Impaired embryonic haematopoiesis yet normal arterial development in the absence of the Notch ligand Jagged1
2008; Springer Nature; Volume: 27; Issue: 13 Linguagem: Inglês
10.1038/emboj.2008.113
ISSN1460-2075
AutoresAlexandre Robert‐Moreno, Jordi Guiu, Cristina Ruiz‐Herguido, Manuel Enrique Lanas López, Julia Inglés‐Esteve, L. Riera, Alex J. Tipping, Tariq Enver, Elaine Dzierzak, Thomas Gridley, Lluı́s Espinosa, Anna Bigas,
Tópico(s)Acute Myeloid Leukemia Research
ResumoArticle5 June 2008free access Impaired embryonic haematopoiesis yet normal arterial development in the absence of the Notch ligand Jagged1 Àlex Robert-Moreno Àlex Robert-Moreno Centre Oncologia Molecular, IDIBELL, Gran Via km 2.7 Hospitalet, Barcelona, Spain These authors contributed equally to this workPresent address: Universitat Pompeu Fabra, Barcelona, Spain Search for more papers by this author Jordi Guiu Jordi Guiu Centre Oncologia Molecular, IDIBELL, Gran Via km 2.7 Hospitalet, Barcelona, Spain These authors contributed equally to this work Search for more papers by this author Cristina Ruiz-Herguido Cristina Ruiz-Herguido Centre Oncologia Molecular, IDIBELL, Gran Via km 2.7 Hospitalet, Barcelona, Spain Search for more papers by this author M Eugenia López M Eugenia López Centre Oncologia Molecular, IDIBELL, Gran Via km 2.7 Hospitalet, Barcelona, Spain Search for more papers by this author Julia Inglés-Esteve Julia Inglés-Esteve Centre Oncologia Molecular, IDIBELL, Gran Via km 2.7 Hospitalet, Barcelona, Spain Search for more papers by this author Lluis Riera Lluis Riera Centre Oncologia Molecular, IDIBELL, Gran Via km 2.7 Hospitalet, Barcelona, SpainPresent address: Diferenciació i Cancer, Center de Regulació Genòmica (CRG-PRBB), Barcelona, Spain Search for more papers by this author Alex Tipping Alex Tipping MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford, UK Search for more papers by this author Tariq Enver Tariq Enver MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford, UK Search for more papers by this author Elaine Dzierzak Elaine Dzierzak Department of Cell Biology and Genetics, Erasmus University Medical Center, Rotterdam, The Netherlands Search for more papers by this author Thomas Gridley Thomas Gridley The Jackson Laboratory, Bar Harbor, ME, USA Search for more papers by this author Lluis Espinosa Lluis Espinosa Centre Oncologia Molecular, IDIBELL, Gran Via km 2.7 Hospitalet, Barcelona, Spain These authors contributed equally to this work Search for more papers by this author Anna Bigas Corresponding Author Anna Bigas Centre Oncologia Molecular, IDIBELL, Gran Via km 2.7 Hospitalet, Barcelona, Spain These authors contributed equally to this work Search for more papers by this author Àlex Robert-Moreno Àlex Robert-Moreno Centre Oncologia Molecular, IDIBELL, Gran Via km 2.7 Hospitalet, Barcelona, Spain These authors contributed equally to this workPresent address: Universitat Pompeu Fabra, Barcelona, Spain Search for more papers by this author Jordi Guiu Jordi Guiu Centre Oncologia Molecular, IDIBELL, Gran Via km 2.7 Hospitalet, Barcelona, Spain These authors contributed equally to this work Search for more papers by this author Cristina Ruiz-Herguido Cristina Ruiz-Herguido Centre Oncologia Molecular, IDIBELL, Gran Via km 2.7 Hospitalet, Barcelona, Spain Search for more papers by this author M Eugenia López M Eugenia López Centre Oncologia Molecular, IDIBELL, Gran Via km 2.7 Hospitalet, Barcelona, Spain Search for more papers by this author Julia Inglés-Esteve Julia Inglés-Esteve Centre Oncologia Molecular, IDIBELL, Gran Via km 2.7 Hospitalet, Barcelona, Spain Search for more papers by this author Lluis Riera Lluis Riera Centre Oncologia Molecular, IDIBELL, Gran Via km 2.7 Hospitalet, Barcelona, SpainPresent address: Diferenciació i Cancer, Center de Regulació Genòmica (CRG-PRBB), Barcelona, Spain Search for more papers by this author Alex Tipping Alex Tipping MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford, UK Search for more papers by this author Tariq Enver Tariq Enver MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford, UK Search for more papers by this author Elaine Dzierzak Elaine Dzierzak Department of Cell Biology and Genetics, Erasmus University Medical Center, Rotterdam, The Netherlands Search for more papers by this author Thomas Gridley Thomas Gridley The Jackson Laboratory, Bar Harbor, ME, USA Search for more papers by this author Lluis Espinosa Lluis Espinosa Centre Oncologia Molecular, IDIBELL, Gran Via km 2.7 Hospitalet, Barcelona, Spain These authors contributed equally to this work Search for more papers by this author Anna Bigas Corresponding Author Anna Bigas Centre Oncologia Molecular, IDIBELL, Gran Via km 2.7 Hospitalet, Barcelona, Spain These authors contributed equally to this work Search for more papers by this author Author Information Àlex Robert-Moreno1,5, Jordi Guiu1,5, Cristina Ruiz-Herguido1, M Eugenia López1, Julia Inglés-Esteve1, Lluis Riera1, Alex Tipping2, Tariq Enver2, Elaine Dzierzak3, Thomas Gridley4, Lluis Espinosa1,5 and Anna Bigas 1,5 1Centre Oncologia Molecular, IDIBELL, Gran Via km 2.7 Hospitalet, Barcelona, Spain 2MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford, UK 3Department of Cell Biology and Genetics, Erasmus University Medical Center, Rotterdam, The Netherlands 4The Jackson Laboratory, Bar Harbor, ME, USA 5These authors contributed equally to this work *Corresponding author. Centre Oncologia Molecular, IDIBELL, Gran Via Km 2.7, Hospitalet, Barcelona 08907, Spain. Tel.: +932 607 404; Fax: +932 607 426; E-mail: [email protected] The EMBO Journal (2008)27:1886-1895https://doi.org/10.1038/emboj.2008.113 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Specific deletion of Notch1 and RBPjκ in the mouse results in abrogation of definitive haematopoiesis concomitant with the loss of arterial identity at embryonic stage. As prior arterial determination is likely to be required for the generation of embryonic haematopoiesis, it is difficult to establish the specific haematopoietic role of Notch in these mutants. By analysing different Notch-ligand-null embryos, we now show that Jagged1 is not required for the establishment of the arterial fate but it is required for the correct execution of the definitive haematopoietic programme, including expression of GATA2 in the dorsal aorta. Moreover, successful haematopoietic rescue of the Jagged1-null AGM cells was obtained by culturing them with Jagged1-expressing stromal cells or by lentiviral-mediated transduction of the GATA2 gene. Taken together, our results indicate that Jagged1-mediated activation of Notch1 is responsible for regulating GATA2 expression in the AGM, which in turn is essential for definitive haematopoiesis in the mouse. Introduction Haematopoietic cells originate from different embryonic sites during development and are generated in close association with the vascular endothelium. Some of the best characterized haematopoietic sites in the embryo include the yolk sac (Yoder and Hiatt, 1997; Yoder et al, 1997a, 1997b), the intra-embryonic para-aortic splanchnopleura (P-sp)/AGM (aorta-gonad-mesonephros) (Medvinsky and Dzierzak, 1996; Cumano et al, 2001), umbilical and vitelline arteries (de Bruijn et al, 2000), allantois (Zeigler et al, 2006; Dieterlen-Lievre, 2007) and placenta (Gekas et al, 2005; Ottersbach and Dzierzak, 2005). The AGM region generates the first adult repopulating haematopoietic stem cells (HSCs) but it is as yet unclear whether some or all these tissues can de novo generate such cells. It is thought that niche-dependent signals can influence the properties of the presumptive haematopoieitc cells as they emerge and function to meet the haematopoietic needs of the embryo. Thus, a variety of haematopoietic cells (as defined by functional assays) emerge, including myeloid progenitors, multipotent progenitors and in vivo repopulating HSC. The Notch pathway is involved in the regulation of cell-fate decisions by cell–cell interactions in a variety of developmental systems, including haematopoiesis. In adult haematopoiesis, the best characterized function of Notch is in the development of T (Radtke et al, 1999) and B cells (Pui et al, 1999; Tanigaki et al, 2002) (reviewed in Radtke et al, 2004). In addition, Notch is likely to participate in other aspects of haematopoietic homoeostasis including stem cell maintenance as shown by the ability to immortalize pluripotent haematopoietic cells (Varnum-Finney et al, 2000). It has been shown that germline mutant embryos deficient for Notch1 and RBPjκ cannot generate intra-embryonic HSC in different organisms, whereas no major haematopoietic defects have been found in the yolk sac haematopoiesis of these mutants (Kumano et al, 2003; Burns et al, 2005; Robert-Moreno et al, 2005, 2007). Moreover, Notch1-deficient ES-cell derived chimaeras never contributed to adult haematopoiesis but did contribute to yolk sac-derived haematopoiesis (Hadland et al, 2004). Several studies in zebrafish also support a role for Notch signalling in definitive but not primitive haematopoiesis (Burns et al, 2005; Gering and Patient, 2005). Although these observations suggest that Notch is involved in the generation of the definitive HSC in the embryo, in vivo experiments in which Notch1 or Jagged1 was conditionally deleted in the adult by the interferon-inducible Mx-cre failed to support the idea that Notch has a similar function in adult HSCs (Radtke et al, 1999; Mancini et al, 2005). In addition, a conclusive proof for the haematopoietic Notch function in the embryo remains elusive, mainly because of the abnormal arterial development of all the analysed Notch mutants. The onset of the haematopoietic programme in the aortic endothelial-like cells is characterized by the transcriptional activation of the Runx1 and GATA2 genes, which are required for the formation of definitive HSC (Tsai et al, 1994; Okuda et al, 1996; North et al, 2002; Ling et al, 2004). We previously described that GATA2 was a direct target of Notch in the AGM region (Robert-Moreno et al, 2005), and data on haematopoietic specification in zebrafish support Runx1 as a downstream factor of the genetic cascade initiated by Notch signalling (Burns et al, 2005). However, there is no evidence that Runx1 is a direct target of Notch. To further identify and characterize the genetic cascade involving Notch and its role in haematopoietic specification, we examined the putative haematopoietic defects on embryos deficient for Jagged1, Jagged2 or Delta4, the three ligands that are present in the dorsal aorta of the mid-gestation embryo. Notch ligands Jagged1, Jagged2 and Delta4 are known to interact and activate all Notch receptors (Shimizu et al, 2000; Shutter et al, 2000). Targeted disruption of each of these genes results in embryonic (Jagged1 and Delta4) or perinatal (Jagged2) lethality, suggesting that all have essential, non-redundant functions (Jiang et al, 1998; Xue et al, 1999; Duarte et al, 2004; Krebs et al, 2004). In vitro cultures with Jagged1 (Varnum-Finney et al, 1998; Karanu et al, 2000), Delta1 (Varnum-Finney et al, 2003) and Delta4 (Dando et al, 2005) have been used for expansion of haematopoietic progenitors or stem cells with the purpose of improving cell therapy protocols. However, to date there is no proof for the involvement of Notch ligands in the embryonic haematopoiesis and maintenance of adult haematopoietic progenitors as well as the individual contribution of these ligands in vivo. Here, we describe that the Notch ligand Jagged1 is directly involved in activating Notch in AGM haematopoietic cells, non-cell autonomously. This results in the transcriptional activation of GATA2 in these cells and the formation of the HSC and their progeny. Results Heterogeneous expression of Notch family members within the aortic haematopoietic clusters Previously, we showed that Notch1, Notch4, Jagged1, Jagged2 and Delta4 are expressed in the mid-gestation AGM region in the mouse embryo (Robert-Moreno et al, 2005). We further studied the expression pattern of these molecules in the haematopoietic clusters emerging from the aortic endothelium at E9.5–10.5 (20–40sp). We found that Notch1 is expressed in most of the cells of the haematopoietic clusters similar to Jagged1 expression. In contrast, Notch4 is strongly decreased in the clustered cells compared with the surrounding endothelium (Figure 1A). Profound heterogeneity was found in the expression patterns of Jagged2 and Delta4 ligands in these structures. Jagged2 was heterogeneously expressed within the cells of a single cluster, whereas Delta4 was expressed in individual clusters and completely absent in others. Detection of activated Notch1 (Figure 1B) with specific antibody recognizing cleaved-Notch1 (Souilhol et al, 2006; Del Monte et al, 2007), concomitant with the presence of the Notch target genes hes1, hrt1 and GATA2 mRNA (Figure 1C) further indicates that Notch signalling is active and functional in the cells of the haematopoietic clusters in the aorta. These results together with the haematopoietic defects observed in the Notch1 and RBPjκ-null embryos (Kumano et al, 2003; Robert-Moreno et al, 2005) support the notion that Notch signalling has a pivotal function in the embryonic haematopoietic development. Figure 1.Heterogeneous expression of Notch family members within the aortic haematopoietic clusters. (A) Expression of the indicated genes in the AGM of E10.5 embryos by WISH. Transversal sections of dorsal aorta in a dorsal-to-ventral orientation ( × 400). Details of clustered cells are in the right panels. (B) Immunofluorescence staining of anti-N1ICv (green) overlayed with DAPI (blue) in the AGM region. Details of clusters are in the lower panels. (C) WISH of Notch target genes in clustered cells budding from the dorsal aorta. Download figure Download PowerPoint Altered haematopoiesis in the AGM of Jagged1 but not Jagged2 mutant embryos To examine the contribution of the different Notch ligands on embryonic haematopoiesis in vivo, we took advantage of the Ly-6A–GFP (Sca-1-GFP) transgenic mice (de Bruijn et al, 2002). These mice express GFP in the first HSCs, in cells of the haematopoietic clusters and haemogenic endothelium of the E10 aorta and have been used to quantitate haematopoietic clusters in normal and GATA2 mutant embryos (Ling et al, 2004). We crossed Jag1+/Δ or Jag2+/Δ (Jiang et al, 1998; Xue et al, 1999) with Ly-6A–GFP mice and determined the number of GFP+ cells in the haematopoietic clusters and endothelium of the aorta of precisely timed embryos. We found similar numbers of GFP+ cells in the AGM of E10–11 Jag2Δ/Δ/Ly-6A–GFP embryos compared with their wild-type littermates. Conversely, the number of GFP+ cells found in the aortic endothelium of Jag1Δ/Δ/Ly-6A–GFP embryos was reduced at least 50% at three different developmental stages (Figure 2A and B), suggesting an important role for Jagged1 in embryonic haematopoiesis. Although the Notch ligand Delta4 was also expressed in the aorta, the strong vascular defects of the Delta4+/− mutants precluded this study (Duarte et al, 2004; Krebs et al, 2004). Figure 2.Altered haematopoiesis in the AGM of Jagged1 but not Jagged2 mutant embryos. (A) Precisely timed E10.5–11 Ly-6A–GFP, Jag1Δ/Δ/Ly-6A–GFP or Jag2Δ/Δ/Ly-6A–GFP embryos were sectioned and the number of GFP+ cells lining the dorsal aorta was counted. Representative photographs from these embryos are shown. The orientation is dorsal-to-ventral ( × 400). (B) Bars represent the number of GFP+ cells found in 100 μm of AGM aorta from three different Jag1Δ/Δ/Ly-6A–GFP or Jag2Δ/Δ/Ly-6A–GFP embryos compared with their wild-type littermates. (C) Bars represent the number of CFC from Jag1Δ/Δ or Jag2Δ/Δ E10.5 embryos compared with their wild-type littermates. (D) Percent of CD45+ cells obtained in liquid culture of AGM cells from Jag1+/+ and Jag1Δ/Δ. (E) Fold increase in the percent of CD45+ cells when cells are cultured on the OP9 cells. (F) Percent of CD45+ cells obtained from Jag1+/+ and Jag1Δ/Δ AGM cells cultured without stroma or on Jag1+/+ or Jag1Δ/Δ MEF. (G) Expression levels of different Notch ligands in the OP9 cells compared with Jag1+/+ and Jag1Δ/Δ fibroblast and (H) in dissected E10.5 AGM from littermates of different Jag1 genotypes. Download figure Download PowerPoint To further examine the influence of Notch ligands on haematopoietic development, haematopoietic progenitors in the different mutants were quantitated by colony-forming cell (CFC) assays. AGM tissues from E10–11 Jag1Δ/Δ embryos contained a severely reduced number of total haematopoietic progenitors (Figure 2C) and all progenitor types (myeloid, erythroid and mix colonies) were reduced (data not shown) compared with both Jag1+/+ and Jag1+/Δ. In contrast, we did not detect any variation in the number and type of CFC in Jagged2 mutant AGMs. These results are in agreement with the absence of Ly-6A–GFP+ cells in the aorta of Jagged1 but not Jagged2 mutant embryos. The capacity of Jag1Δ/Δ AGM tissues to generate haematopoietic cells was tested in liquid cultures. In the absence of stromal cells, a strong reduction in the percentage of CD45+ cells generated from Jag1Δ/Δ AGMs was observed as compared with the wild-type or heterozygous mutants (from 80% in Jag1+/+ to 20% in Jag1Δ/Δ) as determined by flow cytometry (Figure 2D). However, we identified a profound heterogeneity between different littermate null embryos with a range from 0 to 35% of CD45+ cells generated after liquid culture. This indicates variation in the penetrance of the Jagged1 deficiency (0–10% high penetrance and 10–35% low penetrance). We next co-cultured these cells with the OP9 stromal cell line that endogenously express Jagged1 (Figure 2G), to test whether the haematopoietic defect observed in the Jagged1 embryos was cell autonomous. When Jag1Δ/Δ AGM cells were co-cultured with OP9 stroma, the percentage of CD45+ cells generated increased up to four-fold (in both high and low penetrance null embryos) as compared with cells cultured without OP9, whereas we did not find any change in Jag1+/+ and Jag1+/Δ AGM cell co-cultures (Figure 2E). Recovery of the CD45+ population is concomitant with an increase in GATA2 expression in these cells (Supplementary Figure 1). Overexpression of Jagged1 in OP9 cells did not further increase the recovery of CD45+ cells obtained from Jag1Δ/Δ AGMs (data not shown). To further confirm that Jagged1 was responsible for the haematopoietic recovery observed in the co-culture experiments, we isolated Jag1+/+ or Jag1Δ/Δ murine embryonic fibroblast (MEF) (from E10.5 embryos) and used these cells as stromal layer. We observed a partial recovery in the number of CD45+ haematopoietic cells when Jag1Δ/Δ AGM cells were plated on Jag1+/+ MEF but not on Jag1Δ/Δ MEF cells (Figure 2F). Taken together, these results indicate that Jagged1 expression in the aorta is required in a non-haematopoietic cell autonomous manner for haematopoietic cell development. Interestingly, we found comparable levels of Jagged2 and Delta4 in both MEFs (Figure 2G) and AGM from Jag1Δ/Δ (Figure 2H) embryos indicating that Jagged1 deficiency cannot be rescued by other Notch ligands. Arterial fate is not affected in the Jagged1 mutant embryos Notch function is required for aorta specification in different organisms (Lawson et al, 2001; Duarte et al, 2004; Krebs et al, 2004). As arterial determination precedes the haematopoietic cluster emergence in the mouse AGM, we tested whether the haematopoietic defects in the Jagged1 mutant embryos were due to a failure in the arterial programme. The expression of ephrinB2, which distinguishes arteries from veins before any structural, physiologic or functional distinctions (Wang et al, 1998; Adams et al, 1999) was examined by immunostaining of E10.5 AGM sections. We found that ephrinB2 is expressed in the arteries, including the aorta, of the Jag1Δ/Δ embryos similar to the wild type and it is absent from the venous network (Figure 3A). To further confirm that arterial determination is not affected in these mutants, we tested the expression pattern of other arterial markers such as CD44 and α-smooth muscle actin (SMA). We found comparable expression of CD44 and SMA in Jag1+/+ and Jag1Δ/Δ (Figure 3A). Taken together, these results indicate that Jagged1-mediated activation of Notch is not required for arterial specification. Figure 3.Arterial fate is not affected in the Jagged1 mutant embryos. (A, B) Sections from Jag1+/+ and Jag1Δ/Δ E10.5–11 embryos showing expression of Jag1, EfnB2, CD44, SMA and CD31 by immunofluorescence or VE-cad by WISH. Confocal images for Jag1, EfnB2 and CD44 merged with the Nomarsky image are shown ( × 630). Detection of VE-cadherin (WISH) and immunofluorescence of SMA (merged with DAPI) and CD31 were obtained in Olympus BX60 at × 400. Download figure Download PowerPoint Previous studies of aortas from Notch1 and RBPjk mutant embryos showed an increase in the number of endothelial cells concomitant with a reduction in haematopoietic cells, suggesting a switch in the balance between endothelial and haematopoietic cell fates from a common precursor. To analyse whether the endothelial lineage was favoured in the Jagged1 mutants, AGM sections from wild-type and mutant embryos were examined for the expression of endothelial markers CD31 and VE-cadherin. Increased expression levels of both endothelial markers were observed in the Jag1Δ/Δ embryos compared with wild-type littermates (Figure 3B). Moreover, the aortic endothelial monolayer expressing CD31 and VE-cadherin lining the dorsal aorta displays, in these mutants, an abnormal architecture that resembles a multi-stratified endothelium. This phenotype is reminiscent of the one observed in the RBPjκ mutant embryos (Robert-Moreno et al, 2005). Altogether these results indicate that the haematopoietic phenotype of the Jagged1 mutant embryos is not due to defective arterial specification, but instead suggest a defect in haematopoietic determination from the haemogenic endothelium of the AGM region. GATA2 expression is compromised in Jagged1 mutants Although we have previously shown that GATA2 is a direct target of Notch1 signalling, we next examined whether the lack of Jagged1 or Jagged2 influences GATA2 expression in the aortic endothelium and haematopoietic clusters. The expression of Runx1, another pivotal haematopoietic transcription factor in the emergence of the aortic haematopoietic clusters, was also examined by in situ hybridization. E10.5–11 Jag1Δ/Δ showed a complete lack of GATA2 expression in the aorta (Figure 4A). Interestingly, only a few cells expressing Runx1 were found in three out of six mutant embryos. Expression of Runx1 and GATA2 was also absent from E9.5 Delta4−/− embryos (data not shown). In contrast, both transcription factors were expressed in E10.5–11 Jag2Δ/Δ embryos and their expression patterns were similar to those found in wild-type littermates (Figure 4). However, some reduction in the total number of GATA2-expressing cells was found in the AGM region of these embryos (Table I). Figure 4.GATA2 expression is compromised in Jagged1 mutants. (A) WISH for the haematopoietic transcription factors GATA2 and Runx1 in the aortic endothelium of E10.5–11 wild types, Jag1Δ/Δ or Jag2Δ/Δ embryos. Somite pair precisely timed embryos were used to compare. The orientation is dorsal-to-ventral ( × 400). (B) Graphs represent the percentage of embryos showing expression of the indicated genes from total analysed embryos. (C) Chromatin IP with the indicated antibodies from six pooled E10.5 dissected AGMs. PCR amplification of the precipitates (left) and relative fold enrichment of the GATA2 promoter as detected by qPCR (right) are shown. Download figure Download PowerPoint Table 1. Number of GATA2- and Runx1-positive cells per AGM as detected by WISH in the endothelium of the dorsal aorta in different Jagged1 and Jagged2 mutants Jagged1wt Jagged1Δ/Δ Somite pairs Genotype Cells/AGM Somite pairs Cells/AGM GATA2 expression 33 +/Δ 80 33 0 35 +/+ 120 35 3 35 +/+ 197 35 0 38 +/Δ 428 38 3 38 0 Runx1 expression 34 +/+ 259 34 0 35 +/Δ 151 35 2 38 +/+ 165 35 0 35 56 35 113 38 62 Jagged2wt Jagged2Δ/Δ GATA2 expression 38 +/Δ 246 37 94 40 +/+ 177 38 89 38 175 40 85 Runx1 expression 35 +/+ 49 35 61 38 +/+ 233 35 79 38 158 As a direct target for Notch1 in the AGM, we hypothesized that recruitment of Notch1 to the GATA2 promoter may be affected and this may influence GATA2 expression in the Jagged1 mutant embryos. By chromatin IP assay followed by qPCR analysis, we detected Notch1 recruitment on Jag1+/Δ E10.5 AGM cells, whereas no Notch1 recruitment was detected in the GATA2 promoter of Jag1Δ/Δ AGM at the same developmental stage (35–40 sp) (Figure 4C). Ectopic GATA2 expression rescues Jagged1 mutant haematopoiesis As shown in Figure 2, haematopoietic cells cannot be properly generated from Jag1Δ/Δ AGM in liquid cultures unless exogenous Jagged1 signalling is provided. As GATA2 expression is downstream of Notch activation in the AGM and it is absent in the haematopoietic clusters from Jagged1-null embryos, we next tested whether enforced expression of GATA2 on E10.5 AGM cells was sufficient to rescue Jag1Δ/Δ haematopoietic defects in vitro. Single collagenase-treated AGMs from Jag1+/+ (n=2), Jag1+/Δ (n=2) and Jag1Δ/Δ (n=3) embryos were infected with either pHRGFP or pHRhGATA2 lentivirus and maintained in liquid culture. After 7 days, cultures were assayed for the percentage of CD45+ cells by flow cytometry. We detected variable infection efficiency as measured by GFP+ (20–50%) in both wild-type and null AGM cells. We did not observe any difference in the percentage of haematopoietic cells (CD45+) in Jag1+/+, Jag1+/Δ AGM cells transduced with GATA2 compared with the control within the GFP+ population. However, a 2- to 3-fold enrichment in the percentage of CD45+ cells was consistently observed in the Jag1Δ/Δ cultures (either from high penetrance or low penetrance embryos). Thus, enforced expression of GATA2 was sufficient to partially rescue the generation of haematopoietic cells from Jag1Δ/Δ dissected AGMs (Figure 5A and B). Figure 5.Ectopic GATA2 expression rescues Jagged1 mutant haematopoiesis. (A) Analysis of CD45+ cells by flow cytometry in the GFP+ population obtained from E10.5 AGM cultures transduced with control pHRGFP and pHRhGATA2 lentivirus. Representative Jag1+/+ and two Jag1Δ/Δ with different penetrance are shown. (B) Graph represents the relative fold increase in the percent of CD45+ cells obtained in the GATA2- compared with GFP-transduced Jag1Δ/Δ cultures. (C) Flow cytometry analysis of one representative of three cultures of AGM cells transduced with the indicated lentivirus and incubated with DMSO or DAPT. The percentage of CD45+ cells within the GFP+ population is indicated. (D) Bars represent the average and standard deviation of CFCs obtained from three different cultures. Download figure Download PowerPoint To further demonstrate that GATA2 is able to rescue the haematopoietic Notch signalling defect avoiding the putative previous defects in Jag1Δ/Δ during AGM development, we used the γ-secretase inhibitor DAPT to block Notch activation in the AGM cultures and transduced with pHRGFP or pHRhGATA2 lentivirus. After 7 days, we found a decrease in the percentage of CD45+ cells in the control-transduced cells treated with DAPT compared to DMSO-treated cultures (P=0.05). Interestingly, this effect was abrogated by GATA2 expression (Figure 5C). Moreover, a strong inhibitory effect of DAPT (100-fold) was observed on the number of progenitors (CFC) that was significantly recovered in GATA2-transduced cells (P=0.002) (Figure 5D). Consistent with the higher effect of Notch inhibition on CFC assay, we found that DAPT preferentially affects the number of ckit+CD45+ progenitors compared with total CD45+ cells in AGM cultures (data not shown). Altogether our results indicate that Jagged1-mediated activation of Notch is required for the proper expression of GATA2 in the haematopoietic cells of the embryonic aorta, and that GATA2 is a required haematopoietic effector of Notch signalling in these cells. Discussion Our present work demonstrates that the Notch ligand Jagged1 is responsible for the Notch activation that permits the proper execution of the haematopoietic programme in the dorsal aorta of the AGM region. This occurs, at least partially, through GATA2, a direct transcriptional target of Notch1. We have previously shown that Delta4, Jagged1 and Jagged2 are expressed in the mid-gestation aortic endothelium; therefore, they were all candidates to activate Notch in the AGM (Robert-Moreno et al, 2005). We have now focused on studying the loss-of-function mutant embryos for Jagged1 (Xue et al, 1999), Jagged2 (Jiang et al, 1998) and Delta4 (Duarte et al, 2004; Krebs et al, 2004) to identify the putative haematopoietic Notch ligand. We found that Jagged1, but not Jagged2, is responsible for Notch activation in haematopoietic cells in the dorsal aorta. Different Notch ligands, including Jagged1, had been previously shown to influence HSCs in vitro and Jagged1 may be an important component of the osteoblastic niche for maintaining adult HSC in the bone marrow (Calvi et al, 2003). Our results demonstrate for the first time that the Notch ligand Jagged1 is required for the embryonic haematopoietic development in vivo. Although prelimin
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