* Division of Hematology/Oncology, Childrens Hospital, Department of Pediatrics and Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115, USA
Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
Department of Cell Biology, Complutense University, 28040 Madrid, Spain
Author for correspondence (e-mail: zon{at}rascal.med.harvard.edu)
Accepted 30 October 2001
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SUMMARY |
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Key words: Primitive hematopoiesis, Embryonic, Definitive, Stem cell, Zebrafish, bloodless, Non-cell autonomous, scl, gata1
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INTRODUCTION |
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Murine primitive hematopoiesis begins in the extra-embryonic mesoderm of the yolk sac around embryonic day 7.5 (E7.5) (Dzierzak and Medvinsky, 1995; Palis et al., 1999
; Robb, 1997
). In birds, primitive hematopoiesis initiates in yolk sac blood islands that arise in the posterior area opaca at the early somite stage (Dieterlen-Lievre, 1997
; Lassila et al., 1982
; Peault, 1996
; Szenberg, 1977
; Zagris, 1986
). In amphibians such as Xenopus, blood is first formed in the intra-embryonic ventral mesoderm and migrates to form a V-shaped hematopoietic blood island (Kelley et al., 1994
; Rollins-Smith and Blair, 1990
). In teleosts (bony fish) such as zebrafish, primitive hematopoiesis begins in the intra-embryonic mesodermal tissue called the intermediate cell mass (ICM), which is formed by medial migration of cells in the bilateral lateral plate mesoderm (Detrich et al., 1995
; Thompson et al., 1998
).
Several studies suggest that the first definitive hematopoietic cells arise from the yolk sac (Godin et al., 1995; Palis et al., 1999
; Wong et al., 1986
). Studies that correlate stem cell activity from in vitro clonal assays with expression of genes such as scl and gata1 demonstrate that primitive hematopoiesis takes place in the murine yolk sac, where definitive hematopoietic cells also originate (Palis et al., 1999
). In mice, yolk sac cells from day 9 embryos can provide long-term multi-lineage reconstitution, which is capable of contributing to mature peripheral blood, thymus, spleen, and bone marrow lymphoid, myeloid and erythroid cell types (Yoder et al., 1997
). Subsequently, the site of mouse definitive hematopoiesis moves to the aorta-gonad-mesonephros (AGM) region (Medvinsky and Dzierzak, 1996
; Medvinsky et al., 1993
; Muller et al., 1994
). Multi-potential hematopoietic progenitors have also been detected in murine para-aortic splanchnopleura (Dieterlen-Lievre and Le Douarin, 1993
; Godin et al., 1993
).
Factors important for the development of both primitive and definitive hematopoiesis such as Scl, Lmo2, Gata1, Gata2 and Flk1 (Kdr) have been described (Pevny et al., 1995; Porcher et al., 1996
; Robb et al., 1996
; Shalaby et al., 1997
; Shivdasani et al., 1995
; Tsai et al., 1994
; Warren et al., 1994
). Genes required specifically for definitive but not primitive hematopoiesis, such as Myb, Kit, Slf (Pou3f4), Tel, Aml1 (Runx1), Cbfb and Epo have also been identified (Lin et al., 1996
; Mucenski et al., 1991
; Ogawa et al., 1993
; Okada et al., 1998
; Sasaki et al., 1996
; Wang et al., 1997
; Wang et al., 1996
). However, a specific regulator of primitive hematopoiesis has not been reported. A primitive-specific hematopoietic defect in mammals would probably present with in utero lethality, and would not survive to demonstrate normal definitive hematopoiesis. By contrast, zebrafish is uniquely suited to uncover embryonic bloodless phenotypes, as severely anemic zebrafish embryos can be raised to adulthood in laboratory conditions (Brownlie et al., 1998
; Liao et al., 2000a
).
The development of hematopoietic precursors is regulated by both extrinsic and intrinsic cues. Several studies describe the role of secreted growth factors such as bmp4 and stem cell factor (Steel) on the induction of blood and regulation of the hematopoietic stem cells, respectively (Mead and Zon, 1998; Whetton and Spooncer, 1998
). Key intrinsic factors such as transcription factors Scl and Lmo2 are required for the formation of hematopoietic stem cells, and Gata1 is required for the differentiation of stem cells along the erythroid lineage (Orkin, 1996
). In zebrafish, scl has been demonstrated to be sufficient for specifying hematopoietic progenitors (Gering et al., 1998
). Overexpression of scl can rescue blood and endothelial cells in the cloche (clo) mutant, which specifically lacks those two cell types (Liao et al., 1998
). With respect to hematopoiesis, the clo gene appears to act in a non-cell autonomous manner in the differentiation of embryonic blood cells, where reciprocal transplantation experiments show that wild-type donor cells were less likely to express gata1 in a mutant environment (Parker and Stainier, 1999
). In addition, co-transplantation experiments show that clo is required cell autonomously in subsequent proliferation of embryonic blood cells, as wild-type donor cells always contribute a greater number of blood cells than mutant donor cells in the wild-type host.
We report our characterization of bloodless (bls), a dominant zebrafish mutation producing embryos that are severely anemic at the earliest time point that circulation can be detected. Analysis of the expression of early hematopoietic genes show that decreased number of primitive hematopoietic cells are formed from the lateral plate mesoderm. Those blood precursors that are formed fail to differentiate and undergo apoptosis. In addition to an absence of blood cells during embryogenesis, bls mutants also exhibit delayed initiation of lymphopoiesis. However, primitive macrophages develop normally in bls, illustrating distinct developmental regulation of erythroid and myeloid lineages during embryogenesis. Overexpression studies with bmp4, scl and gata1 suggest that bls may regulate primitive hematopoietic cell differentiation or survival, potentially by regulating the expression of scl. Cell transplant experiments between wild-type and bls mutant animals suggest that the bls gene is required in a non-cell autonomous manner for primitive hematopoiesis. Despite the lack of blood cells for the first 4 days of life, hematopoiesis recovers in bls mutants as the definitive blood program replaces the primitive wave. We present the first description of a primitive-specific mutant phenotype and show that the gene product is required in a non-cell autonomous manner for embryonic hematopoiesis, potentially regulating scl expression.
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MATERIALS AND METHODS |
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RNA in situ hybridization, biotin-dextran label detection and immunohistochemistry
In situ hybridization and riboprobe synthesis were performed as described (Schulte-Merker et al., 1992), with modifications (Liao, 1998
). Antisense riboprobes to cmyb, draculin, flk1, gata1, gata2, ikaros, ntl, rag1, scl, shh, spt and tbx6 have been described previously (Detrich et al., 1995
; Griffin et al., 1998
; Herbomel et al., 1999
; Hug et al., 1997
; Liao et al., 1998
; Thompson et al., 1998
; Willett et al., 1999
). Digoxigenin or fluorescein-labeled riboprobes were detected with alkaline phosphatase conjugated anti-digoxigenin or anti-fluorescein antibody, respectively. Alkaline phosphatase substrates used to yield crimson, blue and purple are Vector Red, Vector Blue and BCIP/NBT, respectively (Vector Laboratories). To detect biotin-dextran labeled cells, the Vectastain peroxidase kit was used, where colors red and blue were developed using Vector NovaRed and VIP, respectively (Vector Laboratories). Whole-mount immunohistochemistry was performed essentially as described previously (Schulte-Merker et al., 1992
) with anti-HCK-1 (1:100) followed by anti-rabbit HRP (1:300).
Acridine Orange and o-dianisidine staining
Acridine Orange staining of live embryos was performed as described, at five-somite, 15-somite, 23 hpf and 36 hpf (Seiler and Nicolson, 1999). Staining of hemoglobin by o-dianisidine was performed as previously described (Detrich et al., 1995
). In brief, unfixed embryos were dechorionated and stained for 15 minutes in the dark, with a solution consisting of o-dianisidine (0.6 mg/ml), 0.01 M sodium acetate (pH 4.5), 0.65% hydrogen peroxide and 40% (vol/vol) ethanol. Embryos for histological sections were treated with acetone and embedded in Epon-Araldite (Polysciences) plastic resin, for histological sections.
Plasmid micro-injection and expression constructs
Micro-injection of plasmid DNA was performed essentially as described (Westerfield, 1993), using a Nikon pico-injector and a Narishige micro-manipulator. The full-length bmp4 was a gift from Masataka Nikaido (Hokkaido University, Sapporo, Japan) and directionally cloned into EcoRI and XhoI of pCS2+ for overexpression. The expression construct for tolloid (pCS2:Ztld-3'MT) was a gift from Patrick Blader (IGBMC, Strasbourg, France), and gata1 (pCS2+) a gift from Sue Lyons (NIH, Bethesda, MD). The expression constructs for scl and GFP control has been described (Liao et al., 1998
). Plasmid DNA expression constructs were purified (Qiagen), quantified spectrophometrically, and diluted to 100 ng/µl in sterile double distilled H2O. Expression constructs of GFP control, bmp4, tolloid, scl and gata1 were micro-injected into wild-type, bls and clo embryos at the two- or four-cell stage. Approximately 50-80 pg of DNA was injected into the blastomeres of each embryo. Embryos were fixed at 23 hpf and analyzed by in situ hybridization using scl, gata1 or globin antisense riboprobes.
Cell transplantation
Cell transplant studies were carried out as described, with modifications herein specified (Westerfield, 1993). Donor cells were labeled with 1:1 mixture of 5% rhodamine and 5% biotin-dextran, resuspended in 0.2 M KCl. Micro-injection of embryos was performed from one- to four-cell stage, in 1x Danieus media. Donor and host embryos were dechorionated at the 16-cell stage with pronase treatment (4 mg/ml of pronase in 1x Danieus buffer), for exactly 2 minutes, followed by careful and thorough rinses in 1x Danieus buffer. Pronase dechorionation was performed in agarose (1.2% agarose in 1x Danieus) coated petri-dishes, as were all subsequent steps of embryo manipulation. Wild-type embryos used in transplant studies were derived from mating between wild-type AB fish. Mutant embryos used were derived from mating between bls homozygote adults, guaranteeing a clutch of embryos with mutant genotype. At the sphere stage, donor cells (25-40) were transplanted into the margin of sphere stage host animals. Donor and host animals were fixed at 23 hpf for in situ hybridization analysis and detection of biotin-dextran-labeled donor cells.
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Results |
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Analysis of early hematopoietic markers in bls
At the five-somite stage, expression of scl in the lateral plate mesoderm marks the specification of primitive hematopoietic progenitors (Gering et al., 1998; Liao et al., 1998
; Mead et al., 1998
). By 23 hpf, scl is expressed in the ICM (region overlying the yolk tube and extending slightly caudally, forming a wedge) and an anatomically distinct tailbud derived population, referred to as posterior ICM (Detrich et al., 1995
). Expression of scl was greatly reduced in the lateral plate mesoderm of bls mutants, suggesting that the bls gene product participates in the specification of hematopoietic progenitor cells (Fig. 2A). From 18-somite to 23 hpf, scl expression in the ICM of bls mutants decreased progressively, until only a few cells were detectable in the posterior ICM at 23 hpf (Fig. 2A, arrowhead with asterisk).
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Transcripts of gata2 were detected in the wedge region of the anterior ICM and the posterior ICM at 23 hpf, albeit qualitatively reduced when compared with wild type (Fig. 2C, arrowhead and arrowhead with asterisk). With the reduced expression of gata2 in bls, it was difficult to determine by double RNA in situ whether the gata2-expressing cells co-expressed scl. As gata2 is expressed in both blood and endothelial progenitors, the reduced transcript level could be attributable to decreased number of hematopoietic but not endothelial cells. Additionally, transcripts of the zebrafish ikaros gene were detected in the hematopoietic progenitors of the ICM (Fig. 2C). Similar to observations of scl and gata1 expression, ikaros expression was absent in the ICM of bls mutants at 23 hpf.
As the ICM also includes cells that differentiate into the embryonic angioblasts, we examined flk1 expression in bls (Pardanaud et al., 1996). Expression of flk1 delineates the embryonic dorsal aorta, axial vein and inter-somitic vessels at 23 hpf (Fig. 2C). Expression of flk1 in the lateral plate mesoderm at eight somites was also unaffected (data not shown). This finding is corroborated by visual inspection of bls mutants, which showed morphologically intact vasculature.
Defective hematopoietic progenitors undergo apoptosis in bls
The expression of scl but not gata1 at 18 somites suggests that the hematopoietic precursors that were specified early in embryogenesis of bls mutants failed to undergo normal differentiation and to maintain gata1 expression. It has been shown that hematopoietic precursors defective in gata1 expression undergo apoptosis (Weiss and Orkin, 1995). Consistent with this, staining of apoptotic cells with Acridine Orange demonstrated increased cell death in the ICM of bls mutants at 15 somites and 23 hpf (Fig. 3A). In addition to the ICM hematopoietic defects, bls embryos also exhibited marked apoptosis of cells lining the dorsal trunk and tailbud margins (Fig. 3A, arrowheads with asterisk).
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Normal myelopoiesis in bls
With the striking deficiency of primitive hematopoietic progenitors in bls, we next examined whether primitive myelopoiesis is also affected by the bls mutation. Myelopoiesis during zebrafish embryogenesis consists of expansion and differentiation of primitive macrophages (Herbomel et al., 1999). Unlike primitive erythroid cells that arise from the ICM, zebrafish primitive macrophages arise from a distinct rostral anlage that is derived from the anterior paraxial mesoderm (Herbomel et al., 1999
; Bennet et al., 2001
; Parichy et al., 2000
). Primitive macrophages originating from the anterior paraxial mesoderm express genes such as pu.1 (spi1), cmyb and draculin (dra). In addition, cells in the ICM also express cmyb and dra but not pu.1 (Fig. 4). When the expression of these genes were examined in bls mutants, it was evident that the primitive macrophages were not affected by the mutation. Expression of pu.1, cmyb and dra were at wild-type levels in the primitive macrophages of bls mutants (Fig. 4).
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Initiation of lymphopoiesis is delayed in bls
The thymic organs form by 65 hpf and are populated with rag1-expressing lymphocytes (Hansen and Zapata, 1998; Trede and Zon, 1998
). Expression of rag1 is absent in bls embryos at 4.5 dpf (Fig. 5A). Thymic cytology revealed normal appearing thymic epithelium in bls and absence of lymphoblasts (Fig. 5B, arrowhead with asterisk). Interestingly, if mutant animals were raised to 7.5 dpf and then analyzed with rag1 in situ hybridization, lymphoid cells can be detected in the thymi (Fig. 5A, arrowhead with asterisk). Likewise, histology of thymi of wild-type and bls mutants appear similar at 7.5 dpf, populated with lymphocytes (Fig. 5B, arrowheads).
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Cell transplantation studies
To determine whether bls acts in a cell autonomous or non-cell autonomous manner, reciprocal cell transplantation experiments were carried out between wild-type and bls mutant animals (Table 2). Analysis of gata1 expression in bls hosts carrying either bls or wild-type donor cells failed to detect any hematopoietic progenitors in the ICM (Fig. 7). By contrast, when bls mutant donor cells were transplanted into wild-type hosts, gata1-expressing hematopoietic progenitors derived from the mutant donor cells were found in the ICM (Fig. 7, white arrowheads). These studies demonstrate that bls acts in a non-cell autonomous manner, such that the mutant cells are competent to differentiate and express gata1 in a wild-type environment.
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DISCUSSION |
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The bls gene is required for the differentiation of primitive blood cells
Hematopoietic genes such as scl and gata1 are expressed in the zebrafish lateral plate mesoderm as early as five somites, evidence that hematopoietic precursors have been specified by that time point (Detrich et al., 1995; Gering et al., 1998
; Liao et al., 1998
; Thompson et al., 1998
). The analysis of scl and gata1 expression in bls mutants show that the number of hematopoietic cells derived from the lateral plate mesoderm is greatly reduced. Meanwhile, expression of flk1 is unaffected by bls, and the vasculature is intact.
As embryogenesis progresses in bls, the number of scl- and gata1-expressing cells decreases. By 23 hpf, no scl or gata1 transcripts can be detected in the ICM and only a handful of cells expressing scl can be found in the posterior ICM. Concurrent with the decreasing number of scl- and gata1-expressing cells, apoptosis is noted in the ICM. Furthermore, the persistence of gata2 and scl and the absence of gata1 and ikaros in the anterior ICM of the mutant animal suggests that these cells are able to express early hematopoietic genes, but fail to differentiate and express genes associated with progressive hematopoietic lineage differentiation. These observations suggest that the hematopoietic progenitors that do form in bls fail to undergo normal differentiation and undergo apoptosis (Weiss and Orkin, 1995). Of note, scl, gata1 and flk1 transcripts are absent in the lateral plate mesoderm of clo mutants (Liao et al., 1997
, Liao et al., 1998
). Unlike clo, some scl-positive hematopoietic progenitors are specified in the lateral plate mesoderm. Therefore, bls appears to regulate, but is not absolutely required, for the specification of scl-expressing blood progenitors. Subsequently, bls is involved in maintaining scl and gata1 expression during differentiation of the embryonic blood cells.
Role of bls in embryonic lymphoid and myeloid development
Analysis of macrophage markers pu.1, cmyb and draculin demonstrate that primitive macrophages are produced at wild-type levels in the bilateral rostral paraxial mesoderm. This suggests that distinct hematopoietic programs regulate the development of primitive macrophages from the anterior paraxial mesoderm, and the elaboration of primitive hematopoietic progenitors from the ICM. These different regulatory signals are anatomically segregated, where the ICM may receive signals from the trunk paraxial mesoderm, whereas the rostral macrophage anlage is influenced by anterior cues.
The delay in lymphopoiesis in bls suggests that either the gene is required in the development of lymphoblasts, or that the ICM hematopoietic progenitors contribute to the initiation of lymphopoiesis. Moreover, studies in mice underscore the symbiotic dependence between thymic epithelium and lymphoblasts in their mutual development (Manley, 2000; Nehls et al., 1996
; Ritter and Boyd, 1993
). It is not clear whether this interdependence is also present in the development of lymphoblasts and thymic epithelium in zebrafish. Further work in lineage tracing and electron-microscopic analysis of the thymus in bls mutants will better characterize the lymphoid defect.
The posterior ICM of zebrafish embryo
Unlike gata1 and ikaros, the expression of scl and gata2 is not restricted to the anterior ICM and is also found in the posterior ICM at 23 hpf. Fate-mapping experiments have shown that cells of the posterior ICM arise as a result of complex migration from the extending tailbud (Kanki and Ho, 1997). Among vertebrates, scl and gata2 are expressed in both hematopoietic and endothelial cells, whereas gata1 and ikaros are expressed in only hematopoietic cells (Georgopoulos et al., 1992
; Orkin, 1995
). Additionally, the mutant clo lacks scl expression in both the anterior and posterior ICM, and fails to produce hematopoietic or endothelial cells. Last, endothelial markers such as hhex, flk1 and fli1 are highly expressed in the posterior ICM (Liao et al., 1997
; Liao et al., 2000b
; Thompson et al., 1998
). Taken together, these observations suggest that the posterior ICM represents endothelial tissue and not hematopoietic progenitors. Therefore, expression of scl in the posterior ICM of bls is consistent with its phenotype of compromised hematopoiesis but intact vasculogenesis.
Non-cell autonomous requirement for bls in regulating primitive hematopoiesis
Cell transplantation studies showed that bls donor cells were able to contribute to gata1-expressing cells in the ICM of wild-type hosts. Conversely, bls hosts did not support the differentiation of wild-type donor cells to gata1-expressing cells. Given the non-cell autonomous action of the bls gene, one might speculate that the gene product could be a secreted factor or a cell surface receptor required for proper development of primitive hematopoietic cells. The dominant but incompletely penetrant aspect of the bls mutation supports a secreted factor hypothesis, where the proposed cytokine may be limiting during embryogenesis, manifesting in a haplo-insufficient phenotype.
The clo mutant has combined defect in hematopoietic and endothelial progenitors, and the clo gene is thought to function at the level of the hemangioblast, a proposed transient bi-potential cell that gives rise to endothelial and hematopoietic lineages (Liao et al., 1997; Stainier et al., 1995
). Overexpression of scl and hhex can lead to partial rescue of hematopoietic progenitors in clo mutants (Liao et al., 1998
; Liao et al., 2000b
). Interestingly, the clo gene appears to act non-cell autonomously early in the differentiation of embryonic blood cells, before the expression of gata1 (Parker and Stainier, 1999
). Therefore, the relationships between scl, hhex, clo and bls in early hematopoiesis deserve further investigation.
Overexpression studies in Xenopus have shown that bmp4 expands ventral mesoderm and induces the expression of scl (Mead et al., 1998). Overexpression of tolloid leads to ectopic maintenance of endogenous bmp4, and leads to ventralized phenotype with expanded gata1-expressing cells (Blader et al., 1997
). Although the number of cells expressing scl is increased in the posterior ICM of bmp4 ventralized tails of bls mutants, no scl-expressing cells were observed in the anterior ICM. Only rare gata1-expressing cells were seen in the anterior ICM of bmp4-injected embryos (1.8% of injected embryos, Table 1). Ventralization of the ICM by bmp4 in clo also did not rescue scl- or gata1-expressing cells. Failure of bmp4 to rescue hematopoietic cells in the anterior ICM suggests that the bls and clo gene products are required for hematopoiesis at a stage downstream of ventral mesoderm induction.
By contrast, gata1-expressing cells can be detected in the anterior ICM of bls and clo mutants that have been injected with scl. Either the gata1-positive cells are rescued hematopoietic progenitors or they represent blood cells specified from the lateral plate mesoderm. The percentage of scl injected embryos with gata1-positive blood cells is comparable between bls and clo mutants (48% and 41%, respectively) (Table 1). However, there are much fewer gata1-expressing cells in the anterior ICM of scl-injected bls animals than similarly injected clo mutants (Fig. 6C). This observation implies that bls may play additional roles in maintaining scl expression during embryogenesis, where the absence of the bls gene product attenuates the partial hematopoietic rescue when compared with that observed in clo. This view is consistent with expression analysis of scl and gata1, which show that bls mutants are able to produce some scl-positive blood progenitors in the lateral plate mesoderm, but such cells fail to express gata1 and undergo apoptosis. Furthermore, overexpression of gata1 is not sufficient for rescue of primitive in blood in neither bls nor clo. This suggests that other downstream target genes of bls and scl are necessary for primitive hematopoiesis, and that gata1 represents one such target gene. Taken together with the absence of scl induction by bmp4 in the bls mutant, we propose that bls acts downstream of bmp4 in initiating or maintaining expression of scl.
The unique role of bls as a non-cell autonomous regulator of primitive hematopoiesis exemplifies the utility of zebrafish as a genetic model system for the study of blood development. Possible function of bls as a stromal signal in activating and maintaining scl expression underscores the importance of extrinsic cues during early hematopoietic differentiation. Moreover, the requirement for bls during primitive hematopoiesis but not definitive blood development may provide insight into regulatory cues that differ between embryonic development and adult homeostasis.
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ACKNOWLEDGMENTS |
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REFERENCES |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bennett, C. M., Kanki, J. P., Rhodes, J., Liu, T. X., Paw, B. H., Kieran, M. W., Langenau, D. M., Delahaye-Brown, A., Zon, L. I., Fleming, M. D. and Look, A. T. (2001). Myelopoiesis in the zebrafish, Danio rerio. Blood 98, 643-651.
Blader, P., Rastegar, S., Fischer, N. and Strahle, U. (1997). Cleavage of the BMP-4 antagonist chordin by zebrafish tolloid. Science 278, 1937-1940.
Brownlie, A., Donovan, A., Pratt, S. J., Paw, B. H., Oates, A. C., Brugnara, C., Witkowska, H. E., Sassa, S. and Zon, L. I. (1998). Positional cloning of the zebrafish sauternes gene: a model for congenital sideroblastic anaemia. Nat. Genet. 20, 244-250.[Medline]
Davidson, A. J. and Zon, L. I. (2000). Turning mesoderm into blood: the formation of hematopoietic stem cells during embryogenesis. Curr. Top. Dev. Biol. 50, 45-60.[Medline]
Detrich, H. W., III, Kieran, M. W., Chan, F. Y., Barone, L. M., Yee, K., Rundstadler, J. A., Pratt, S., Ransom, D. and Zon, L. I. (1995). Intraembryonic hematopoietic cell migration during vertebrate development. Proc. Natl. Acad. Sci. USA 92, 10713-10717.[Abstract]
Dieterlen-Lievre, F. (1997). Intraembryonic hematopoietic stem cells. Hematol. Oncol. Clin. North Am. 11, 1149-1171.[Medline]
Dieterlen-Lievre, F. and Le Douarin, N. M. (1993). Developmental rules in the hemtopoietic and immune system of birds: how general are they? Semin. Dev. Biol. 4, 325-332.
Dzierzak, E. and Medvinsky, A. (1995). Mouse embryonic hematopoiesis. Trends Genet. 11, 359-366.[Medline]
Georgopoulos, K., Moore, D. D. and Derfler, B. (1992). Ikaros, an early lymphoid-specific transcription factor and a putative mediator for T cell commitment. Science 258, 808-812.[Medline]
Gering, M., Rodaway, A. R., Gottgens, B., Patient, R. K. and Green, A. R. (1998). The SCL gene specifies haemangioblast development from early mesoderm. EMBO J. 17, 4029-4045.
Godin, I. E., Garcia-Porrero, J. A., Coutinho, A., Dieterlen-Lievre, F. and Marcos, M. A. (1993). Para-aortic splanchnopleura from early mouse embryos contains B1a cell progenitors. Nature 364, 67-70.[Medline]
Godin, I., Dieterlen-Lievre, F. and Cumano, A. (1995). Emergence of multipotent hemopoietic cells in the yolk sac and paraaortic splanchnopleura in mouse embryos, beginning at 8.5 days postcoitus. Proc. Natl. Acad. Sci. USA 92, 773-777.[Abstract]
Griffin, K. J., Amacher, S. L., Kimmel, C. B. and Kimelman, D. (1998). Molecular identification of spadetail: regulation of zebrafish trunk and tail mesoderm formation by T-box genes. Development 125, 3379-3388.
Hansen, J. D. and Zapata, A. G. (1998). Lymphocyte development in fish and amphibians. Immunol. Rev. 166, 199-220.[Medline]
Herbomel, P., Thisse, B. and Thisse, C. (1999). Ontogeny and behaviour of early macrophages in the zebrafish embryo. Development 126, 3735-3745.
Hug, B., Walter, V. and Grunwald, D. J. (1997). tbx6, a Brachyury-related gene expressed by ventral mesendodermal precursors in the zebrafish embryo. Dev. Biol. 183, 61-73.[Medline]
Kanki, J. P. and Ho, R. K. (1997). The development of the posterior body in zebrafish. Development 124, 881-893.
Kelley, C., Yee, K., Harland, R. and Zon, L. I. (1994). Ventral expression of GATA-1 and GATA-2 in the xenopus embryo defines induction of hematopoietic mesoderm. Dev. Biol. 165, 193-205.[Medline]
Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B. and Schilling, T. F. (1995). Stages of embryonic development of the zebrafish. Dev. Dyn. 203, 253-310.[Medline]
Lassila, O., Martin, C., Dieterlen, L. F., Gilmour, D. G., Eskola, J. and Toivanen, P. (1982). Migration of prebursal stem cells from the early chicken embryo to the yolk sac. Scand. J. Immunol. 16, 265-268.[Medline]
Liao, E. C. and Zon, L. I. (1999). Conservation of themes in vertebrate blood development. In Cell Lineage and Fate Determination (ed. S. Moody), pp. 569-582. London: Academic Press.
Liao, W., Bisgrove, B. W., Sawyer, H., Hug, B., Bell, B., Peters, K., Grunwald, D. J. and Stainier, D. Y. R. (1997). The zebrafish gene cloche acts upstream of a flk-1 homologue to regulate endothelial differentiation. Development 124, 381-389.
Liao, E. C., Paw, B. H., Oates, A. C, Pratt, S. J., Postelthwait, J. H. and Zon, L. I. (1998). SCL/tal-1 transcription factor acts downstream of cloche to specify hematopoietic and vascular progenitors in zebrafish. Genes Dev. 12, 621-626.
Liao, E. C., Paw, B. H., Peters, L. L., Zapata, A., Pratt, S. J., Do, C. P., Lieschke, G. and Zon, L. I. (2000a). Hereditary spherocytosis in zebrafish riesling illustrates evolution of erythroid beta-spectrin structure, and function in red cell morphogenesis and membrane stability. Development 127, 5123-5132.
Liao, W., Ho, C. Y., Yan, Y. L., Postlethwait, J. and Stainier, D. Y. (2000b). Hhex and scl function in parallel to regulate early endothelial and blood differentiation in zebrafish. Development 127, 4303-4313.
Lin, C. S., Lim, S. K., DAgati, V. and Costantini, F. (1996). Differential effects of an erythropoietin receptor gene disruption on primitive and definitive erythropoiesis. Genes Dev. 10, 154-164.[Abstract]
Manley, N. R. (2000). Thymus organogenesis and molecular mechanisms of thymic epithelial cell differentiation. Semin. Immunol. 12, 421-428.[Medline]
Mead, P. E. and Zon, L. I. (1998). Molecular insights into early hematopoiesis. Curr. Opin. Hematol. 5, 156-160.[Medline]
Mead, P. E., Kelley, C. M., Hahn, P. S., Piedad, O. and Zon, L. I. (1998). SCL specifies hematopoietic mesoderm in Xenopus embryos. Development 125, 2611-2620.
Medvinsky, A. and Dzierzak, E. (1996). Definitive hematopoiesis is autonomously initiated by the AGM region. Cell 86, 897-906.[Medline]
Medvinsky, A. L., Samoylina, N. L., Muller, A. M. and Dzierzak, E. A. (1993). An early pre-liver intraembryonic source of CFU-S in the developing mouse. Nature 364, 64-67.[Medline]
Mucenski, M. L., McLain, K., Kier, A. B., Swerdlow, S. H., Schreiner, C. M., Miller, T. A., Pietryga, D. W., Scott, W. J., Jr and Potter, S. S. (1991). A functional c-myb gene is required for normal murine fetal hepatic hematopoiesis. Cell 65, 677-689.[Medline]
Muller, A. M., Medvinsky, A., Strouboulis, J., Grosveld, F. and Dzierzak, E. (1994). Development of hematopoietic stem cell activity in the mouse embryo. Immunity 1, 291-301.[Medline]
Nehls, M., Kyewski, B., Messerle, M., Waldschutz, R., Schuddekopf, K., Smith, A. J. and Boehm, T. (1996). Two genetically separable steps in the differentiation of thymic epithelium. Science 272, 886-889.[Abstract]
Oates, A. C., Brownlie, A., Pratt, S. J., Irvine, D. V., Liao, E. C., Paw, B. H., Dorian, K. J., Johnson, S. L., Postlethwait, J. H., Zon, L. I. et al. (1999). Gene duplication of zebrafish JAK2 homologs is accompanied by divergent embryonic expression patterns: only jak2a is expressed during erythropoiesis. Blood 94, 2622-2636.
Ogawa, M., Nishikawa, S., Yoshinaga, K., Hayashi, S., Kunisada, T., Nakao, J., Kina, T., Sudo, T. and Kodama, H. (1993). Expression and function of c-Kit in fetal hemopoietic progenitor cells: transition from the early c-Kit-independent to the late c-Kit-dependent wave of hemopoiesis in the murine embryo. Development 117, 1089-1098.
Okada, H., Watanabe, T., Niki, M., Takano, H., Chiba, N., Yanai, N., Tani, K., Hibino, H., Asano, S., Mucenski, M. L. et al. (1998). AML1/ embryos do not express certain hematopoiesis-related gene transcripts including those of the PU.1 gene. Oncogene 17, 2287-2293.[Medline]
Orkin, S. H. (1995). Transcription factors and hematopoietic development. J. Biol. Chem. 270, 4955-4958.
Orkin, S. H. (1996). Development of the hematopoietic system. Curr. Opin. Genet. Dev. 6, 597-602.[Medline]
Palis, J., Robertson, S., Kennedy, M., Wall, C. and Keller, G. (1999). Development of erythroid and myeloid progenitors in the yolk sac and embryo proper of the mouse. Development 126, 5073-5084.
Pardanaud, L., Luton, D., Prigent, M., Bourcheix, L.-M., Catala, M. and Dierterlen-Lievre, F. (1996). Two distinct endothelial lineages in ontogeny, one of them related to hemopoiesis. Development 122, 1363-1371.
Parichy, D. M., Ransom, D. G., Paw, B., Zon, L. I. and Johnson, S. L. (2000). An orthologue of the kit-related gene fms is required for development of neural crest-derived xanthophores and a subpopulation of adult melanocytes in the zebrafish, Danio rerio. Development 127, 3031-3044.
Parker, L. and Stainier, D. Y. (1999). Cell-autonomous and non-autonomous requirements for the zebrafish gene cloche in hematopoiesis. Development 126, 2643-2651.
Peault, B. (1996). Hematopoietic stem cell emergence in embryonic life: developmental hematology revisited. J. Hematother. 5, 369-378.[Medline]
Pevny, L., Lin, C.-S., DAgati, V., Simon, M. C., Orkin, S. H. and Costantini, F. (1995). Development of hematopoietic cells lacking transcription factor GATA-1. Development 121, 163-172.
Porcher, C., Wojciech, S., Rockwell, K., Fujiwara, Y., Alt, F. W. and Orkin, S. H. (1996). The T cell leukemia oncoprotein SCL/tal-1 is essential for development of all hematopoietic lineages. Cell 86, 1-20.[Medline]
Ritter, M. A. and Boyd, R. L. (1993). Development in the thymus: it takes two to tango. Immunol. Today 14, 462-469.[Medline]
Robb, L. (1997). Hematopoiesis: origin pinned down at last? Curr. Biol. 7, R10-R12.[Medline]
Robb, L., Elwood, N. J., Elefanty, A. G., Kontgen, F., Li, R., Barnett, L. D. and Begley, C. G. (1996). The scl gene product is required for the generation of all hematopoietic lineages in adult mouse. EMBO J. 15, 4123-4129.[Abstract]
Robertson, S., Kennedy, M. and Keller, G. (1999). Hematopoietic commitment during embryogenesis. Ann. New York Acad. Sci. 872, 9-15.
Rollins-Smith, L. A. and Blair, P. (1990). Contribution of ventral blood island mesoderm to hematopoiesis in postmetamorphic and metamorphosis-inhibited xenopus laevis. Dev. Biol. 142, 178-183.[Medline]
Sasaki, K., Yagi, H., Bronson, R. T., Tominaga, K., Matsunashi, T., Deguchi, K., Tani, Y., Kishimoto, T. and Komori, T. (1996). Absence of fetal liver hematopoiesis in mice deficient in transcriptional coactivator core binding factor beta. Proc. Natl. Acad. Sci. USA 93, 12359-12363.
Schulte-Merker, S., Ho, R. K., Hermann, B. G. and Nusslein-Volhard, C. (1992). Then protein product of the zebrafish homologue of the mouse T gene is expressed in nuclei of the germ ring and the notochord of the early embryo. Development 116, 1021-1032.
Seiler, C. and Nicolson, T. (1999). Defective calmodulin-dependent rapid apical endocytosis in zebrafish sensory hair cell mutants. J. Neurobiol. 41, 424-434.[Medline]
Shalaby, F., Ho, J., Stanford, W., K.-D., F., Schuch, A., Schwartz, L., Bernstein, A. and Rossant, J. (1997). A requirement for Flk1 in primitive and definitive hematopoiesis and vasculogenesis. Cell 89, 981-990.[Medline]
Shivdasani, R. A., Mayer, E. L. and Orkin, S. H. (1995). Absence of blood formation in mice lacking the T-cell leukaemia oncoprotein tal-1/SCL. Nature 373, 432-434.[Medline]
Stainier, D. Y. R., Weinstein, B. M., Deitrich, H. W., III, Zon, L. I. and Fishman, M. C. (1995). cloche, an early acting zebrafish gene, is required by both the endothelial and hematopoietic lineages. Development 121, 3141-3150.
Szenberg, A. (1977). Ontogeny of myelopoietic precursor cells in the chicken embryo. Adv. Exp. Med. Biol. 88, 3-11.[Medline]
Thompson, M. A., Ransom, D. G., Pratt, S. J., MacLennan, H., Kieran, M. W., Detrich, H. W., Vail, B., Huber, T. L., Paw, B. H. and Zon, L. I. (1998). Characterization of primitive and definitive waves of hematopoiesis in wild-type and mutant zebrafish. Dev. Biol. 197, 248-269.[Medline]
Trede, N. S. and Zon, L. I. (1998). Development of T-cells during fish embryogenesis. Dev. Comp. Immunol. 22, 253-263.[Medline]
Tsai, F.-Y., Keller, G., Kuo, F. C., Weiss, M., Chen, J., Rosenblatt, M., Alt, F. W. and Orkin, S. H. (1994). An early hematopoietic defect in mice lacking the transcription factor GATA-2. Nature 371, 221-226.[Medline]
Wang, L. C., Kuo, F., Fujiwara, Y., Gilliland, D. G., Golub, T. R. and Orkin, S. H. (1997). Yolk sac angiogenic defect and intra-embryonic apoptosis in mice lacking the Ets-related factor TEL. EMBO J. 16, 4374-4383.
Wang, Q., Stacy, T., Binder, M., Martin-Padilla, M., Sharpe, A. H. and Speck, N. A. (1996). Disruption of the CBFa2 gene causes necrosis and hemorrhaging in the central nervous system, and blocks definitive hematopoiesis. Proc. Natl. Acad. Sci. USA 93, 3444-3449.
Warren, A. J., College, W. H., Carlton, M. B. L., Evans, M. J., Smith, A. J. H. and Rabbits, T. H. (1994). The oncogenic cysteine-rich LIM domain protein Rbtn2 is essential for erythroid development. Cell 78, 45-57.[Medline]
Weiss, M. J. and Orkin, S. H. (1995). Transcription factor GATA-1 permits survival and maturation of erythroid precursors by preventing apoptosis. Proc. Natl. Acad. Sci. USA 92, 9623.[Abstract]
Westerfield, M. (1993). The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish (Brachydanio rerio). Eugene, OR: University of Oregon Press.
Whetton, A. D. and Spooncer, E. (1998). Role of cytokines and extracellular matrix in the regulation of haemopoietic stem cells. Curr. Opin. Cell Biol. 10, 721-726.[Medline]
Willett, C. E., Cortes, A., Zuasti, A. and Zapata, A. G. (1999). Early hematopoiesis and developing lymphoid organs in the zebrafish. Dev. Dyn. 214, 323-336.[Medline]
Wong, P. M., Chung, S. W., Chui, D. H. and Eaves, C. J. (1986). Properties of the earliest clonogenic hemopoietic precursors to appear in the developing murine yolk sac. Proc. Natl. Acad. Sci. USA 83, 3851-3854.[Abstract]
Yoder, M. C., Hiatt, K. and Mukherjee, P. (1997). In vivo repopulating hematopoietic stem cells are present in the murine yolk sac at day 9.0 postcoitus. Proc. Natl. Acad. Sci. USA 94, 6776-6780.
Zagris, N. (1986). Communication between primary endoderm and mesoderm for erythroblast differentiation in early chick blastoderm. Exp. Cell Biol. 54, 170-174.[Medline]
Zon, L. I. (1995). Developmental biology of hematopoiesis. Blood 86, 2876-2891.