (2.1 fold), and platelets (11 fold). Flow cytometric analysis of spleen cells demonstrated significant decreases in myeloid cells (Gr-1+; Gr-1+/Mac-1+; 4 fold) and megakaryocytes (CD41+; 14 fold), as well as in c-Kit+ hematopoietic progenitors, without changes in lymphoid cell populations. Bone sections from rare surviving adult mice exhibited hematopoietic cell depletion. In addition, analysis of bone marrow revealed nine-fold decreases in lin-/Sca-1+/c-Kit+ cells; colony forming assays revealed that no hematopoietic progenitors grew from the KO bone marrow, compared to an average of 54 colonies per mouse from the wild-type (WT) mice. We therefore analyzed the development of the hematopoietic system. To do this, we cultured fetal liver cells in semisolid media that supported the proliferation and differentiation of multipotential and lineage-committed hematopoietic progenitors. There were significant decreases in the numbers of erythroid (BFU-E, 10-fold), granulocyte- macrophage (CFU-GM, 32-fold), granulocyte macrophage/macrophage (CFU-GM/M, 14-fold), and multipotential (CFU-GEMM, 14-fold) progenitor cells obtained from Zfp36l2 KO fetal liver cells at embryonic day (E) 14.5 as compared to WT cells from littermates. There were also statistically significant decreases in these progenitors in heterozygous mice compared to WT, suggesting a gene dosage effect. Similar studies of yolk sacs from E11.5 mice revealed significant decreases in myeloid (CFU-GM, 2-fold) and multipotential (CFU-GEMM, 1.7-fold) progenitors in the KO yolk sacs. Primitive hematopoiesis was unaffected, as assessed by in vitro colony forming assays with E8-8.25 yolk sac cells. Competitive reconstitution experiments demonstrated that Zfp36l2 KO fetal cells from E14.5 mice were markedly defective in reconstituting the hematopoietic system of lethally irradiated recipients after 1, 2 and 6 months of follow-up; engraftment rates for these dates were, respectively, 45% (WT) vs. 8% (KO); 65% vs. 3%; and 86% vs. 1%. These studies demonstrated that the development of the definitive hematopoietic system was severely adversely affected in the Zfp36l2 KO mice. This led to the hypothesis that elimination of the RNA destabilizing protein ZFP36L2 leads to the accumulation of one or more transcripts that are toxic to hematopoiesis. To explore this hypothesis, microarray analyses were performed on RNA samples from fetal liver at E14.5. We identified 239 significantly elevated transcripts in the KO samples, including several possible candidates for direct binding targets for ZFP36L2. In addition, 175 transcripts were significantly down-regulated in the KO fetal livers, in many cases in pathways of hematopoiesis or platelet development and function. These results are currently being validated by a variety of functional biochemical approaches, and are being supplemented by analogous microarray assays using transcripts from E11.5 yolk sacs. These data establish Zfp36l2 as a critical modulator of definitive hematopoiesis, and suggest a novel regulatory pathway involving control of mRNA stability in the life cycle of hematopoietic stem and progenitor cells.