Histone methylation is an important regulator of gene expression; its coordinated activity is critical in complex developmental processes such as hematopoiesis. Disruptor of telomere silencing 1-like (DOT1L) is a unique histone methyltransferase that specifically methylates histone H3 at lysine 79. We analyzed Dot1L-mutant mice to determine influence of this enzyme on embryonic hematopoiesis. Mutant mice developed more slowly than wild-type embryos and died between embryonic days 10.5 and 13.5, displaying a striking anemia, especially apparent in small vessels of the yolk sac. Further, a severe, selective defect in erythroid, but not myeloid, differentiation was observed. Erythroid progenitors failed to develop normally, showing retarded progression through the cell cycle, accumulation during G 0 /G 1 stage, and marked increase in apoptosis in response to erythroid growth factors. GATA2, a factor essential for early erythropoiesis, was significantly reduced in Dot1L-deficient cells, whereas expression of PU.1, a transcription factor that inhibits erythropoiesis and promotes myelopoiesis, was increased. These data suggest a model whereby DOT1L-dependent lysine 79 of histone H3 methylation serves as a critical regulator of a differentiation switch during early hematopoiesis, regulating steady-state levels of GATA2 and PU.1 transcription, thus controlling numbers of circulating erythroid and myeloid cells. IntroductionAmong the first differentiated cell types to emerge in the developing mammalian embryo are the blood cells. In the mouse, the process of blood development, hematopoiesis, begins at approximately embryonic day 7.0-7.5 (E7.0-E7.5), when cells originating in the primitive streak migrate to the site of yolk sac formation. 1 By E7.5, the cells coalesce into blood islands, where they mature, proliferate, and differentiate. 2 These early hematopoietic progenitors, termed primitive erythroid colony-forming cells, are nucleated red cells, which express primitive globins and can carry oxygen to nourish the developing embryo on the initiation of blood flow after E8.5. 1,[3][4][5] The presence of these primitive progenitors is transient, peaking in numbers at E8.0 and disappearing by E9.0, 2 whereas the progeny erythrocytes persist throughout gestation. 6 After E8.5, a second wave of hematopoietic progenitors emerges from a variety of sites, including the vasculature about the aorta-gonadmesonephros and the yolk sac. These cells enter the circulation and migrate to the developing fetal liver. There, they proliferate and undergo "definitive" maturation, giving rise to multiple adult hematopoietic lineages, including mature, enucleated erythrocytes. 7 This multistep process of hematopoiesis and the fate decisions of the developing cells are regulated by the precisely controlled, sequential induction and silencing of gene expression in response to a variety of growth and differentiation factors. 8 The identity of the cell-type specific genes that direct differentiation, the factors controlling their expression, and...

BackgroundThe signaling cascades that direct the morphological differentiation of the vascular system during early embryogenesis are not well defined. Several signaling pathways, including Notch and VEGF signaling, are critical for the formation of the vasculature in the mouse. To further understand the role of Notch signaling during endothelial differentiation and the genes regulated by this pathway, both loss-of-function and gain-of-function approaches were analyzed in vivo.ResultsConditional transgenic models were used to expand and ablate Notch signaling in the early embryonic endothelium. Embryos with activated Notch1 signaling in the vasculature displayed a variety of defects, and died soon after E10.5. Most notably, the extraembryonic vasculature of the yolk sac displayed remodeling differentiation defects, with greatly enlarged lumens. These phenotypes were distinct from endothelial loss-of-function of RBPJ, a transcriptional regulator of Notch activity. Gene expression analysis of RNA isolated from the yolk sac endothelia of transgenic embryos indicated aberrant expression in a variety of genes in these models. In particular, a variety of secreted factors, including VEGF and TGF-β family members, displayed coordinate expression defects in the loss-of-function and gain-of-function models.ConclusionsMorphological analyses of the in vivo models confirm and expand the understanding of Notch signaling in directing endothelial development, specifically in the regulation of vessel diameter in the intra- and extraembryonic vasculature. Expression analysis of these in vivo models suggests that the vascular differentiation defects may be due to the regulation of key genes through the Notch-RBPJ signaling axis. A number of these genes regulated by Notch signaling encode secreted factors, suggesting that Notch signaling may mediate remodeling and vessel diameter in the extraembryonic yolk sac via autocrine and paracrine cell communication. We propose a role for Notch signaling in elaborating the microenvironment of the nascent arteriole, suggesting novel regulatory connections between Notch signaling and other signaling pathways during endothelial differentiation.

Histone methylation of H3 is catalyzed by at least two families of proteins, the PRMT family and the SET family. Recent studies have found a new histone H3 methyltransferase without a SET domain named Dot1L (disrupter of telomere silencing 1-like). To investigate the function of Dot1L, a Dot1L deficient mouse line was generated by injection of murine embryonic stem (ES) cells containing a gene trap into C57BL/6 female blastocysts (clone #RRR032; Bay Genomics, San Francisco, CA). Fibroblasts from Dot1L-homozygous mutant embryos were completely defective in their ability to methylate lysine 79 of histone H3. All knockout embryos died by embryonic day 13. The knockout embryos showed significant reductions in blood and blood vessel formation. Further, colony assays revealed a defect primarily in early erythropoiesis. The early erythroid precursor cells were defective in their ability to respond to erythropoietin, and the cells were more susceptible to early apoptosis after stimulation. We conclude that Dot1L plays an important role in early hematopoiesis, primarily affecting early erythroid precursor cells.