A gene trap screen reveals the expression of the transcription factor Gfi1.1 in haemogenic endothelial cells of the Zebrafish embryo

In vertebrates, haematopoiesis occurs in two waves. The primitive wave gives rise to transient myeloid and erythroid cells whereas the definitive wave generates haematopoietic stem cells (HSCs), which maintain the blood system throughout life. These HSCs are able to self-renew and to give rise to progenitors that differentiate into mature cells of all blood lineages. Little is known about the cellular origin and molecular programming of HSCs. This knowledge is useful to generate HSCs in vitro from embryonic stem cells or induced pluripotent cells. In zebrafish, HSCs form in the intermediate cell mass (ICM), in the trunk of the embryo. Here, they develop dorsal to the primitive red blood cells and in close association with the ventral wall of the dorsal aorta (DA). Like their mammalian counterparts, they express the transcription factors runx-1 and c-myb. As in other vertebrates, zebrafish HSCs are thought to arise from the haemogenic endothelium in the ventral wall of the DA. A signalling cascade that involves the Hedgehog, Vascular endothelial growth factor (Vegf) and Notch signalling pathways is needed for arterial specification of the DA and for HSC formation. Short-term lineage tracing experiments showed that cells in the ventral wall of the DA first seed the tail mesenchyme (caudal haematopoietic tissue, CHT) through blood circulation before they seed the final sites of haematopoiesis, the thymus and kidney in the adult fish. Here, we conducted a tol2-transposon based gene trap vector screen with eGFP as the reporter gene with the aim to label nascent HSCs in vivo and to identify novel genes involved in haematopoiesis. We obtained 174 transgenic lines with tissue-specific eGFP expression in non-haematopoietic and haematopoietic tissues. We identified two lines with marker gene expression in haematopoietic cells. One of the transgenic lines, I-551:eGFP, showed reporter gene expression in primitive red blood cells and in endothelial cells in the ventral wall of the DA at 25 hours post fertilization (hpf). Using inverse PCR we identified the trapped gene in I-551:eGFP as gfi1.1, the homolog of the mouse Growth independence factor 1 (Gfi1), a transcriptional repressor expressed in HSCs. Here, we present results that the transgenic line Gfi1.1:eGFP enables us to follow emerging haematopoietic progenitors from the ventral wall of the dorsal aorta in their subsequent migration to the CHT, before they seed the final haematopoietic sites, the kidney and thymus. We show that Gfi1.1:eGFP expression is restricted to the ventral wall of the dorsal aorta by combining the transgenic line with endothelial and aorta-specific transgenic lines. We further demonstrate that the endothelial expression of the eGFP in the aorta is dependent on the vegf and notch signalling pathway and co-localizes to cells which also express the transcription factors runx1 and c-myb. When Gfi1.1:eGFP embryos are injected with the runx1 morpholino (MO), gfi1.1 expression in haemogenic endothelial cells initially occurs, which indicates that initial gfi1.1 expression is independent of runx1. But a reduction in the number of eGFP positive cells is observed at 50 hpf in the CHT. Using time-lapse imaging, we were able to visualize gfi1.1 positive cells detaching from the haemogenic endothelium. This observation indicates that gfi1.1 positive cells in the CHT are derived from the haemogenic endothelium and therefore nascent HSCs. We therefore strongly suggest that this transgenic line labels haemogenic endothelial cells at 26hpf. The transgenic line Gfi1.1:eGFP therefore provides a tool for studying HSC development since its expression labels the emergence of nascent HSCs from haemogenic endothelial cells and continues to be expressed in larval and adult haematopoietic sites

A gene trap transposon eliminates haematopoietic expression of zebrafish Gfi1aa, but does not interfere with haematopoiesis

A transposon-mediated gene trap screen identified the zebrafish line qmc551 that expresses a GFP reporter in primitive erythrocytes and also in haemogenic endothelial cells, which give rise to haematopoietic stem and progenitor cells (HSPCs) that seed sites of larval and adult haematopoiesis. The transposon that mediates this GFP expression is located in intron 1 of the gfi1aa gene, one of three zebrafish paralogs that encode transcriptional repressors homologous to mammalian Gfi1 and Gfi1b proteins. In qmc551 transgenics, GFP expression is under the control of the endogenous gfi1aa promoter, recapitulates early gfi1aa expression and allows live observation of gfi1aa promoter activity. While the transposon integration interferes with the expression of gfi1aa mRNA in haematopoietic cells, homozygous qmc551 fish are viable and fertile, and display normal primitive and definitive haematopoiesis. Retained expression of Gfi1b in primitive erythrocytes and upregulation of Gfi1ab at the onset of definitive haematopoiesis in homozygous qmc551 carriers, are sufficient to allow normal haematopoiesis. This finding contradicts previously published morpholino data that suggested an essential role for zebrafish Gfi1aa in primitive erythropoiesi

Identification of novel regulators of developmental hematopoiesis using Endoglin regulatory elements as molecular probes.

Enhancers are the primary determinants of cell identity, and specific promoter/enhancer combinations of Endoglin (ENG) have been shown to target blood and endothelium in the embryo. Here, we generated a series of embryonic stem cell lines, each targeted with reporter constructs driven by specific promoter/enhancer combinations of ENG, to evaluate their discriminative potential and value as molecular probes of the corresponding transcriptome. The Eng promoter (P) in combination with the -8/+7/+9-kb enhancers, targeted cells in FLK1 mesoderm that were enriched for blast colony forming potential, whereas the P/-8-kb enhancer targeted TIE2+/c-KIT+/CD41- endothelial cells that were enriched for hematopoietic potential. These fractions were isolated using reporter expression and their transcriptomes profiled by RNA-seq. There was high concordance between our signatures and those from embryos with defects at corresponding stages of hematopoiesis. Of the 6 genes that were upregulated in both hemogenic mesoderm and hemogenic endothelial fractions targeted by the reporters, LRP2, a multiligand receptor, was the only gene that had not previously been associated with hematopoiesis. We show that LRP2 is indeed involved in definitive hematopoiesis and by doing so validate the use of reporter gene-coupled enhancers as probes to gain insights into transcriptional changes that facilitate cell fate transitions.National Health and Medical Research Council of Australia, Australian Research Council, Dr Tom Bee Stem Cell Research Fund, Cancer Research UK, Biotechnology and Biological Sciences Research Council, Leukaemia and Lymphoma Research, The Leukaemia and Lymphoma Society, core support grants by the Wellcome Trust to the Cambridge Institute for Medical Research and Wellcome Trust - MRC Cambridge Stem Cell Institute (Grant IDs: R01 HL04880, P015PO1HL32262-32, 5P30 DK49216, 5R01 DK53298, 5U01 HL10001-05, R24 DK092760)This is the author accepted manuscript. The final version is available from the American Society of Hematology via http://dx.doi.org/10.1182/blood-2016-02-69787

GFI1 proteins regulate stem cell formation in the AGM

In vertebrates, the first haematopoietic stem cells (HSCs) with multi-lineage and long-term repopulating potential arise in the AGM (aorta-gonad-mesonephros) region. These HSCs are generated from a rare and transient subset of endothelial cells, called haemogenic endothelium (HE), through an endothelial-to-haematopoietic transition (EHT). Here, we establish the absolute requirement of the transcriptional repressors GFI1 and GFI1B (growth factor independence 1 and 1B) in this unique trans-differentiation process. We first demonstrate that Gfi1 expression specifically defines the rare population of HE that generates emerging HSCs. We further establish that in the absence of GFI1 proteins, HSCs and haematopoietic progenitor cells are not produced in the AGM, revealing the critical requirement for GFI1 proteins in intra-embryonic EHT. Finally, we demonstrate that GFI1 proteins recruit the chromatin-modifying protein LSD1, a member of the CoREST repressive complex, to epigenetically silence the endothelial program in HE and allow the emergence of blood cells.We thank the staff at the Advanced Imaging, animal facility, Molecular Biology Core facilities and Flow Cytometry of CRUK Manchester Institute for technical support and Michael Lie-A-Ling and Elli Marinopoulou for initiating the DamID-PIP bioinformatics project. We thank members of the Stem Cell Biology group, the Stem Cell Haematopoiesis groups and Martin Gering for valuable advice and critical reading of the manuscript. Work in our laboratory is supported by the Leukaemia and Lymphoma Research Foundation (LLR), Cancer Research UK (CRUK) and the Biotechnology and Biological Sciences Research Council (BBSRC). SC is the recipient of an MRC senior fellowship (MR/J009202/1).This is the author accepted manuscript. The final version is available from NPG via http://dx.doi.org/10.1038/ncb327

A gene trap screen reveals the expression of the transcription factor Gfi1.1 in haemogenic endothelial cells of the Zebrafish embryo

In vertebrates, haematopoiesis occurs in two waves. The primitive wave gives rise to transient myeloid and erythroid cells whereas the definitive wave generates haematopoietic stem cells (HSCs), which maintain the blood system throughout life. These HSCs are able to self-renew and to give rise to progenitors that differentiate into mature cells of all blood lineages. Little is known about the cellular origin and molecular programming of HSCs. This knowledge is useful to generate HSCs in vitro from embryonic stem cells or induced pluripotent cells. In zebrafish, HSCs form in the intermediate cell mass (ICM), in the trunk of the embryo. Here, they develop dorsal to the primitive red blood cells and in close association with the ventral wall of the dorsal aorta (DA). Like their mammalian counterparts, they express the transcription factors runx-1 and c-myb. As in other vertebrates, zebrafish HSCs are thought to arise from the haemogenic endothelium in the ventral wall of the DA. A signalling cascade that involves the Hedgehog, Vascular endothelial growth factor (Vegf) and Notch signalling pathways is needed for arterial specification of the DA and for HSC formation. Short-term lineage tracing experiments showed that cells in the ventral wall of the DA first seed the tail mesenchyme (caudal haematopoietic tissue, CHT) through blood circulation before they seed the final sites of haematopoiesis, the thymus and kidney in the adult fish. Here, we conducted a tol2-transposon based gene trap vector screen with eGFP as the reporter gene with the aim to label nascent HSCs in vivo and to identify novel genes involved in haematopoiesis. We obtained 174 transgenic lines with tissue-specific eGFP expression in non-haematopoietic and haematopoietic tissues. We identified two lines with marker gene expression in haematopoietic cells. One of the transgenic lines, I-551:eGFP, showed reporter gene expression in primitive red blood cells and in endothelial cells in the ventral wall of the DA at 25 hours post fertilization (hpf). Using inverse PCR we identified the trapped gene in I-551:eGFP as gfi1.1, the homolog of the mouse Growth independence factor 1 (Gfi1), a transcriptional repressor expressed in HSCs. Here, we present results that the transgenic line Gfi1.1:eGFP enables us to follow emerging haematopoietic progenitors from the ventral wall of the dorsal aorta in their subsequent migration to the CHT, before they seed the final haematopoietic sites, the kidney and thymus. We show that Gfi1.1:eGFP expression is restricted to the ventral wall of the dorsal aorta by combining the transgenic line with endothelial and aorta-specific transgenic lines. We further demonstrate that the endothelial expression of the eGFP in the aorta is dependent on the vegf and notch signalling pathway and co-localizes to cells which also express the transcription factors runx1 and c-myb. When Gfi1.1:eGFP embryos are injected with the runx1 morpholino (MO), gfi1.1 expression in haemogenic endothelial cells initially occurs, which indicates that initial gfi1.1 expression is independent of runx1. But a reduction in the number of eGFP positive cells is observed at 50 hpf in the CHT. Using time-lapse imaging, we were able to visualize gfi1.1 positive cells detaching from the haemogenic endothelium. This observation indicates that gfi1.1 positive cells in the CHT are derived from the haemogenic endothelium and therefore nascent HSCs. We therefore strongly suggest that this transgenic line labels haemogenic endothelial cells at 26hpf. The transgenic line Gfi1.1:eGFP therefore provides a tool for studying HSC development since its expression labels the emergence of nascent HSCs from haemogenic endothelial cells and continues to be expressed in larval and adult haematopoietic sites.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Notch Signaling in HSC Emergence: When, Why and How

The hematopoietic stem cell (HSC) sustains blood homeostasis throughout life in vertebrates. During embryonic development, HSCs emerge from the aorta-gonads and mesonephros (AGM) region along with hematopoietic progenitors within hematopoietic clusters which are found in the dorsal aorta, the main arterial vessel. Notch signaling, which is essential for arterial specification of the aorta, is also crucial in hematopoietic development and HSC activity. In this review, we will present and discuss the evidence that we have for Notch activity in hematopoietic cell fate specification and the crosstalk with the endothelial and arterial lineage. The core hematopoietic program is conserved across vertebrates and here we review studies conducted using different models of vertebrate hematopoiesis, including zebrafish, mouse and in vitro differentiated Embryonic stem cells. To fulfill the goal of engineering HSCs in vitro, we need to understand the molecular processes that modulate Notch signaling during HSC emergence in a temporal and spatial context. Here, we review relevant contributions from different model systems that are required to specify precursors of HSC and HSC activity through Notch interactions at different stages of development

In the spotlight: the role of TGFβ signalling in haematopoietic stem and progenitor cell emergence

Haematopoietic stem and progenitor cells (HSPCs) sustain haematopoiesis by generating precise numbers of mature blood cells throughout the lifetime of an individual. In vertebrates, HSPCs arise during embryonic development from a specialised endothelial cell population, the haemogenic endothelium (HE). Signalling by the Transforming Growth Factor β (TGFβ) pathway is key to regulate haematopoiesis in the adult bone marrow, but evidence for a role in the formation of HSPCs has only recently started to emerge. In this review, we examine recent work in various model systems that demonstrate a key role for TGFβ signalling in HSPC emergence from the HE. The current evidence underpins two seemingly contradictory views of TGFβ function: as a negative regulator of HSPCs by limiting haematopoietic output from HE, and as a positive regulator, by programming the HE towards the haematopoietic fate. Understanding how to modulate the requirement for TGFβ signalling in HSC emergence may have critical implications for the generation of these cells in vitro for therapeutic use

A gene trap transposon eliminates haematopoietic expression of zebrafish Gfi1aa, but does not interfere with haematopoiesis.

A transposon-mediated gene trap screen identified the zebrafish line qmc551 that expresses a GFP reporter in primitive erythrocytes and also in haemogenic endothelial cells, which give rise to haematopoietic stem and progenitor cells (HSPCs) that seed sites of larval and adult haematopoiesis. The transposon that mediates this GFP expression is located in intron 1 of the gfi1aa gene, one of three zebrafish paralogs that encode transcriptional repressors homologous to mammalian Gfi1 and Gfi1b proteins. In qmc551 transgenics, GFP expression is under the control of the endogenous gfi1aa promoter, recapitulates early gfi1aa expression and allows live observation of gfi1aa promoter activity. While the transposon integration interferes with the expression of gfi1aa mRNA in haematopoietic cells, homozygous qmc551 fish are viable and fertile, and display normal primitive and definitive haematopoiesis. Retained expression of Gfi1b in primitive erythrocytes and up-regulation of Gfi1ab at the onset of definitive haematopoiesis in homozygous qmc551 carriers, are sufficient to allow normal haematopoiesis. This finding contradicts previously published morpholino data that suggested an essential role for zebrafish Gfi1aa in primitive erythropoiesis

GFI1 proteins regulate stem cell formation in the AGM

In vertebrates, the first haematopoietic stem cells (HSCs) with multi-lineage and long-term repopulating potential arise in the AGM (aorta-gonad-mesonephros) region. These HSCs are generated from a rare and transient subset of endothelial cells, called haemogenic endothelium (HE), through an endothelial-to-haematopoietic transition (EHT). Here, we establish the absolute requirement of the transcriptional repressors GFI1 and GFI1B (growth factor independence 1 and 1B) in this unique trans-differentiation process. We first demonstrate that Gfi1 expression specifically defines the rare population of HE that generates emerging HSCs. We further establish that in the absence of GFI1 proteins, HSCs and haematopoietic progenitor cells are not produced in the AGM, revealing the critical requirement for GFI1 proteins in intra-embryonic EHT. Finally, we demonstrate that GFI1 proteins recruit the chromatin-modifying protein LSD1, a member of the CoREST repressive complex, to epigenetically silence the endothelial program in HE and allow the emergence of blood cells.We thank the staff at the Advanced Imaging, animal facility, Molecular Biology Core facilities and Flow Cytometry of CRUK Manchester Institute for technical support and Michael Lie-A-Ling and Elli Marinopoulou for initiating the DamID-PIP bioinformatics project. We thank members of the Stem Cell Biology group, the Stem Cell Haematopoiesis groups and Martin Gering for valuable advice and critical reading of the manuscript. Work in our laboratory is supported by the Leukaemia and Lymphoma Research Foundation (LLR), Cancer Research UK (CRUK) and the Biotechnology and Biological Sciences Research Council (BBSRC). SC is the recipient of an MRC senior fellowship (MR/J009202/1).This is the author accepted manuscript. The final version is available from NPG via http://dx.doi.org/10.1038/ncb327

The Polycomb-associated factor PHF19 controls hematopoietic stem cell state and differentiation

Phf19, a Polycomb subunit, controls hematopoietic stem cells identity and differentiation. Adult hematopoietic stem cells (HSCs) are rare multipotent cells in bone marrow that are responsible for generating all blood cell types. HSCs are a heterogeneous group of cells with high plasticity, in part, conferred by epigenetic mechanisms. PHF19, a subunit of the Polycomb repressive complex 2 (PRC2), is preferentially expressed in mouse hematopoietic precursors. Here, we now show that, in stark contrast to results published for other PRC2 subunits, genetic depletion of Phf19 increases HSC identity and quiescence. While proliferation of HSCs is normally triggered by forced mobilization, defects in differentiation impede long-term correct blood production, eventually leading to aberrant hematopoiesis. At molecular level, PHF19 deletion triggers a redistribution of the histone repressive mark H3K27me3, which notably accumulates at blood lineage-specific genes. Our results provide novel insights into how epigenetic mechanisms determine HSC identity, control differentiation, and are key for proper hematopoiesis