| Abstract |
Erf is a ubiquitously expressed transcriptional repressor regulated by MAPK via nucleo-cytoplasmic shuttling. In its non-phosphorylated form, Erf is nuclear and active as a repressor, while phosphorylation by Erks in the nucleus, leads to cytoplasmic export and inactivation. In its active nuclear form, Erf blocks cell proliferation and arrests cells at G0/G1 phase in a cell type-specific manner. Moreover, Erk signaling is essential to “transform” the cell cycle inhibitory and proapoptotic action of TGF on epithelial cells, into a growth promoting and migratory response, rendering it as repressor of epithelial mesenchymal transition. Erf is also able to suppress ets- and ras-induced tumorigenicity in fibroblasts and Ewing’s Sarcoma cells.
However, little is known for the role of Erf in development. Total elimination of Erf in mice leads to lethality at embryonic day E10.5 due to placental defects. Erf knockout embryos fail to undergo chorioallantoic fusion and labyrinth development. To bypass placenta defects and study the role of Erf in the embryo proper, we employed epiblast conditional mice. Here, we show that Erf is required in all three waves of embryo hematopoiesis.
The first hematopoietic cells are formed in blood islands in the mouse embryo and have arisen from extraembryonic mesoderm at the neural plate stage of development at embryonic day 7.5 (E7.5) and are called the primitive wave. These primitive erythroblasts that come from the yolk sac comprise the predominant cells in the early circulating blood, resulting to erythroid, macrophage and megakaryocyte cell lineage in bloodstream. Shortly after, at embryonic day E8.25, a second wave is produced in the yolk sac, called the definitive wave and is consisted of erythroid and myeloid progenitors (EMPs). It migrates in bloodstream and finally colonize the fetal liver at embryonic day E10.5. A third wave of hematopoiesis that emerges from the aorta – gonad – mesonephros (AGM) region at E10.5, also migrates in the beginning to the fetal liver and finally the bone marrow, where it comprises the adult hematopoietic stem cells (HSC). Even though HSCs can be found in yolk sac, the AGM seems to be the major source of the adult HSCs.
12
Mice lacking Erf in embryo proper exhibited severe anemia and died around embryonic day (E) 14.5. Erf-/- embryos had reduced peripheral blood cells from E9.5 onwards that precipitated at E14.5. Elimination of Erf resulted in both reduced and more immature blood cells at E9.5-10.5 based on hematoxylin and eosin assays in yolks sacs, counts of circulating blood cells and real-time for transcription levels of globins in circulating blood. These data indicate a defect in the primitive hematopoietic wave. In addition, reduced erythroid-myeloid progenitors and colony forming cells could be detected at peripheral blood at E10.5 based on flow cytometry and colony assays, indicating a defect in the yolk sac derived definitive hematopoietic progenitors. Liver hematopoiesis was also impaired with decreased numbers of precursor and mature cells and inefficient differentiation of immature erythroid precursors. Assays with specific markers for flow cytometry and colony assays confirmed the inefficient differentiation of erythroblasts. Finally, elimination of Erf appeared to be necessary for hematopoietic stem cell maintenance or differentiation, as evident by their impaired repopulation ability.
Moreover, we show that reduced levels of Erf cause craniosynostosis in human and mice. Erf mutations were found in humans with craniosynostosis and their significance was studied with cloning of Erf mutations and luciferase assays. Moreover, Runx2, a possible target of Erf in osteogenesis found by Chip-seq, was studied by co-transfection of Erf and Runx2 followed by luciferase assay. It would thus appear that Erf affects the efficiency of the entire hematopoiesis and osteogenesis. To that extend, Erf could serve as an appealing intervention target.
|
The role of erf in mouse development
