Endomucin, a CD34-like sialomucin, marks hematopoietic stem cells throughout development

To detect as yet unidentified cell-surface molecules specific to hematopoietic stem cells (HSCs), a modified signal sequence trap was successfully applied to mouse bone marrow (BM) CD34−c-Kit+Sca-1+Lin− (CD34−KSL) HSCs. One of the identified molecules, Endomucin, is an endothelial sialomucin closely related to CD34. High-level expression of Endomucin was confined to the BM KSL HSCs and progenitor cells, and, importantly, long-term repopulating (LTR)–HSCs were exclusively present in the Endomucin+CD34−KSL population. Notably, in the yolk sac, Endomucin expression separated multipotential hematopoietic cells from committed erythroid progenitors in the cell fraction positive for CD41, an early embryonic hematopoietic marker. Furthermore, developing HSCs in the intraembryonic aorta-gonad-mesonephros (AGM) region were highly enriched in the CD45−CD41+Endomucin+ fraction at day 10.5 of gestation (E10.5) and in the CD45+CD41+Endomucin+ fraction at E11.5. Detailed analyses of these fractions uncovered drastic changes in their BM repopulating capacities as well as in vitro cytokine responsiveness within this narrow time frame. Our findings establish Endomucin as a novel cell-surface marker for LTR-HSCs throughout development and provide a powerful tool in understanding HSC ontogeny

Essential and Instructive Roles of GATA Factors in Eosinophil Development

GATA transcription factors are major regulators of hematopoietic and immune system. Among GATA factors, GATA-1, GATA-2, and GATA-3 play crucial roles in the development of erythroid cells, hematopoietic stem, and progenitor cells, and T helper type 2 (Th2) cells, respectively. A high level of GATA-1 and GATA-2 expression has been observed in eosinophils, but their roles in eosinophil development remain uncertain both in vitro and in vivo. Here we show that enforced expression of GATA-1 in human primary myeloid progenitor cells completely switches myeloid cell fate into eosinophils. Expression of GATA-1 exclusively promotes development and terminal maturation of eosinophils. Functional domain analyses revealed that the COOH-terminal finger is essential for this capacity while the other domains are dispensable. Importantly, GATA-1–deficient mice failed to develop eosinophil progenitors in the fetal liver. On the other hand, GATA-2 also showed instructive capacity comparable to GATA-1 in vitro and efficiently compensated for GATA-1 deficiency in terms of eosinophil development in vivo, indicating that proper accumulation of GATA factors is critical for eosinophil development. Taken together, our findings establish essential and instructive roles of GATA factors in eosinophil development. GATA-1 and GATA-2 could be novel molecular targets for therapeutic approaches to allergic inflammation

Reciprocal Roles for CCAAT/Enhancer Binding Protein (C/EBP) and PU.1 Transcription Factors in Langerhans Cell Commitment

Myeloid progenitor cells give rise to a variety of progenies including dendritic cells. However, the mechanism controlling the diversification of myeloid progenitors into each progeny is largely unknown. PU.1 and CCAAT/enhancing binding protein (C/EBP) family transcription factors have been characterized as key regulators for the development and function of the myeloid system. However, the roles of C/EBP transcription factors have not been fully identified because of functional redundancy among family members. Using high titer–retroviral infection, we demonstrate that a dominant-negative C/EBP completely blocked the granulocyte–macrophage commitment of human myeloid progenitors. Alternatively, Langerhans cell (LC) commitment was markedly facilitated in the absence of tumor necrosis factor (TNF)α, a strong inducer of LC development, whereas expression of wild-type C/EBP in myeloid progenitors promoted granulocytic differentiation, and completely inhibited TNFα-dependent LC development. On the other hand, expression of wild-type PU.1 in myeloid progenitors triggered LC development in the absence of TNFα, and its instructive effect was canceled by coexpressed C/EBP. Our findings establish reciprocal roles for C/EBP and PU.1 in LC development, and provide new insight into the molecular mechanism of LC development, which has not yet been well characterized

A KRAB Domain Zinc Finger Protein in Imprinting and Disease

International audienc

Somatic Donor Cell Type Correlates with Embryonic, but Not Extra-Embryonic, Gene Expression in Postimplantation Cloned Embryos

<div><p>The great majority of embryos generated by somatic cell nuclear transfer (SCNT) display defined abnormal phenotypes after implantation, such as an increased likelihood of death and abnormal placentation. To gain better insight into the underlying mechanisms, we analyzed genome-wide gene expression profiles of day 6.5 postimplantation mouse embryos cloned from three different cell types (cumulus cells, neonatal Sertoli cells and fibroblasts). The embryos retrieved from the uteri were separated into embryonic (epiblast) and extraembryonic (extraembryonic ectoderm and ectoplacental cone) tissues and were subjected to gene microarray analysis. Genotype- and sex-matched embryos produced by <i>in vitro</i> fertilization were used as controls. Principal component analysis revealed that whereas the gene expression patterns in the embryonic tissues varied according to the donor cell type, those in extraembryonic tissues were relatively consistent across all groups. Within each group, the embryonic tissues had more differentially expressed genes (DEGs) (>2-fold vs. controls) than did the extraembryonic tissues (<i>P</i><1.0×10^–26). In the embryonic tissues, one of the common abnormalities was upregulation of <i>Dlk1</i>, a paternally imprinted gene. This might be a potential cause of the occasional placenta-only conceptuses seen in SCNT-generated mouse embryos (1–5% per embryos transferred in our laboratory), because dysregulation of the same gene is known to cause developmental failure of embryos derived from induced pluripotent stem cells. There were also some DEGs in the extraembryonic tissues, which might explain the poor development of SCNT-derived placentas at early stages. These findings suggest that SCNT affects the embryonic and extraembryonic development differentially and might cause further deterioration in the embryonic lineage in a donor cell-specific manner. This could explain donor cell-dependent variations in cloning efficiency using SCNT.</p></div

Expression of LOCKs-related genes in SCNT-derived embryos.

<p>Raw intensity values of LOCKs-related genes at E3.5 (blastocysts) and E6.5. The data set at E3.5 was from a previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076422#pone.0076422-Inoue1" target="_blank">[12]</a> (accession number: GSE23181). At E3.5, all the LOCKs genes examined were strongly repressed in SCNT-derived blastocysts compared with those in control IVF-derived blastocysts. At E6.5, after implantation, the genes were downregulated in the embryonic tissue and no significant differences were found between the SCNT- and IVF-derived embryos. By contrast, in the extraembryonic tissues, <i>Xlr</i> genes (e.g., <i>Xlr4b</i>, <i>Xlr3c</i>, <i>Xlr5a</i> and <i>Xlr5c</i>) remained active after implantation and in the SCNT-derived samples the expression levels were restored to nearly normal. Bla, blastocysts; Em, embryonic samples; Ex, extraembryonic samples; CC, cumulus cell-derived clone; SC, Sertoli cell-derived clone.</p

Fold change analysis of gene expression profiles of embryonic and extraembryonic samples.

<p>Fold change analysis with a cutoff of f >2 identified the number of differentially expressed genes (DEGs) in SCNT-derived samples compared with IVF-derived controls. (<b>A</b>) Histogram showing the distribution of DEGs classified by the relative values compared with the IVF-derived controls. The embryonic samples included more DEGs (901–4642) than the extraembryonic samples (272–592). This tendency is depicted by the narrower and higher distribution patterns of the extraembryonic samples. (<b>B</b>) The cumulative numbers of up- and downregulated DEGs shown side-by-side based on fold changes. Dark and light bars represent up- and downregulated DEGs, respectively. In the embryonic tissues, large fold changes occurred predominantly with the upregulated DEGs, whereas in the extraembryonic tissues such changes occurred with the downregulated DEGs. *No corresponding gene.</p

Expression levels of genes important for early placentation.

<p>Genes important for maintenance of undifferentiated trophoblast cells (<i>Cdx2</i>, <i>Esrrb</i> and <i>Eomes</i>) were downregulated while those essential for differentiation into giant cells (<i>Hand1</i>) were upregulated. * <i>P</i><0.05, ** <i>P</i><0.01 (compared with the corresponding IVF-derived controls). “†” indicates a tendency for down- or upregulation, but not with a statistical significance (<i>P</i><0.10).</p