Characterization of GATA-1+ hemangioblastic cells in the mouse embryo
2006; Springer Nature; Volume: 26; Issue: 1 Linguagem: Inglês
10.1038/sj.emboj.7601480
ISSN1460-2075
AutoresTomomasa Yokomizo, Satoru Takahashi, Naomi Mochizuki, Takashi Kuroha, Masatsugu Ema, Asami Wakamatsu, Ritsuko Shimizu, Osamu Ohneda, Motomi Osato, Hitoshi Okada, Toshihisa Komori, Minetaro Ogawa, Shin-Ichi Nishikawa, Yoshiaki Ito, Masayuki Yamamoto,
Tópico(s)Congenital heart defects research
ResumoArticle7 December 2006free access Characterization of GATA-1+ hemangioblastic cells in the mouse embryo Tomomasa Yokomizo Tomomasa Yokomizo Institute of Basic Medical Sciences and Center for TARA, University of Tsukuba, Tsukuba, Japan Search for more papers by this author Satoru Takahashi Corresponding Author Satoru Takahashi Institute of Basic Medical Sciences and Center for TARA, University of Tsukuba, Tsukuba, Japan Search for more papers by this author Naomi Mochizuki Naomi Mochizuki Institute of Basic Medical Sciences and Center for TARA, University of Tsukuba, Tsukuba, Japan Search for more papers by this author Takashi Kuroha Takashi Kuroha Institute of Basic Medical Sciences and Center for TARA, University of Tsukuba, Tsukuba, Japan Search for more papers by this author Masatsugu Ema Masatsugu Ema Institute of Basic Medical Sciences and Center for TARA, University of Tsukuba, Tsukuba, Japan Search for more papers by this author Asami Wakamatsu Asami Wakamatsu Institute of Basic Medical Sciences and Center for TARA, University of Tsukuba, Tsukuba, Japan Search for more papers by this author Ritsuko Shimizu Ritsuko Shimizu Institute of Basic Medical Sciences and Center for TARA, University of Tsukuba, Tsukuba, Japan Search for more papers by this author Osamu Ohneda Osamu Ohneda Institute of Basic Medical Sciences and Center for TARA, University of Tsukuba, Tsukuba, Japan JST-ERATO Environmental Response Project, University of Tsukuba, Tsukuba, Japan Search for more papers by this author Motomi Osato Motomi Osato Institute of Molecular and Cell Biology and Oncology Research Institute, Proteos, Singapore, Singapore Search for more papers by this author Hitoshi Okada Hitoshi Okada Cancer Institute, Kami-ikebukuro, Toshima-ku, Tokyo, Japan Search for more papers by this author Toshihisa Komori Toshihisa Komori Division of Cell Biology, Department of Developmental and Reconstructive Medicine, Nagasaki University Graduate School of Biomedical Sciences, Sakamoto, Nagasaki, Japan Search for more papers by this author Minetaro Ogawa Minetaro Ogawa Department of Cell Differentiation, Institute of Molecular Embryology and Genetics, Kumamoto University, Minatojima-minamicho, Chuo-ku, Kobe, Japan Search for more papers by this author Shin-Ichi Nishikawa Shin-Ichi Nishikawa Riken Center for Developmental Biology, Minatojima-minamicho, Chuo-ku, Kobe, Japan Search for more papers by this author Yoshiaki Ito Yoshiaki Ito Institute of Molecular and Cell Biology and Oncology Research Institute, Proteos, Singapore, Singapore Search for more papers by this author Masayuki Yamamoto Corresponding Author Masayuki Yamamoto Institute of Basic Medical Sciences and Center for TARA, University of Tsukuba, Tsukuba, Japan JST-ERATO Environmental Response Project, University of Tsukuba, Tsukuba, Japan Search for more papers by this author Tomomasa Yokomizo Tomomasa Yokomizo Institute of Basic Medical Sciences and Center for TARA, University of Tsukuba, Tsukuba, Japan Search for more papers by this author Satoru Takahashi Corresponding Author Satoru Takahashi Institute of Basic Medical Sciences and Center for TARA, University of Tsukuba, Tsukuba, Japan Search for more papers by this author Naomi Mochizuki Naomi Mochizuki Institute of Basic Medical Sciences and Center for TARA, University of Tsukuba, Tsukuba, Japan Search for more papers by this author Takashi Kuroha Takashi Kuroha Institute of Basic Medical Sciences and Center for TARA, University of Tsukuba, Tsukuba, Japan Search for more papers by this author Masatsugu Ema Masatsugu Ema Institute of Basic Medical Sciences and Center for TARA, University of Tsukuba, Tsukuba, Japan Search for more papers by this author Asami Wakamatsu Asami Wakamatsu Institute of Basic Medical Sciences and Center for TARA, University of Tsukuba, Tsukuba, Japan Search for more papers by this author Ritsuko Shimizu Ritsuko Shimizu Institute of Basic Medical Sciences and Center for TARA, University of Tsukuba, Tsukuba, Japan Search for more papers by this author Osamu Ohneda Osamu Ohneda Institute of Basic Medical Sciences and Center for TARA, University of Tsukuba, Tsukuba, Japan JST-ERATO Environmental Response Project, University of Tsukuba, Tsukuba, Japan Search for more papers by this author Motomi Osato Motomi Osato Institute of Molecular and Cell Biology and Oncology Research Institute, Proteos, Singapore, Singapore Search for more papers by this author Hitoshi Okada Hitoshi Okada Cancer Institute, Kami-ikebukuro, Toshima-ku, Tokyo, Japan Search for more papers by this author Toshihisa Komori Toshihisa Komori Division of Cell Biology, Department of Developmental and Reconstructive Medicine, Nagasaki University Graduate School of Biomedical Sciences, Sakamoto, Nagasaki, Japan Search for more papers by this author Minetaro Ogawa Minetaro Ogawa Department of Cell Differentiation, Institute of Molecular Embryology and Genetics, Kumamoto University, Minatojima-minamicho, Chuo-ku, Kobe, Japan Search for more papers by this author Shin-Ichi Nishikawa Shin-Ichi Nishikawa Riken Center for Developmental Biology, Minatojima-minamicho, Chuo-ku, Kobe, Japan Search for more papers by this author Yoshiaki Ito Yoshiaki Ito Institute of Molecular and Cell Biology and Oncology Research Institute, Proteos, Singapore, Singapore Search for more papers by this author Masayuki Yamamoto Corresponding Author Masayuki Yamamoto Institute of Basic Medical Sciences and Center for TARA, University of Tsukuba, Tsukuba, Japan JST-ERATO Environmental Response Project, University of Tsukuba, Tsukuba, Japan Search for more papers by this author Author Information Tomomasa Yokomizo1, Satoru Takahashi 1, Naomi Mochizuki1, Takashi Kuroha1, Masatsugu Ema1, Asami Wakamatsu1, Ritsuko Shimizu1, Osamu Ohneda1,2, Motomi Osato3, Hitoshi Okada4, Toshihisa Komori5, Minetaro Ogawa6, Shin-Ichi Nishikawa7, Yoshiaki Ito3 and Masayuki Yamamoto 1,2 1Institute of Basic Medical Sciences and Center for TARA, University of Tsukuba, Tsukuba, Japan 2JST-ERATO Environmental Response Project, University of Tsukuba, Tsukuba, Japan 3Institute of Molecular and Cell Biology and Oncology Research Institute, Proteos, Singapore, Singapore 4Cancer Institute, Kami-ikebukuro, Toshima-ku, Tokyo, Japan 5Division of Cell Biology, Department of Developmental and Reconstructive Medicine, Nagasaki University Graduate School of Biomedical Sciences, Sakamoto, Nagasaki, Japan 6Department of Cell Differentiation, Institute of Molecular Embryology and Genetics, Kumamoto University, Minatojima-minamicho, Chuo-ku, Kobe, Japan 7Riken Center for Developmental Biology, Minatojima-minamicho, Chuo-ku, Kobe, Japan *Corresponding authors: Institute of Basic Medical Sciences and Center for TARA, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba 305-8575, Japan. Tel.: +81 29 853 7516; Fax: +81 29 853 6965; E-mail: [email protected] or Tel.: +81 29 853 6158; Fax: +81 29 853 7318; E-mail: [email protected] The EMBO Journal (2007)26:184-196https://doi.org/10.1038/sj.emboj.7601480 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Hemangioblasts are thought to be one of the sources of hematopoietic progenitors, yet little is known about their localization and fate in the mouse embryo. We show here that a subset of cells co-expressing the hematopoietic marker GATA-1 and the endothelial marker VE-cadherin localize on the yolk sac blood islands at embryonic day 7.5. Clonal analysis demonstrated that GATA-1+ cells isolated from E7.0–7.5 embryos include a common precursor for hematopoietic and endothelial cells. Moreover, this precursor possesses primitive and definitive hematopoietic bipotential. By using a transgenic complementation rescue approach, GATA-1+ cell-derived progenitors were selectively restored in Runx1-deficient mice. In the rescued mice, definitive erythropoiesis was recovered but the rescued progenitors did not display multilineage hematopoiesis or intra-aortic hematopoietic clusters. These results provide evidence of the presence of GATA-1+ hemangioblastic cells in the extra-embryonic region and also their functional contribution to hematopoiesis in the embryo. Introduction During mouse development, successive phases of hematopoiesis occur in several anatomic sites (reviewed by Speck et al, 2002). Although the origins of blood cells is still controversial, a tight developmental relationship between hematopoietic and endothelial lineages has been recognized (reviewed by Nishikawa, 2001; Choi, 2002; Dieterlen-Lievre et al, 2002). The first blood cells (primitive erythrocytes) and endothelial cells are observed within the blood islands of yolk sac at embryonic day 7.5 (E7.5) (Haar and Ackerman, 1971). This feature has led to a hypothesis that there is a transient common precursor for hematopoietic and endothelial cells, the hemangioblast (Sabin, 1920; Murray, 1932). Since it is difficult to catch a narrow time window of development in vivo and to obtain experimentally sufficient number of cells from early embryo, an in vitro model system has been developed as an alternative approach for finding hemangioblasts. The embryonic stem (ES) cell differentiation system identified a putative hemangioblast, termed the blast colony-forming cell (BL-CFC), which gives rise to primitive and definitive hematopoietic cells and endothelial cells (Kennedy et al, 1997; Choi et al, 1998). Recently, the in vivo counterpart of BL-CFC was also detected in mouse primitive streak at E7.0–7.5 (Huber et al, 2004). However, the relationship between BL-CFC and yolk sac definitive progenitors, previously reported by several groups, is unknown (Yoder et al, 1997; Nishikawa et al, 1998b; Palis et al, 1999). Another type of close association between hematopoietic and endothelial cells has been observed in a later stage intraembryonic region. At the microscopic level, a cluster of hematopoietic cells, candidates of hematopoietic stem cells (HSCs) or progenitors, has been found in the aorta–gonad–mesonephros (AGM) region, which attach to the ventral aspect of the endothelium (Garcia-Porrero et al, 1995; Tavian et al, 1996; Yoshida et al, 1998; Bertrand et al, 2005). Using a tracing technique, the continuity between endothelial cells and hematopoietic cells has been proven in chick and murine embryos (Jaffredo et al, 1998; Sugiyama et al, 2003). Furthermore, VE-cadherin+ endothelial cells isolated from mouse embryos can generate hematopoietic cells including lymphoid lineages on OP9 stromal cells (Nishikawa et al, 1998b; Fraser et al, 2002), suggesting that a subset of definitive hematopoietic cells originate directly from hematogenic endothelial cells. Consistent with this, HSC activity was detected in the endothelium of the dorsal aorta, emphasizing that the origin of HSCs resides in endothelial cells (de Bruijn et al, 2002). The transcription factor GATA-1 is a central regulator of erythroid gene transcription (reviewed by Ferreira et al, 2005). GATA-1 expression in the extra-embryonic mesoderm before the establishment of blood islands was demonstrated by in situ analysis (Silver and Palis, 1997), suggesting that GATA-1 gene expression begins in the very early stages of hematopoiesis. Using an in vitro ES cell differentiation system, GATA-1 was shown to be a good marker of mesodermal cells, which possess hematopoietic activity (Robertson et al, 2000; Fujimoto et al, 2001). Previously, the transcription regulatory domain that directs both the primitive and definitive erythroid cell-specific expression of GATA-1 was identified and referred to as the GATA-1 gene hematopoietic regulatory domain or G1-HRD (Onodera et al, 1997). We also confirmed that the G1-HRD is sufficient to recapitulate the expression of the GATA-1 gene in extra-embryonic mesoderm and hematopoietic mesoderm cells generated from ES cells, as well as in erythroid cells (Onodera et al, 1997; Fujimoto et al, 2001; Shimizu et al, 2001). These observations imply that it is technically feasible to express any effector gene in hematopoietic mesoderm cells using the G1-HRD. Runx1/AML1 encodes the DNA-binding subunit of a heterodimeric transcription factor complex named polyoma enhancer binding protein 2 (PEBP2)/core-binding factor (CBF) (reviewed by Ito, 1999). Homozygous disruption of Runx1 results in embryonic lethality secondary to a complete block in fetal liver definitive hematopoiesis (Okuda et al, 1996; Wang et al, 1996; Okada et al, 1998). Previous analyses revealed that there are no hematopoietic cell clusters on endothelial cells in Runx1-deficient embryos and further, that sorted VE-cadherin+ endothelial cells from E10.5 embryos do not generate hematopoietic colonies (North et al, 1999; Yokomizo et al, 2001). These results suggested that Runx1 is required for the generation of definitive hematopoietic cells from endothelial cells. We show here that GATA-1+ cells at E7.0–7.5 have both hematopoietic and endothelial potential. As the GATA-1-like expression of Runx1 is also detectable in the extra-embryonic mesoderm and blood islands before the formation of intra-aortic clusters (North et al, 1999; Lacaud et al, 2002), we generated transgenic mouse lines expressing Runx1 under the control of the G1-HRD and performed a complementation rescue experiment of Runx1 function. As expected, definitive hematopoiesis in the compound mutant embryos was partially rescued. However, intra-aortic clusters were absent, indicating that only GATA-1+ cell-derived progenitors were restored in Runx1-deficient mice. The rescued progenitors did not have the properties of HSC. These data demonstrate that GATA-1 expression marks hemangioblastic cells in the extra-embryonic region and that these cells display restricted hematopoietic potential in vivo. Results Identification of GATA-1 and VE-cadherin double-positive cells in yolk sac blood Islands In early mouse ontogeny, GATA-1 expression is observed in the extra-embryonic mesoderm and blood islands of the yolk sac (Onodera et al, 1997; Silver and Palis, 1997). Since the function of GATA-1 at this stage has not been well characterized, we examined the expression pattern of GATA-1 using transgenic mouse lines expressing green fluorescent protein (GFP) under the control of the G1-HRD (Nishimura et al, 2000). At E7.5 (early headfold stage; Downs and Davies, 1993), GATA-1 expression is restricted to the blood islands of the yolk sac (Supplementary Figure S1). Almost all cells within the blood islands were positive for GFP (Figure 1A(a, d and h)). The GFP expression pattern overlapped with that of endogenous GATA-1 as detected with anti-GATA-1 antibody (Figure 1A(e and f)). These results indicate that the transgenic GFP reporter can effectively monitor GATA-1+ cells in the early embryonic stage. Figure 1.GATA-1+ cells in blood islands coexpress VE-cadherin. (A) Immunohistochemical analysis of G1-HRD-GFP transgenic embryo at E7.5 (early headfold stage, EHF). (a) Fluorescence microscopic analysis shows GFP+ cells are present in the blood islands. (b) Section of (a). The blue color represents staining with Hoechst 33342. (c–j) Serial sections of the blood island depicted in the boxed region in (b). Hoechst dye staining is shown in (c) and (g). Staining with anti-GFP (d and h), anti-GATA-1 (e) and anti-VE-cadherin (i) antibodies are also shown. Merged images of GFP and GATA-1 (f) and GFP and VE-cadherin (j). (B) FACS analysis of G1-HRD-GFP transgenic embryos. Note that most GFP+ cells are positive for VE-cadherin+ (highlighted by a red circle) at E7.5. (C) Immunohistochemical analysis of G1-HRD-GFP transgenic embryo at E10.5. Transverse section of AGM region, stained with anti-GFP (green) and anti-PECAM-1 (red) antibodies. The dorsal aspect is upward. (b) This is higher magnification of the area within (a) indicated by the box. GFP+ cells (white arrows) are observed in circulating cells, not in endothelial cells or hematopoietic cell clusters (white arrowheads). Abbreviations: ex, extra-embryonic region; em, embryonic region; bi, blood island; ve, visceral endoderm; me, mesothelium; da, dorsal aorta. Scale bars: (A) 50 μm; (C) 100 μm. Download figure Download PowerPoint To our surprise, these GATA-1+ cells co-expressed VE-cadherin (Figure 1A(i)), a known endothelial cell marker in midgestation (Nishikawa et al, 1998b). The expression profiles of GFP and VE-cadherin merged nicely (Figure 1A(j)). This observation was further confirmed by fluorescence activated cell sorter (FACS) analysis (Figure 1B). In E7.5 embryos, almost all GFP+ cells were VE-cadherin+ (Figure 1B, highlighted by a red circle). By contrast, the expression of GATA-1 and VE-cadherin became mutually exclusive after E8.5 both in the yolk sac and embryo proper (Figure 1B, right panels). At E10.5, GATA-1 expression in the AGM region was predominantly observed in erythroid cells and not in PECAM-1+ endothelial cells (Figure 1C), in agreement with our previous analysis (Onodera et al, 1997). PECAM-1 was also expressed in blood island GFP+ cells at E7.5 (Supplementary Figure S2; Ema et al, 2006). Although the morphology of the blood island GFP+ cells at E7.5 did not coincide with that of a typical endothelial cell (not shown), these results demonstrate that hematopoietic and endothelial markers are co-expressed in blood island cells and suggest that these cells might be early precursors before or during commitment to the hematopoietic or endothelial lineage. Definitive hematopoietic potential of GATA-1+ cells It was previously demonstrated that VE-cadherin+ cells derived from embryos or ES cells have definitive hematopoietic potential (Nishikawa et al, 1998a, 1998b). To further characterize the blood island GATA-1+ cells, they were sorted from E7.5 G1-HRD-GFP transgenic mouse embryos. Approximately 5% of these cells were GFP+ and these cells were efficiently recovered (Figure 2A). GFP+ cells expressed the transcription factors GATA-1, GATA-2, Runx1 and SCL, all of which are known to be important for hematopoiesis (Figure 2B). To test the functional potential of the recovered GFP+ fraction, cells were cultured on OP9 stromal cells (Figure 2C(a and b)). GFP+ cells were capable of generating enucleated erythroid cells and mature myeloid cells (Figure 2C(c)). Also CD45 and c-Kit hematopoietic progenitor cells, as well as erythroid (Ter119) and myeloid (Mac-1 and Gr-1) cells, were recovered from these cultures (Figure 2C(d)). In contrast, GFP− cells from the E7.5 embryos (early or late headfold stage) did not produce any hematopoietic cell colonies, even when plated at 5000 cells per well (Figure 2C(b) and D), indicating that the definitive hematopoietic potential was enriched in the GATA-1+ cell fraction in E7.5 blood islands. Whereas, colony forming potential was still found in the GFP+ fraction at E8.25, the potential shifted to the GFP− fraction after E8.5 (Figure 2D). The period in which the GFP+ fraction contained progenitor cells with definitive hematopoietic potential correlated with the presence of VE-cadherin-expressing cells (Figure 1B). Figure 2.Definitive hematopoietic potential resides in GATA-1+ cells at E7.5. (A) FACS profile of the cells derived from E7.5 G1-HRD-GFP transgenic embryos. GFP+ and GFP− cells were sorted (upper panel, right) and re-analyzed (lower panels). (B) RT–PCR analysis of the expression of hematopoietic transcription factor mRNAs in GFP+ and GFP− cells sorted from E7.5 embryos. (C) Definitive hematopoietic potential of GFP+ cells derived from E7.5 G1-HRD-GFP transgenic embryo. Both GFP+ and GFP− cells were cultured on an OP9 stromal cell layer in the presence of SCF, IL-3, G-CSF and Epo. A phase-contrast microscopic photograph was taken after 4 days of culture (a and b). After 9 days of culture, the cells formed in the culture of GFP+ cells were harvested and analyzed for morphology (c) and the expression of surface markers (d). Erythrocytes, granulocytes and monocytes/macrophages were identified. Scale bar: 200 μm. (D) Definitive hematopoietic potential of GFP+ and GFP− cells sorted from embryos of different stages. The frequency of hematopoietic colony development was examined with the OP9 stromal cell culture. 500 cells were cultured per well under the same conditions as described in (C). The generation of hematopoietic colonies was judged by their morphology after 7 days of culture. Stages determined by morphological appearance are presented in parentheses. LB–EHF represents a mixture of LB and EHF stage embryos. Abbreviations: LB, late bud; EHF, early headfold; LHF, late headfold; sp, somite pairs. Download figure Download PowerPoint Clonal analysis of GATA-1+ cells sorted from E7.0–7.5 embryos The presence of cells co-expressing the hematopoietic marker GATA-1 and the endothelial marker VE-cadherin suggested that GATA-1+ cells might include the common precursor, the hemangioblast. To test this possibility, we performed a single cell deposition assay. GFP+ cells isolated from G1-HRD-GFP transgenic mouse embryos at E7.5 (early headfold and late headfold stages) were deposited into individual wells of a 96-well plate. After 1 day of culture, colonies of round cells appeared in almost 30% of wells. These cells were positive for βH1 globin, and morphologically identical to primitive erythrocytes (Supplementary Figure S3), indicating that the majority of GATA-1+ cells at E7.5 are committed primitive erythroid progenitors. After 2 days of culture, another type of colony appeared in 1.04% (6/576) of wells. These colonies expanded rapidly and generated definitive hematopoietic cells including erythrocytes, granulocytes and monocytes/macrophages (Figure 3A). Immunostaining with anti-PECAM-1 antibody was used to identify endothelial cell colonies in day 7 cultures. Among 576 cells, one cell (0.17%, three experiments) gave rise to both hematopoietic and endothelial cells. This shows that a portion of GATA-1+ cells at E7.5 has hemangioblastic activity, although at a very low frequency. Thus, we did an examination at an earlier stage, E7.0. Similar to the E7.5 embryo, GFP+ cells at E7.0 were observed in the extra-embryonic region and co-expressed endothelial markers, VE-cadherin and PECAM-1 (Figure 3B(a); Ema et al, 2006). These GFP+ cells were isolated from mid-streak to no bud stage embryos and cultured with OP9 stromal cells. After 2 days of culture, we found densely packed cell clusters in some wells (Figure 3B(b)). These clusters expanded and generated round cells around themselves (Figure 3B(c and d)). After 4 days, cell clusters were harvested and divided into two fractions. One fraction was seeded on an OP9 stromal layer and the other was subjected to methylcellulose culture to test for primitive erythroid progenitors. The culture remaining after harvest was also tested for endothelial cell colony forming potential. Among 720 cells, 5 cells (0.69%, five experiments) displayed bipotential definitive hematopoietic and endothelial potential (Figure 3B(e, f, and g)). Moreover, clusters could generate primitive erythroid colonies, which were comparable to those from E7.5 embryos (Figure 3B(h–k)) and were positive for βH1 globin (Figure 3B(l)). No hemangioblastic activity was observed in the GFP− fraction, even when up to 6600 cells were investigated. These results clearly demonstrate that a single GATA-1+ cell can differentiate into primitive and definitive hematopoietic and endothelial cells. Figure 3.Single GATA-1+ cells can differentiate into primitive and definitive hematopoietic cells and endothelial cells. GFP+ cells isolated from G1-HRD-GFP transgenic mouse were deposited into individual wells of a 96-well plate and cultured with OP9 stromal cells in the presence of SCF, IL-3, G-CSF, Epo, VEGF and Ang-1. (A) Analysis of GFP+ cells sorted from E7.5 (early to late headfold stage) embryos. A phase-contrast microscopic photograph was taken after 3 days of culture (a). After 7 days of culture, the cells formed in the culture of GFP+ cells were harvested and analyzed for morphology (b). Erythrocytes, granulocytes and monocytes/macrophages were identified. (B) Analysis of GFP+ cells sorted from E7.0 (mid-streak to no bud stage) embryos. (a) Representative picture of E7.0 embryo (no bud/early bud stage). Abbreviations: ex, extra-embryonic region; em, embryonic region. A densely packed cluster was detected at day 2 of culturing (b, arrow) and grown to a larger size (c), with the generation of round cells around it (d). Note that the shape of the colony is different from that of a typical colony formed in E7.5 culture (A(a)). After 4 days of culture, the progeny of the cluster was replated onto fresh OP9 stromal cells (e) and in methylcellulose (h). The culture remaining after harvesting was also investigated for the detection of endothelial cell colonies (g). (e and f) Erythrocytes, granulocytes and monocytes/macrophages were identified in the replated dish after an additional 3 days of culture. (g) Endothelial cell colonies formed on OP9 stromal cells were visualized by immunostaining with anti-PECAM-1 antibody. (h–l) Generation of primitive erythroid colonies from clusters. Morphology of primitive erythroid colony generated from replated clusters (h and i) was similar to those from E7.5 embryo (j and k). Inset in (h) shows a GFP-expressing colony detected by fluorescence microscopy. (l) RT–PCR analysis of globin gene expression in the colonies. Both βH1 and βmajor globins were detected in the colonies derived from the clusters (lane 4). Peripheral blood from E10.5 embryo, primitive erythroid colonies from E7.5 embryo (primitive erythrocytes, lanes 1 and 2, respectively) and BFU-E from E10.5 yolk sac (definitive erythrocytes, lane 3) were used as controls. Scale bars: (Aa, Bb, Be, and Bg) 100 μm; (Ab, Bf, Bh, and Bi) 25 μm. Download figure Download PowerPoint GATA-1+ cell-specific expression of Runx1 in a Runx1−/− background The data so far suggest that a portion of definitive hematopoietic progenitors may be generated from GATA-1+ hemangioblastic cells in the yolk sac. To visualize the emergence of these progenitors, immunohistochemical staining for CD45 was performed (Nishikawa et al, 1998b; Takakura et al, 2000). CD45+ cells localized to the blood island region that was positively marked with the G1-HRD-LacZ transgene (Supplementary Figure S4), supporting the notion that the blood islands of the yolk sac contain precursors of definitive hematopoiesis. To elucidate the fate of definitive hematopoietic progenitors derived from GATA-1+ hemangioblastic cells, we attempted to generate a mouse composed only of this population of definitive progenitors. We postulated that in a Runx1 null background where definitive hematopoiesis does not commence, the G1-HRD directed expression of Runx1 would rescue definitive hematopoiesis. To this end, we generated G1-HRD-Runx1 transgenic mice (Figure 4A; Supplementary Figure S5) and crossed them with Runx1 mutant mice. Figure 4.Rescue of GATA-1+ cell-derived progenitors in Runx1−/− embryos. (A) Strategy for the rescue of GATA-1+ cell-derived progenitors in Runx1−/− embryos. The G1-HRD-Runx1 transgene contains the 3.9-kb sequence 5′ of the IE exon, the IE exon itself, the first intron, and a part of the second exon of the mouse GATA-1 gene in front of Runx1 cDNA. The initiation Met codon in the second exon was replaced by a unique NotI site (shown as N) for subsequent cloning. Restriction enzyme sites are B, BamHI; E, EcoRI; N, NotI; S, SacI. (B) Macroscopic appearance of G1-HRD-Runx1 transgene-rescued embryos. Embryos with each genotype at E12.5 (a–c) and E18.5 (d and e) are shown. A higher magnification picture of the skin of transgene-rescued embryo (f) reveals micro-hemorrhages in the skin. (C) Histological analysis of wild type and Runx1−/−∷Runx1-Tg+ embryos at E17.5. Hematoxylin and eosin staining of peripheral blood from wild type (a) and Runx1−/−∷Runx1-Tg+ (b) embryos are shown. Note that numerous enucleated erythrocytes are present in the Runx1−/−∷Runx1-Tg+ embryos. Panels (c) and (d) show sections stained with anti-βmajor globin antibody or anti-εy globin antibody. Hematoxylin and eosin-stained livers of wild type (e) and Runx1−/−∷Runx1-Tg+ (f) embryos. (D) FACS analysis of E14.5 fetal liver cells from wild type and Runx1−/−∷Runx1-Tg+ embryos. Download figure Download PowerPoint E12.5 Runx1−/− embryos showed the expected lethality and massive hemorrhaging in the central nervous system (Figure 4B(c)). By contrast, the embryos compound for the G1-HRD-Runx1 transgene (Runx1-Tg) were viable, showed no such morphological defects (Figure 4B(b)) and we could not distinguish the compound mutant embryos from wild-type embryos (Figure 4B(a)). To identify whether Runx1−/−∷Runx1-Tg+ embryos can complete embryogenesis, we analyzed newborn pups. Of 52 newborn mice, no live Runx1-null pups were found (an expected number of pups was 6.5). Examination at late-gestational stages revealed that Runx1−/−∷Runx1-Tg+ embryos begin to die at E14.5 (Supplementary Table S1). At E18.5, Runx1−/−∷Runx1-Tg+ pups showed flushing and massive hemorrhaging (Figure 4B(e)). Histological analysis revealed general bleeding within the central nervous system, subarachnoid space, urinary bladder, subcutaneous area and peritoneal cavity of these rescued embryos (not shown). Microaneurysms were also observed in the skin (Figure 4B(f)). Vascular abnormality in Runx1−/−∷Runx1-Tg+ mice at E18.5 was more prominent than that in Runx1-null pups at E12.5 (Supplementary Figure S6). Thus, G1-HRD directed Runx1-Tg expression rescues the Runx1 deficient hemorrhaging phenotype and embryonic lethality up to E18.5. Partial restoratio
Referência(s)