Ectopic Runx1 expression rescues Tal-1-deficiency in the generation of primitive and definitive hematopoiesis.
ABSTRACT: The transcription factors SCL/Tal-1 and AML1/Runx1 control the generation of pluripotent hematopoietic stem cells (pHSC) and, thereby, primitive and definitive hematopoiesis, during embryonic development of the mouse from mesoderm. Thus, Runx1-deficient mice generate primitive, but not definitive hematopoiesis, while Tal-1-deficient mice are completely defective. Primitive as well as definitive hematopoiesis can be developed "in vitro" from embryonic stem cells (ESC). We show that wild type, as well as Tal-1(-/-) and Runx1(-/-) ESCs, induced to differentiation, all expand within 5 days to comparable numbers of Flk1(+) mesodermal cells. While wild type ESCs further differentiate to primitive and definitive erythrocytes, to c-fms(+)Gr1(+)Mac1(+) myeloid cells, and to B220(+)CD19(+) B- and CD4(+)/CD8(+) T-lymphoid cells, Runx1(-/-) ESCs, as expected, only develop primitive erythrocytes, and Tal-1(-/-) ESCs do not generate any hematopoietic cells. Retroviral transduction with Runx1 of Runx1(-/-) ESCs, differentiated for 4 days to mesoderm, rescues definitive erythropoiesis, myelopoiesis and lymphopoiesis, though only with 1-10% of the efficiencies of wild type ESC hematopoiesis. Surprisingly, Tal-1(-/-) ESCs can also be rescued at comparably low efficiencies to primitive and definitive erythropoiesis, and to myelopoiesis and lymphopoiesis by retroviral transduction with Runx1. These results suggest that Tal-1 expression is needed to express Runx1 in mesoderm, and that ectopic expression of Runx1 in mesoderm is sufficient to induce primitive as well as definitive hematopoiesis in the absence of Tal-1. Retroviral transduction of "in vitro" differentiating Tal-1(-/-) and Runx1(-/-) ESCs should be a useful experimental tool to probe selected genes for activities in the generation of hematopoietic progenitors "in vitro", and to assess the potential transforming activities in hematopoiesis of mutant forms of Tal-1 and Runx1 from acute myeloid leukemia and related tumors.
Project description:A genomic variant in the human ADTRP [androgen-dependent tissue factor (TF) pathway inhibitor (TFPI) regulating protein] gene increases the risk of coronary artery disease, the leading cause of death worldwide. TFPI is the TF pathway inhibitor that is involved in coagulation. Here, we report that adtrp and tfpi form a regulatory axis that specifies primitive myelopoiesis and definitive hematopoiesis, but not primitive erythropoiesis or vasculogenesis. In zebrafish, there are 2 paralogues for adtrp (i.e., adtrp1 and adtrp2). Knockdown of adtrp1 expression inhibits the specification of hemangioblasts, as shown by decreased expression of the hemangioblast markers, etsrp, fli1a, and scl; blocks primitive hematopoiesis, as shown by decreased expression of pu.1, mpo, and l-plastin; and disrupts the specification of hematopoietic stem cells (definitive hematopoiesis), as shown by decreased expression of runx1 and c-myb However, adtrp1 knockdown does not affect erythropoiesis during primitive hematopoiesis (no effect on gata1 or h-bae1) or vasculogenesis (no effect on kdrl, ephb2a, notch3, dab2, or flt4). Knockdown of adtrp2 expression does not have apparent effects on all markers tested. Knockdown of adtrp1 reduced the expression of tfpi, and hematopoietic defects in adtrp1 morphants were rescued by tfpi overexpression. These data suggest that the regulation of tfpi expression is one potential mechanism by which adtrp1 regulates primitive myelopoiesis and definitive hematopoiesis.-Wang, L., Wang, X., Wang, L., Yousaf, M., Li, J., Zuo, M., Yang, Z., Gou, D., Bao, B., Li, L., Xiang, N., Jia, H., Xu, C., Chen, Q., Wang, Q. K. Identification of a new adtrp1-tfpi regulatory axis for the specification of primitive myelopoiesis and definitive hematopoiesis.
Project description:The transcription factor Runx1 (AML1) is a central regulator of hematopoiesis and is required for the formation of definitive hematopoietic stem cells (HSCs). Runx1 is alternatively expressed from two promoters: the proximal (P2) prevails during primitive hematopoiesis, while the distal (P1) dominates in definitive HSCs. Although some transcription factor binding sites and cis-regulatory elements have been identified, a mechanistic explanation for the alternative promoter usage remains elusive. We investigated DNA methylation of known Runx1 cis-elements at stages of hematopoietic development in vivo and during differentiation of murine embryonic stem cells (ESCs) in vitro. In vivo, we find loss of methylation correlated with the primitive to definitive transition at the P1 promoter. In vitro, hypomethylation, acquisition of active chromatin modifications, and increased transcriptional activity at P1 are promoted by direct interaction with HOXB4, a transcription factor that confers definitive repopulation status on primitive hematopoietic progenitors. These data demonstrate a novel role for DNA methylation in the alternative promoter usage at the Runx1 locus and identify HOXB4 as a direct activator of the P1 promoter. This epigenetic signature should serve as a novel biomarker of HSC potential in vivo, and during ESC differentiation in vitro.
Project description:The molecular program controlling hematopoietic differentiation is not fully understood. Here, we describe a family of zebrafish genes that includes a novel hematopoietic regulator, draculin-like 3 (drl.3). We found that drl.3 is expressed in mesoderm-derived hematopoietic cells and is retained during erythroid maturation. Moreover, drl.3 expression correlated with erythroid development in gata1a- and spi1b-depleted embryos. Loss-of-function analysis indicated that drl.3 plays an essential role in primitive erythropoiesis and, to a lesser extent, myelopoiesis that is independent of effects on vasculature, emergence of primitive and definitive progenitor cells and cell viability. While drl.3 depletion reduced gata1a expression and inhibited erythroid development, enforced expression of gata1a was not sufficient to rescue erythropoiesis, indicating that the regulation of hematopoiesis by drl.3 extends beyond control of gata1a expression. Knockdown of drl.3 increased the proportion of less differentiated, primitive hematopoietic cells without affecting proliferation, establishing drl.3 as an important regulator of primitive hematopoietic cell differentiation.
Project description:Shifting sites of blood cell production during development is common across widely divergent phyla. In zebrafish, like other vertebrates, hematopoietic development has been roughly divided into two waves, termed primitive and definitive. Primitive hematopoiesis is characterized by the generation of embryonic erythrocytes in the intermediate cell mass and a distinct population of macrophages that arises from cephalic mesoderm. Based on previous gene expression studies, definitive hematopoiesis has been suggested to begin with the generation of presumptive hematopoietic stem cells (HSCs) along the dorsal aorta that express c-myb and runx1. Here we show, using a combination of gene expression analyses, prospective isolation approaches, transplantation, and in vivo lineage-tracing experiments, that definitive hematopoiesis initiates through committed erythromyeloid progenitors (EMPs) in the posterior blood island (PBI) that arise independently of HSCs. EMPs isolated by coexpression of fluorescent transgenes driven by the lmo2 and gata1 promoters exhibit an immature, blastic morphology and express only erythroid and myeloid genes. Transplanted EMPs home to the PBI, show limited proliferative potential, and do not seed subsequent hematopoietic sites such as the thymus or pronephros. In vivo fate-mapping studies similarly demonstrate that EMPs possess only transient proliferative potential, with differentiated progeny remaining largely within caudal hematopoietic tissue. Additional fate mapping of mesodermal derivatives in mid-somitogenesis embryos suggests that EMPs are born directly in the PBI. These studies provide phenotypic and functional analyses of the first hematopoietic progenitors in the zebrafish embryo and demonstrate that definitive hematopoiesis proceeds through two distinct waves during embryonic development.
Project description:Retinoic acid (RA) is known to regulate definitive myelopoiesis but its role in vertebrate primitive myelopoiesis remains unclear. Here we report that zebrafish primitive myelopoiesis is restricted by RA in a dose dependent manner mainly before 11 hpf (hours post fertilization) when anterior hemangioblasts are initiated to form. RA treatment significantly reduces expressions of anterior hemangioblast markers scl, lmo2, gata2 and etsrp in the rostral end of ALPM (anterior lateral plate mesoderm) of the embryos. The result indicates that RA restricts primitive myelopoiesis by suppressing formation of anterior hemangioblasts. Analyses of ALPM formation suggest that the defective primitive myelopoiesis resulting from RA treatment before late gastrulation may be secondary to global loss of cells for ALPM fate whereas the developmental defect resulting from RA treatment during 10-11 hpf should be due to ALPM patterning shift. Overexpressions of scl and lmo2 partially rescue the block of primitive myelopoiesis in the embryos treated with 250 nM RA during 10-11 hpf, suggesting RA acts upstream of scl to control primitive myelopoiesis. However, the RA treatment blocks the increased primitive myelopoiesis caused by overexpressing gata4/6 whereas the abolished primitive myelopoiesis in gata4/5/6 depleted embryos is well rescued by 4-diethylamino-benzaldehyde, a retinal dehydrogenase inhibitor, or partially rescued by knocking down aldh1a2, the major retinal dehydrogenase gene that is responsible for RA synthesis during early development. Consistently, overexpressing gata4/6 inhibits aldh1a2 expression whereas depleting gata4/5/6 increases aldh1a2 expression. The results reveal that RA signaling acts downstream of gata4/5/6 to control primitive myelopoiesis. But, 4-diethylamino-benzaldehyde fails to rescue the defective primitive myelopoiesis in either cloche embryos or lycat morphants. Taken together, our results demonstrate that RA signaling restricts zebrafish primitive myelopoiesis through acting downstream of gata4/5/6, upstream of, or parallel to, cloche, and upstream of scl.
Project description:Overexpression of miRNA, miR-24, in mouse hematopoietic progenitors increases monocytic/ granulocytic differentiation and inhibits B cell development. To determine if endogenous miR-24 is required for hematopoiesis, we antagonized miR-24 in mouse embryonic stem cells (ESCs) and performed in vitro differentiations. Suppression of miR-24 resulted in an inability to produce blood and hematopoietic progenitors (HPCs) from ESCs. The phenotype is not a general defect in mesoderm production since we observe production of nascent mesoderm as well as mesoderm derived cardiac muscle and endothelial cells. Results from blast colony forming cell (BL-CFC) assays demonstrate that miR-24 is not required for generation of the hemangioblast, the mesoderm progenitor that gives rise to blood and endothelial cells. However, expression of the transcription factors Runx1 and Scl is greatly reduced, suggesting an impaired ability of the hemangioblast to differentiate. Lastly, we observed that known miR-24 target, Trib3, is upregulated in the miR-24 antagonized embryoid bodies (EBs). Overexpression of Trib3 alone in ESCs was able to decrease HPC production, though not as great as seen with miR-24 knockdown. These results demonstrate an essential role for miR-24 in the hematopoietic differentiation of ESCs. Although many miRNAs have been implicated in regulation of hematopoiesis, this is the first miRNA observed to be required for the specification of mammalian blood progenitors from early mesoderm.
Project description:Runt-related transcription factor 1 (RUNX1) is required for definitive hematopoiesis; however, the functions of most human RUNX1 isoforms are unclear. In particular, the effects of RUNX1-205 (a novel splice variant that lacks exon 6 in comparison with RUNX1b) on human hematopoiesis are not clear. In this study, a human embryonic stem cell (hESC) line with inducible RUNX1-205 overexpression was established. Analyses of these cells revealed that induction of RUNX1-205 overexpression at early stage did not influence the induction of mesoderm but blocked the emergence of CD34+ cells, and the production of hematopoietic stem/progenitor cells was significantly reduced. In addition, the expression of hematopoiesis-related factors was downregulated. However, these effects were abolished when RUNX1-205 overexpression was induced after Day 6 in co-cultures of hESCs and AGM-S3 cells, indicating that the inhibitory effect occurred prior to generation of hemogenic endothelial cells, while the promotive effect could be observed during the late stage of hematopoiesis. This is very similar to that of RUNX1b. Interestingly, the mRNA expression profile of RUNX1-205 during hematopoiesis was distinct from that of RUNX1b, and the protein stability of RUNX1-205 was much higher than that of RUNX1b. Thus, the function of RUNX1-205 in normal and diseased models should be further explored.
Project description:Targeted disruption of the Runx1/ AML1 gene in mice has demonstrated that it is required for the emergence of definitive hematopoietic cells, but that it is not essential for the formation of primitive erythrocytes. These findings led to the conclusion that Runx1 is a stage-specific transcription factor acting only during definitive hematopoiesis. However, the zebrafish and Xenopus homologues of Runx1 have been shown to play roles in primitive hematopoiesis, suggesting that mouse Runx1 might also be involved in the development of primitive lineages. In order to identify differentially expressed genes in runx1-/- primitive erythroid, we carried out microarray analysis. Keywords: genetic modification Overall design: Total RNA from E10.5 wild type and Runx1–/– yolk sacs was extracted, and gene expression patterns were compared.
Project description:Targeted disruption of the Runx1/ AML1 gene in mice has demonstrated that it is required for the emergence of definitive hematopoietic cells, but that it is not essential for the formation of primitive erythrocytes. These findings led to the conclusion that Runx1 is a stage-specific transcription factor acting only during definitive hematopoiesis. However, the zebrafish and Xenopus homologues of Runx1 have been shown to play roles in primitive hematopoiesis, suggesting that mouse Runx1 might also be involved in the development of primitive lineages. In order to identify differentially expressed genes in runx1-/- primitive erythroid, we carried out microarray analysis. Experiment Overall Design: Total RNA from E10.5 wild type and Runx1–/– yolk sacs was extracted, and gene expression patterns were compared.
Project description:The hemangioblast is a multipotential progenitor, which is derived from the mesoderm and can further differentiate into hematopoietic and endothelial lineages. The molecular mechanism governing the specification of hemangioblasts is fundamental to regenerative medicine based on embryonic stem cells for the treatment of various hematologic and vascular diseases. Here we show that aggf1 acts at the top of the genetic regulatory hierarchy in the specification of hemangioblasts in zebrafish. Knockdown of aggf1 expression decreases expression of endothelial cell-specific markers (cdh5, admr) and disrupts primitive hematopoiesis as shown by a decreased number of erythroid cells and reduced expression of gata1 (marker for erythroid progenitors) and pu.1 (myeloid progenitors). Aggf1 knockdown also decreases expression of runx1 and c-myb, indicating that it is required for specification of hematopoietic stem cells (definitive hematopoiesis). Aggf1 knockdown led to dramatically reduced expression of hemangioblast markers fli1, etsrp, lmo2, and scl, and hematopoietic/endothelial defects in aggf1 morphants were rescued by messenger RNA for scl, fli-vp16, or etsrp. Taken together, these data indicate that aggf1 is involved in differentiation of both hematopoietic and endothelial lineages and that aggf1 acts upstream of scl, fli1, and etsrp in specification of hemangioblasts.