Definitive hematopoiesis initiates through a committed erythromyeloid progenitor in the zebrafish embryo.
Journal: 2008/March - Development (Cambridge)
ISSN: 0950-1991
Abstract:
Shifting sites of blood cell production during development is common across widely divergent phyla. In zebrafish, like other vertebrates, hematopoietic development has been roughly divided into two waves, termed primitive and definitive. Primitive hematopoiesis is characterized by the generation of embryonic erythrocytes in the intermediate cell mass and a distinct population of macrophages that arises from cephalic mesoderm. Based on previous gene expression studies, definitive hematopoiesis has been suggested to begin with the generation of presumptive hematopoietic stem cells (HSCs) along the dorsal aorta that express c-myb and runx1. Here we show, using a combination of gene expression analyses, prospective isolation approaches, transplantation, and in vivo lineage-tracing experiments, that definitive hematopoiesis initiates through committed erythromyeloid progenitors (EMPs) in the posterior blood island (PBI) that arise independently of HSCs. EMPs isolated by coexpression of fluorescent transgenes driven by the lmo2 and gata1 promoters exhibit an immature, blastic morphology and express only erythroid and myeloid genes. Transplanted EMPs home to the PBI, show limited proliferative potential, and do not seed subsequent hematopoietic sites such as the thymus or pronephros. In vivo fate-mapping studies similarly demonstrate that EMPs possess only transient proliferative potential, with differentiated progeny remaining largely within caudal hematopoietic tissue. Additional fate mapping of mesodermal derivatives in mid-somitogenesis embryos suggests that EMPs are born directly in the PBI. These studies provide phenotypic and functional analyses of the first hematopoietic progenitors in the zebrafish embryo and demonstrate that definitive hematopoiesis proceeds through two distinct waves during embryonic development.
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Development 134(23): 4147-4156

Definitive hematopoiesis initiates through a committed erythromyeloid progenitor in the zebrafish embryo

INTRODUCTION

The genesis of the blood-forming system is complex, with shifting sites of hematopoiesis occurring during development. The ontogeny of blood cells from multiple hematopoietic organs appears to be a feature common to all organisms with multiple hematopoietic lineages, including both invertebrates and vertebrates (Evans et al., 2003; Hartenstein, 2006). Whereas the specific organs that transiently host blood cell production can be divergent between species, hematopoiesis can be roughly divided into two major waves in vertebrates based on the cell types generated. Primitive hematopoiesis is characterized by a relatively rapid commitment of embryonic mesoderm to monopotent hematopoietic precursors (Keller et al., 1999). These cells give rise to embryonic erythrocytes and macrophages that are respectively required to oxygenate and remodel growing tissues of the embryo (Palis and Yoder, 2001). Definitive hematopoiesis is characterized by the emergence of multipotent hematopoietic stem and progenitor cells (Cumano and Godin, 2007).

The multiple waves of blood cell development have been best studied in the mouse, where hematopoiesis initiates with the formation of primitive erythrocytes and macrophages in the extraembryonic yolk sac (Palis et al., 1999). It is widely believed that definitive hematopoiesis subsequently begins in the midgestation embryo with the formation of HSCs in a region bounded by the aorta, gonads and mesonephros (AGM) (Cumano and Godin, 2007; Dzierzak, 2005). Several recent studies, however, have shown the presence of definitive cell types within the yolk sac (Bertrand et al., 2005b; Palis et al., 1999; Yoder, 1997; Yoder et al., 1997; Yokomizo et al., 2007; Yokota et al., 2006) and placenta (Gekas et al., 2005; Ottersbach and Dzierzak, 2005; Zeigler et al., 2006) that arise either before or concomitant with HSC formation in the AGM region. The lineage relationships and relative contributions of each of these blood cell precursors to later hematopoietic sites, such as the fetal liver, fetal spleen and bone marrow remain to be clarified.

In the zebrafish, primitive hematopoiesis also produces macrophages and erythrocytes. The first functional hematopoietic cells born in the embryo are primitive macrophages. These cells arise from anterior, cephalic mesoderm then migrate onto the yolk syncitial layer before colonizing embryonic tissues (Herbomel et al., 1999). Primitive erythrocytes develop from bilateral stripes of ventral mesoderm that, upon migration to the midline, form a structure termed the intermediate cell mass (ICM) (Detrich et al., 1995; Thompson et al., 1998). Endothelial cells encapsulate this mass of maturing erythroid precursors to form the cardinal vein and, upon initiation of heart contractions at approximately 22 hours post-fertilization (hpf), primitive erythroblasts enter circulation. Based on the appearance of cells expressing HSC-associated genes such as c-myb and runx1 along the ventral wall of the dorsal aorta, definitive hematopoiesis has been presumed to initiate in this zebrafish equivalent of the AGM region between 28–48 hpf (Burns et al., 2002; Kalev-Zylinska et al., 2002; Thompson et al., 1998). Lineage tracing studies have recently shown that cells residing between the aorta and vein could subsequently colonize the thymus and pronephros, the major definitive hematopoietic organs in zebrafish (Jin et al., 2007; Murayama et al., 2006).

The vertebrate AGM is not a hematopoietic organ per se, since differentiation is not observed in this region (Godin et al., 1999). Rather, the differentiation of embryonic HSCs into multiple definitive lineages occurs only after the seeding of other tissues, such as the fetal liver in mammals. Until recently, it was thought that HSCs born in the zebrafish AGM colonized the pronephros to initiate definitive hematopoiesis (Hsia and Zon, 2005). Recent fate mapping studies of Murayama et al. showed that presumptive HSCs targeted along the aorta first migrate to a region in the tail they termed caudal hematopoietic tissue (Murayama et al., 2006). This region has also been referred to as the posterior ICM (Detrich et al., 1995; Thompson et al., 1998), ventral vein region (Liao et al., 1998; Willett et al., 1999) and more conventionally as the posterior blood island (PBI) (Crowhurst et al., 2002; Jin et al., 2007; Kinna et al., 2006; Renshaw et al., 2006; Rombout et al., 2005) based on localization of hematopoietic markers to the ventral portion of the tail immediately caudal to the yolk tube extension. Electron microscopy studies showed that definitive myeloid cells, such as neutrophilic granulocytes, are first detected in this region at 34 hpf (Willett et al., 1999). It is not clear whether these cells migrate to the PBI from other hematopoietic sites, or whether they arise in situ from resident stem or progenitor cells. Murayama and colleagues (Murayama et al., 2006) hypothesized that the PBI is seeded by AGM HSCs to act as a fetal liver equivalent in bridging definitive blood cell production until the pronephros becomes the final hematopoietic site.

In the present study, we describe a previously uncharacterized hematopoietic precursor that arises in the PBI to initiate definitive hematopoiesis. This precursor can be prospectively isolated by flow cytometry as early as 24 hpf based on the expression of fluorescent transgenes. Molecular characterization showed promiscuous expression of erythroid and myeloid genes. Accordingly, functional studies showed that these cells have erythroid and myeloid differentiation potential but lack lymphoid potential. We have therefore termed these cells erythromyeloid progenitors (EMPs). EMPs arise before HSCs were detected in the AGM, and fate mapping studies suggest that they arise directly from lmo2 posterior lateral plate mesoderm (LPM) derivatives. Taken together, these results demonstrate that the EMP serves as a transient progenitor that is born independently of HSCs to initiate definitive hematopoiesis in the developing zebrafish embryo.

MATERIALS AND METHODS

Zebrafish stocks and embryos

Zebrafish were mated, staged and raised as described (Westerfield, 2000) and maintained in accordance with UCSD IACUC guidelines. Transgenic lines Tg(gata1:DsRed) (Traver et al., 2003), Tg(cd41:eGFP) (Lin et al., 2005), Tg(lmo2:DsRed) (Zhu et al., 2005) and Tg(lmo2:eGFP) (Zhu et al., 2005) were used. Tg(mpx:eGFP) animals were generously provided by S. A. Renshaw (Renshaw et al., 2006). Hereafter, transgenic lines will be referred to without the Tg(xxx:xxx) nomenclature for clarity. Embryos were processed as described (Westerfield, 1994).

Whole-mount RNA in situ hybridization

Embryos were treated with PTU, then fixed in 4% or 10% paraformaldehyde (PFA). Whole-mount in situ hybridization was carried out as described (Thisse et al., 1993). The zebrafish cd45 homologue was identified on chromosome 22 (Ensembl #ENSDARG00000030937), and a 1.2kb probe generated from genomic DNA by PCR using the following primers: cd45-FP: AATGAAAAGGCTGTAATCGG; cd45-RP: GTCCTTGTTTTCTTCGCTGC.

Fluorescence in situ hybridization (FISH)

Antisense mRNA probes were prepared as previously reported for gata-1, pu.1 and mpx (Rhodes et al., 2005) using digoxigenin (DIG) or fluorescein (FITC) labeled UTPs according to manufacturer’s instructions (Roche, Palo Alto, CA). Two-color FISH was carried out as previously described (Kosman et al., 2004) with minor modifications. The 90% xylene/10% ethanol wash was omitted and proteinase K treatments were adjusted to 25 minutes for optimal permeabilization. Hybridization with 500ng of DIG-labeled and FITC-labeled probes was performed at 70°C. mpx-FITC probes were detected with a mouse anti-FITC antibody (Roche) followed by a donkey anti-mouse Alexa Fluor 555 antibody (Molecular Probes). Reactions were developed using anti-DIG-horseradish peroxidase (HRP) antibody. gata-1-FITC was detected with a mouse anti-FITC antibody (Roche) followed by a goat anti-mouse-HRP antibody (Molecular Probes, Carlsbad, CA). Alexa Fluor 488 or 594 tyramide substrates were used according to manufacturer’s instructions (Molecular Probes) to further amplify gata-1-DIG and pu.1-DIG or gata-1-FITC signals, respectively. Fluorescence images were acquired on an Olympus Fluoview 1000 confocal microscope at 20X and 40X magnification (Center Valley, PA).

Cell suspension preparation

PTU-treated whole or dissected embryos were dissociated between 24–72 hpf. Embryos were treated with 10mM DTT in E3 medium then transferred to Hanks Balanced Salt Solution (with Calcium) and digested with Liberase Blendzyme II (Sigma Aldrich, St. Louis, MO) for at least 1 hour at 33°C. Cell suspensions were then filtered through 40-μm nylon mesh, washed twice and pelleted by centrifugation at 250 × g for 5 min.

Flow Cytometry

Embryonic cell suspensions were prepared as described above and fluorescence-activated cell sorting (FACS) was performed as previously described (Traver et al., 2003) using a FACS Aria flow cytometer (Becton Dickinson, La Jolla, CA). Data analyses were performed using FlowJo software (TreeStar, Ashland, OR).

Cytology

Blood cells were collected by tail dissection of embryos, then homogenized using the embryonic cell collection protocol described above. Hematopoietic cells were concentrated by cytocentrifugation at 450 g for 5 minutes onto glass slides using a Shandon Cytospin 4 (Thermo Fischer Scientific, Waltham, MA). Slides were then processed through May-Grünwald and Giemsa stains according to manufacturer’s instructions (Fluka, Buchs, Switzerland).

Microscopy

Embryos were imaged using a Leica DMI6000 inverted fluorescent microscope (Wetzlar, Germany), a Hamamatsu C7780 digital camera (Hamamatsu, Japan) and Volocity Acquisition and Restoration software (Improvision, Lexington, MA).

RT-PCR analyses

For RT–PCR analysis, RNA was isolated from cells sorted from lmo2:eGFP and gata1:DsRed dual-positive embryos using TRIZOL (Invitrogen, Philadelphia, PA). 2 to 5 μg of total RNA was reverse transcribed into cDNA using a Superscript III RT-PCR kit (Invitrogen, Philadelphia, PA). The following primers were used: c-mpl-FP: ATGGATCCAGTTTTCATCTGGTGG, c-mpl-RP: TATAGGTAGACGTCACTTGGTGGG; l-plastin-FP: GTCGATGTGGATGGGAACGG, l-plastin-RP: CCTCCTCGGAGTATGAGTGC; cd41-FP:TTACTACGACCTATATCTGGG, cd41-RP: GATGACCTGGACATACTGGG; c-myb-FP:AGTTACTTCCGGGAAGAACCG, c-myb-RP: AGAGCAAGTGGAAATGGCACC; runx1-FP:TTGGGACGCCAAATACGAACC, runx1-RP: ATATCACCAAGGGCAACCACC; ef1a-FP:CGGTGACAACATGCTGGAGG, ef1a-RP: ACCAGTCTCCACACGACCCA. Primers for mpx, pu.1 and gata-1 transcripts were used as described (Hsu et al., 2004).

Hematopoietic cell transplantation

Single cell suspensions from lmo2:eGFP; gata-1:DsRed transgenic embryos and were prepared as described above. EMPs were sorted as described above and transplantated into 36 or 48 hpf wild-type embryos as previously described (Traver et al., 2003).

Fate mapping

1–8 cell stage cd41:eGFP or lmo2:eGFP transgenic embryos were injected with 0.5nl of a 0:1 or 1:1 mix of caged fluorescein-dextran 10,000MW and caged rhodamine-dextran 10,000MW (Molecular Probes, Carlsbad, CA). Uncaging was performed using a 365nm Micropoint laser system (Photonic Instruments, St Charles, IL) routed through the epifluorescence port and 20x objective of a Leica DMI6000 inverted microscope. Ten GFP target cells were uncaged per embryo following laser pulses of 10–20 seconds each. Uncaged embryos injected with rhodamine-dextran were subsequently observed using fluorescence microscopy whereas uncaged FITC was detected by immunohistochemistry, as previously described (Murayama et al., 2006).

Generation of zebrafish kidney stromal (ZKS) cell lines

Kidneys were isolated from wt AB* fish by dissection and bleached for 5 minutes in .000525% Sodium Hypochlorite (Fisher Scientific, Pittsburgh, PA). Tissue was then rinsed in sterile Dulbecco’s Phosphate Buffered Saline (PBS; Mediatech Inc., Herndon, VA) and mechanically dissociated by trituration. Dissociated cells were passed through a .45μM filter (BD Biosciences, San Jose, CA) and discarded. The remaining stromal cells were cultured in 12.5 cm flasks at 32°C, 5% CO2. Cells were maintained until reaching 50–80% confluency, then trypsinized (0.25%; Invitrogen, Grand Island, NY) for 10 minutes at 32°C, and split at 1:3 for expansion.

ZKS cells defined in this report were maintained in a mixture of 50% L-15, 35% DMEM, 15% Ham’s F-12 media (Mediatech Inc.) supplemented with 10% FBS (American Type Culture Collection, Manassas, VA), 2% penicillin/streptomycin (10U/ml stock), 0.1mg/ml gentamicin sulfate, 1% L-glutamine, 150 mg/L sodium bicarbonate, and 1.5% HEPES (All supplements from Mediatech Inc.).

In vitro differentiation assay

EMPs were purified by flow cytometry and plated onto confluent ZKS cells at a density of 1 × 10 cells/well in 12 well plates using 2ml of media/well. For morphological analyses, wells were gently aspirated and 200 μl removed for cytocentrifugation onto glass slides. Cells were stained with May-Grünwald/Giemsa stains as described above. Light microscopy images were obtained using an Olympus BX51 light microscope, Olympus DP70 camera, and Olympus DP Controller software (Center Valley, PA).

Zebrafish stocks and embryos

Zebrafish were mated, staged and raised as described (Westerfield, 2000) and maintained in accordance with UCSD IACUC guidelines. Transgenic lines Tg(gata1:DsRed) (Traver et al., 2003), Tg(cd41:eGFP) (Lin et al., 2005), Tg(lmo2:DsRed) (Zhu et al., 2005) and Tg(lmo2:eGFP) (Zhu et al., 2005) were used. Tg(mpx:eGFP) animals were generously provided by S. A. Renshaw (Renshaw et al., 2006). Hereafter, transgenic lines will be referred to without the Tg(xxx:xxx) nomenclature for clarity. Embryos were processed as described (Westerfield, 1994).

Whole-mount RNA in situ hybridization

Embryos were treated with PTU, then fixed in 4% or 10% paraformaldehyde (PFA). Whole-mount in situ hybridization was carried out as described (Thisse et al., 1993). The zebrafish cd45 homologue was identified on chromosome 22 (Ensembl #ENSDARG00000030937), and a 1.2kb probe generated from genomic DNA by PCR using the following primers: cd45-FP: AATGAAAAGGCTGTAATCGG; cd45-RP: GTCCTTGTTTTCTTCGCTGC.

Fluorescence in situ hybridization (FISH)

Antisense mRNA probes were prepared as previously reported for gata-1, pu.1 and mpx (Rhodes et al., 2005) using digoxigenin (DIG) or fluorescein (FITC) labeled UTPs according to manufacturer’s instructions (Roche, Palo Alto, CA). Two-color FISH was carried out as previously described (Kosman et al., 2004) with minor modifications. The 90% xylene/10% ethanol wash was omitted and proteinase K treatments were adjusted to 25 minutes for optimal permeabilization. Hybridization with 500ng of DIG-labeled and FITC-labeled probes was performed at 70°C. mpx-FITC probes were detected with a mouse anti-FITC antibody (Roche) followed by a donkey anti-mouse Alexa Fluor 555 antibody (Molecular Probes). Reactions were developed using anti-DIG-horseradish peroxidase (HRP) antibody. gata-1-FITC was detected with a mouse anti-FITC antibody (Roche) followed by a goat anti-mouse-HRP antibody (Molecular Probes, Carlsbad, CA). Alexa Fluor 488 or 594 tyramide substrates were used according to manufacturer’s instructions (Molecular Probes) to further amplify gata-1-DIG and pu.1-DIG or gata-1-FITC signals, respectively. Fluorescence images were acquired on an Olympus Fluoview 1000 confocal microscope at 20X and 40X magnification (Center Valley, PA).

Cell suspension preparation

PTU-treated whole or dissected embryos were dissociated between 24–72 hpf. Embryos were treated with 10mM DTT in E3 medium then transferred to Hanks Balanced Salt Solution (with Calcium) and digested with Liberase Blendzyme II (Sigma Aldrich, St. Louis, MO) for at least 1 hour at 33°C. Cell suspensions were then filtered through 40-μm nylon mesh, washed twice and pelleted by centrifugation at 250 × g for 5 min.

Flow Cytometry

Embryonic cell suspensions were prepared as described above and fluorescence-activated cell sorting (FACS) was performed as previously described (Traver et al., 2003) using a FACS Aria flow cytometer (Becton Dickinson, La Jolla, CA). Data analyses were performed using FlowJo software (TreeStar, Ashland, OR).

Cytology

Blood cells were collected by tail dissection of embryos, then homogenized using the embryonic cell collection protocol described above. Hematopoietic cells were concentrated by cytocentrifugation at 450 g for 5 minutes onto glass slides using a Shandon Cytospin 4 (Thermo Fischer Scientific, Waltham, MA). Slides were then processed through May-Grünwald and Giemsa stains according to manufacturer’s instructions (Fluka, Buchs, Switzerland).

Microscopy

Embryos were imaged using a Leica DMI6000 inverted fluorescent microscope (Wetzlar, Germany), a Hamamatsu C7780 digital camera (Hamamatsu, Japan) and Volocity Acquisition and Restoration software (Improvision, Lexington, MA).

RT-PCR analyses

For RT–PCR analysis, RNA was isolated from cells sorted from lmo2:eGFP and gata1:DsRed dual-positive embryos using TRIZOL (Invitrogen, Philadelphia, PA). 2 to 5 μg of total RNA was reverse transcribed into cDNA using a Superscript III RT-PCR kit (Invitrogen, Philadelphia, PA). The following primers were used: c-mpl-FP: ATGGATCCAGTTTTCATCTGGTGG, c-mpl-RP: TATAGGTAGACGTCACTTGGTGGG; l-plastin-FP: GTCGATGTGGATGGGAACGG, l-plastin-RP: CCTCCTCGGAGTATGAGTGC; cd41-FP:TTACTACGACCTATATCTGGG, cd41-RP: GATGACCTGGACATACTGGG; c-myb-FP:AGTTACTTCCGGGAAGAACCG, c-myb-RP: AGAGCAAGTGGAAATGGCACC; runx1-FP:TTGGGACGCCAAATACGAACC, runx1-RP: ATATCACCAAGGGCAACCACC; ef1a-FP:CGGTGACAACATGCTGGAGG, ef1a-RP: ACCAGTCTCCACACGACCCA. Primers for mpx, pu.1 and gata-1 transcripts were used as described (Hsu et al., 2004).

Hematopoietic cell transplantation

Single cell suspensions from lmo2:eGFP; gata-1:DsRed transgenic embryos and were prepared as described above. EMPs were sorted as described above and transplantated into 36 or 48 hpf wild-type embryos as previously described (Traver et al., 2003).

Fate mapping

1–8 cell stage cd41:eGFP or lmo2:eGFP transgenic embryos were injected with 0.5nl of a 0:1 or 1:1 mix of caged fluorescein-dextran 10,000MW and caged rhodamine-dextran 10,000MW (Molecular Probes, Carlsbad, CA). Uncaging was performed using a 365nm Micropoint laser system (Photonic Instruments, St Charles, IL) routed through the epifluorescence port and 20x objective of a Leica DMI6000 inverted microscope. Ten GFP target cells were uncaged per embryo following laser pulses of 10–20 seconds each. Uncaged embryos injected with rhodamine-dextran were subsequently observed using fluorescence microscopy whereas uncaged FITC was detected by immunohistochemistry, as previously described (Murayama et al., 2006).

Generation of zebrafish kidney stromal (ZKS) cell lines

Kidneys were isolated from wt AB* fish by dissection and bleached for 5 minutes in .000525% Sodium Hypochlorite (Fisher Scientific, Pittsburgh, PA). Tissue was then rinsed in sterile Dulbecco’s Phosphate Buffered Saline (PBS; Mediatech Inc., Herndon, VA) and mechanically dissociated by trituration. Dissociated cells were passed through a .45μM filter (BD Biosciences, San Jose, CA) and discarded. The remaining stromal cells were cultured in 12.5 cm flasks at 32°C, 5% CO2. Cells were maintained until reaching 50–80% confluency, then trypsinized (0.25%; Invitrogen, Grand Island, NY) for 10 minutes at 32°C, and split at 1:3 for expansion.

ZKS cells defined in this report were maintained in a mixture of 50% L-15, 35% DMEM, 15% Ham’s F-12 media (Mediatech Inc.) supplemented with 10% FBS (American Type Culture Collection, Manassas, VA), 2% penicillin/streptomycin (10U/ml stock), 0.1mg/ml gentamicin sulfate, 1% L-glutamine, 150 mg/L sodium bicarbonate, and 1.5% HEPES (All supplements from Mediatech Inc.).

In vitro differentiation assay

EMPs were purified by flow cytometry and plated onto confluent ZKS cells at a density of 1 × 10 cells/well in 12 well plates using 2ml of media/well. For morphological analyses, wells were gently aspirated and 200 μl removed for cytocentrifugation onto glass slides. Cells were stained with May-Grünwald/Giemsa stains as described above. Light microscopy images were obtained using an Olympus BX51 light microscope, Olympus DP70 camera, and Olympus DP Controller software (Center Valley, PA).

RESULTS

The posterior blood island is a site of hematopoiesis

We wished to determine when and where the first definitive hematopoietic precursors arise in the zebrafish embryo. Early gene expression studies suggested that the most posterior portion of the ICM contained hematopoietic precursors distinct from the primitive erythroblasts that arise in the ICM (Thompson et al., 1998). To determine what types of blood cells are found in the PBI, we performed whole-mount in situ hybridization (WISH) with a panel of hematopoietic genes at 30 and 36 hpf. We first identified the zebrafish homologue of cd45, which in mammals is expressed in all hematopoietic cell lineages except for erythrocytes. Expression of the zebrafish cd45 gene was observed as early as 30 hpf, and positive cells increased in number by 36 hpf in the ventral part of the tail (Fig. 1A, G). Although some studies have treated l-plastin (also known as lcp1) as a macrophage-specific marker in the zebrafish, it has been shown to be expressed in all murine leukocytes, including hematopoietic progenitors (Arpin et al., 1994; Bertrand et al., 2005a) and in zebrafish granulocytes (Meijer et al., 2007). Expression of l-plastin was observed in a pattern similar to that of cd45 (Fig. 1B, H), suggesting that both genes similarly mark zebrafish leukocytes.

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Genes associated with multilineage hematopoiesis are expressed in the posterior blood island as early as 30 hpf. (A, B) By 30 hpf, expression of the pan-leukocyte markers cd45 and l-plastin are observed throughout the PBI. (G, H) By 36 hpf, the number of cells expressing each gene has increased in the PBI, and expressing cells begin to migrate throughout the embryo. Localized expression of lmo2 is observed in the PBI at 30 hpf (C) and 36 hpf (I). (D, J) Expression of gata-1 is also observed in cells within the vascular plexus of the PBI at both timepoints. (E, F, K, L) Expression of genes associated with myelopoiesis is also observed within the PBI, including pu.1 and mpx. Rare, pu.1 cells are observed at 30 hpf in the PBI (E) that increase in number by 36 hpf (K), whereas mpx expression is not observed until 36 hpf (F, L), consistent with it being a marker of relatively mature myelomonocytic cell types. Lower panels are 20X magnification views of the PBI from upper, 10X whole-embryo photographs.

To further assess the possible presence of hematopoietic progenitors, we analyzed expression of lmo2, gata-1 and pu.1. lmo2 is expressed by both vascular and hematopoietic precursors. In addition to widespread vascular staining in the tail, a small population of lmo2 cells was observed in the PBI (Fig. 1C, I). Similarly, expression of a canonical marker of erythroid potential, gata-1, was observed in small numbers of cells in the PBI at both time points (Fig. 1D, J). These cells did not appear to be within vessels, but rather within the vascular plexus of the PBI. pu.1 is a transcription factor that acts as a master regulator of myeloid commitment (Tenen et al., 1997). We first observed a small population of pu.1 cells in the PBI at 30 hpf that increased in number by 36 hpf (Fig. 1E, K). Myeloperoxidase (mpx) is expressed specifically in cells of the myelomonocytic lineages, and is transcriptionally controlled by Pu.1 (Tenen et al., 1997). Accordingly, pu.1 expression precedes transcription of mpx in the PBI by at least 6 hours, since pu.1 and mpx expression are first detected at 30 and 36 hpf, respectively (Fig. 1E, F, K, L). Expression of all genes appeared to localize to the region between the aorta and the caudal vein, just posterior to the yolk tube extension. Taken together, these data indicate that both erythroid and myeloid cells are present in the PBI at 30 hpf and are consistent with the hypothesis that the PBI is an early site of multilineage hematopoiesis.

We next took a cytological approach to determine if definitive hematopoietic cell types could be recognized morphologically from PBI preparations. Tails were dissected from embryos at time points ranging from 24–72 hpf and single cell suspensions prepared following enzymatic digestion. At 24 hpf, the only hematopoietic lineages observed were erythroblasts and rare monocyte/macrophages (sFig. 1), each likely originating from primitive hematopoiesis. At 30 hpf another cell subset appeared that showed characteristics of early hematopoietic precursors, including blastic morphology, open chromatin structure and basophilic cytoplasm. Eosinophilic and neutrophilic granulocytes were first observed by 36 and 48 hpf, respectively. Cells of the thrombocytic series were first observed at 48 hpf, with mature thrombocytes appearing only after 72 hpf. Finally, cells displaying a lymphoid-like morphology (small, round cells with scant cytoplasm) appeared at 48 hpf. Since circulating lymphocytes are not present at this time, these cells may be HSCs that have immigrated from the AGM region, consistent with the migration hypothesis of Murayama and colleagues (Murayama et al., 2006).

We performed similar analyses of hematopoietic cells present in dissected embryonic trunk and head segments. Before 48 hpf, we did not observe definitive cell types in regions other than the tail (not shown). Collectively, these hematological results demonstrate that multiple lineages of hematopoietic cells are present in the PBI, corroborating our WISH data.

Coexpression of lmo2 and gata-1 defines a novel hematopoietic population in the PBI

One possible explanation for the presence of definitive myeloid and erythroid cells uniquely in the PBI at 30 hpf is that they arise from local progenitor cells. In murine bone marrow, only hematopoietic stem or progenitor cells express a combination of both Lmo2 and Gata-1 (Miyamoto et al., 2002). Since both lmo2 and gata-1 are expressed in the PBI, we analyzed their coexpression in animals carrying lmo2:eGFP and gata-1:DsRed transgenes. As shown in Figure 2A, cells expressing high levels of both transgenes were observed in the PBI from 30–48 hpf. Flow cytometric analysis of double transgenic embryos showed a distinct population of lmo2gata-1 cells (Fig. 2B). This population peaks in number per embryo between 30–36 hpf, the time at which the first myeloid precursors were observed by morphology (sFig. 1).

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Coexpression of lmo2 and gata-1 reveals immature hematopoietic precursors in the PBI. (A) Fluorescence microscopy reveals cells within the vascular plexus of the PBI that expressed both gata-1:DsRed and lmo2:eGFP fluorochromes (arrowheads) in double transgenic animals. Blue boxes superimposed on embryonic photographs in upper panels denote the regions shown at 20× magnification in middle and 40× magnification in lower panels at 30, 36, and 48 hpf. Lower panels show a single, deconvolved Z slice, demonstrating coexpression of each transgene in single cells (arrowheads). (B) Cells coexpressing the gata-1:DsRed and lmo2:eGFP transgenes are prospectively isolated by flow cytometry. Double positive cells peak in number at 30 hpf, with approximately 160 cells per embryo (left panel). (C) Compared to purified primitive erythroblasts sorted by low levels of lmo2:eGFP and high levels of gata-1:DsRed (red gate in 30 hpf plot), cytological staining of purified 30 hpf lmo2gata-1 cells (black gate) showed immature morphologies indicative of early hematopoietic progenitors.

Before 30 hpf, a population of lmo2gata-1 cells was observed by flow cytometry that expressed approximately 15-fold lower levels of GFP than lmo2gata-1 cells (Fig. 2B). Cell sorting showed that the lmo2gata-1 fraction consisted entirely of primitive erythroblasts (Fig. 2C, left panel). Compared to purified primitive erythoblasts, purification of lmo2gata-1 cells showed morphologies characteristic of earlier hematopoietic progenitors (Fig. 2C, middle and right panels). At 30 hpf, two morphological subtypes were present in purified lmo2gata-1 cells. Approximately half of this population displayed morphologies characteristic of immature myelomonocytic progenitors, including asymmetric oval or bean-shaped nuclei with lightly stained cytoplasm containing darker granules (Fig. 2C, middle panel). The remaining half displayed morphologies characteristic of immature erythroid progenitors, including centered, round nuclei with stippled staining patterns and basophilic cytoplasm (Fig. 2C, right panel).

lmo2gata-1 cells express both erythroid and myeloid genes

Hematopoietic progenitor cells frequently express lineage markers for multiple mature cell types, reflecting their multilineage differentiation potential (Hu et al., 1997; Miyamoto et al., 2002). We therefore profiled the gene expression pattern of purified lmo2gata-1 cells. At 30 hpf, we isolated four populations of cells defined by differential expression of lmo2:eGFP and gata-1:dsRed transgenes in double transgenic embryos, including lmo2gata-1, lmo2gata-1, lmo2gata-1, and lmo2gata-1 fractions. In addition to expressing high levels of the erythroid-associated gata-1 reporter gene, RT-PCR analyses showed that purified lmo2gata-1 cells expressed the pan-leukocyte marker, l-plastin, and the myelomonocytic genes, mpx and pu.1 (“LG”, Fig. 3A). By contrast, expression of these genes in purified primitive erythoblasts (lmo2gata-1 cells) was undetectable (“G”, Fig. 3A). This expression pattern suggests that, despite expression of the gata-1:dsRed transgene, the lmo2gata-1 population is not committed to the erythroid lineage. Accordingly, these cells also express scl and runx1, genes associated with early hematopoietic progenitors. Expression of the mpx and runx1 genes was found in the lmo2gata-1 fraction (“L”, Fig. 3A). runx1 is known to be expressed in endothelial cells (Kalev-Zylinska et al., 2002), the most abundant cell type marked by the lmo2:eGFP transgene. The presence of mpx transcripts may be due to low-level expression of the lmo2:eGFP transgene in primitive macrophages (not shown).

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Gene expression profiling of hematopoietic precursors in the PBI suggest multipotency. (A) Cells were purified from 30 hpf embryos by flow cytometry based on expression of gata-1:DsRed and lmo2:eGFP transgenes (DN – Double Negative; L - lmo2:eGFP, gata-1:DsRed; LG - lmo2:eGFP, gata-1:DsRed; G – lmo2:eGFP, gata-1:DsRed+; WKM – Whole Kidney Marrow) and subjected to RT-PCR. (B) FACS analysis shows a population that coexpresses the gata-1:DsRed and mpx:eGFP transgenes at 30 hpf (black gate). (C) Two-color FISH demonstrates that cells within the PBI at 30 hpf coexpress gata-1 and pu.1 (C, upper panels) and gata-1 and mpx (lower panels).

We also analyzed cd41 in each isolated subset, since its expression appears to be one of the first markers of mesoderm commitment to definitive hematopoiesis in mammals (Ferkowicz et al., 2003; Mikkola et al., 2003; Mitjavila-Garcia et al., 2002). In 30 hpf embryos, cd41 expression was only detected within lmo2gata-1 cells. This result supports the hypothesis that lmo2gata-1 cells represent an early definitive hematopoietic progenitor population.

To determine whether individual lmo2gata-1 cells express multiple lineage markers, as would be expected for a hematopoietic progenitor cell, we performed single-cell expression studies by FACS and FISH. To assess myeloerythroid gene expression at the single cell level, we analyzed embryos carrying mpx:eGFP and gata-1:DsRed transgenes. mpx:eGFP transgenic zebrafish were generated recently and shown to express GFP in embryonic granulocytes (Renshaw et al., 2006). In double transgenic embryos, early expression of the mpx:eGFP transgene is largely localized to cells also expressing the gata-1:DsRed transgene (Fig. 3B). We have also observed coexpression of the mpx and gata-1 transcripts in single cells in the PBI using two-color FISH (Fig. 3C). In murine multipotent progenitors, myeloid versus erythroid fate decisions are largely dependent upon the Pu.1 and Gata-1 transcription factors, respectively (Huang et al., 2007; Nerlov et al., 2000; Zhang et al., 2000). We observed coexpression of pu.1 and gata-1 in single cells in the PBI (Fig. 3C). We did not observe coexpression of either pu.1 and gata-1 or mpx and gata-1 in any other anatomical site in the zebrafish embryo (not shown). Taken together, our gene expression analyses strongly support the existence of a definitive, multilineage precursor in the zebrafish PBI.

PBI hematopoietic progenitors lack key characteristics of HSCs

Because lmo2gata-1 cells displayed many of the features of hematopoietic progenitor cells, we wanted to test their homing, proliferative, and differentiation potentials in functional assays. lmo2gata-1 cells were purified by flow cytometry from 36 hpf embryos and transplanted into WT embryonic recipients, either stage-matched or at 48 hpf. Transplanted cells homed to the PBI within 24 hours post-transplantation (Fig. 4A). In contrast to transplanted adult whole kidney marrow (WKM) cells which populate the embryonic thymus and pronephros (Traver et al., 2003), embryonic lmo2gata-1 cells were never observed to seed these hematopoietic organs. Rather, donor-derived cells remained largely within the PBI and were observable for approximately one week following transplantation by fluorescent transgene expression.

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Functional studies demonstrate that gata-1lmo2cd41cells are committed erythromyeloid progenitors. (A) Dissociated gata-1:DsRedlmo2:eGFP cells were purified from 36 hpf embryos by flow cytometry and transplanted into wild-type embryonic recipients. Transplanted cells were observed to home back to the PBI in host animals (right panel, 20X magnification). (B) In vivo fate mapping studies were performed by laser activation of caged rhodamine in cd41cells in the 44 hpf AGM or 40 hpf PBI. Presumptive AGM HSCs were targeted as positive controls for thymus colonization (lower panels; outlined crescent-shaped structure). Boxed yellow and blue regions in upper panel denote close up areas shown in left panels for the AGM and right panels for the PBI, respectively. All animals are shown in lateral views, with heads oriented to the left and dorsal sides up. Dotted line at the left edge of lower boxes denotes outline of the eye for orientation. (C) Short-term culture of lmo2gata-1 cells atop kidney stromal cells demonstrates erythroid (E) and myeloid (M) differentiation potential.

Several reports have demonstrated that rare, definitive hematopoietic precursors arise in the mammalian yolk sac prior to circulation (Bertrand et al., 2005b; Ferkowicz et al., 2003; Palis et al., 1999; Yokomizo et al., 2007; Yokota et al., 2006). These precursors express Cd41 (Ferkowicz et al., 2003; Mikkola et al., 2003; Yokota et al., 2006), which appears to be the first marker of definitive hematopoietic commitment from mesoderm. Since cd41 was detected only within lmo2gata-1 cells in 30–36 hpf embryos, we analyzed expression of cd41:eGFP in the PBI of transgenic embryos. GFP expression was detected as early as 26 hpf, and increased in intensity and in number of cells labeled over time (not shown). The location, appearance, and proliferative kinetics of cd41 cells closely matched those of lmo2gata-1 cells, suggesting that cd41 expression represents another, independent method of detecting definitive hematopoietic precursors in the PBI.

Use of a single, GFP marker enabled lineage-tracing studies. We performed lineage tracing experiments in living embryos by laser targeting cd41:eGFP cells to activate caged rhodamine molecules. By 40 hpf, many GFP cells were found within the vascular plexus of the PBI (Fig. 4B). Rhodamine was uncaged in ten cd41:eGFP cells in the PBIs of 30 transgenic animals. 30/30 animals displayed robust proliferation of targeted cells and distribution of their progeny throughout the PBI when analyzed at 4 days post-uncaging (Fig. 4B). 0/30 animals, however, showed labeled progeny in the thymus (Fig. 4B, Table 1), suggesting that cd41:eGFP cells in the PBI are not HSCs.

Table 1

Fate Mapping of Hematopoietic Precursors by Laser Uncaging

Laser Activation of Caged Rhodamine/FITC in Transgenic cd41:eGFP Cells
TissueStagenMarked Progeny in PBIMarked Progeny in Thymus
PBI40 hpf30300
44 hpf40405
48 hpf771

AGM44 hpf865
48 hpf885
Laser Activation of Caged Rhodamine/FITC in Transgenic lmo2:eGFP Cells
TissueFixationnMarked Progeny in PBIMarked Progeny in Trunk

Posterior30 hpf850
Medial30 hpf808

In each experiment, 10 GFP cells were laser targeted to uncage a combination of Rhodamine and FITC. Marked progeny were followed in living animals by Rhodamine fluorescence and were detected at noted timepoints following fixation and antibody visualization. Cells targeted in lmo2:eGFP animals were either in medial GFP stripes (bounded by somites 1–10) or at the posterior most end of the GFP expression domain.

As a positive control for thymic immigration, we performed similar experiments targeting putative HSCs in the AGM region. We previously reported that cd41:eGFP cells are observed between the dorsal aorta and cardinal vein in the zebrafish equivalent of the AGM (Lin et al., 2005). Based on the similarities to other vertebrate AGMs, the regional fate-mapping results of Murayama et al. (Murayama et al., 2006), and the finding that Cd41 marks embryonic HSCs in the mouse (Bertrand et al., 2005a), we hypothesized that cd41:eGFP expression in the AGM may mark the first HSCs to arise in the zebrafish embryo. We therefore performed similar lineage tracing studies by laser targeting ten cd41 cells in the AGM. Unlike cd41 cells in the 40 hpf PBI which never populated the thymus, 10/16 embryos where cd41 cells were targeted in the AGM between 44–48 hpf showed thymic colonization (Fig. 4B). The progeny of AGM cells were also observed to colonize the PBI, but we never observed the progeny of PBI targeted cd41 cells to colonize the AGM, thymus or pronephros.

Our transplantation and fate mapping experiments demonstrated that hematopoietic progenitors in the PBI lack lymphoid potential, a hallmark of HSCs. PBI progenitors did, however, display transient but relatively robust proliferation potential. To assess the differentiation potential of these cells, we developed in vitro culture assays using a new zebrafish kidney stromal (ZKS) cell line. lmo2gata-1 cells were purified from 30 hpf embryos and deposited onto ZKS layers. Aliquots were removed daily and analyzed for cellular morphology. Compared to the immature morphology of uncultured cells (Fig. 2C), lmo2gata-1 cells rapidly differentiated into erythroid and myelomonocytic cell fates upon 2 days of culture (Fig. 4C). An approximately equivalent ratio of erythroid to myelomonocytic cells was observed on day 2 (sFig. 2). By day 4 of culture myelomonocytic cells predominated, with most erythroid cells presumably having differentiated and died (Fig. 4C, sFig. 2). Based on morphological analyses, cultured lmo2gata-1 cells generated multiple myeloid lineages, including neutrophilic granulocytes, monocytes and macrophages, whereas parallel experiments using purified lmo2gata-1 primitive erythroblasts showed only erythroid progeny (not shown).

Taken together, these functional assays demonstrate that lmo2gata-1cd41 cells in the PBI represent a population of cells with both erythroid and myeloid differentiation potential. Their proliferation capacity is limited both in transplantation experiments and in vitro, and their progeny remain largely within in the PBI. Furthermore, they lack lymphoid potential. We therefore term these cells erythromyeloid progenitors (EMPs).

EMPs arise independently of HSCs from caudal, lmo2 mesoderm

Collectively, our data suggest that lmo2gata-1, or cd41 cells in the PBI before 40 hpf are committed erythromyeloid progenitors that lack self-renewal and multilineage differentiation abilities. Our fate mapping experiments also suggest that cd41 cells in the nascent AGM region represent the first HSCs born in the embryo. Although EMPs appear to arise before presumptive HSCs are observed in the AGM, it is possible that EMPs derive from HSCs. To determine whether EMPs are born directly in the PBI or migrate from the AGM, we performed uncaging experiments in mid-somitogenesis embryos. Approximately 10 lmo2 cells were targeted in either the medial (bounded by somites 1–10; the region that will later contain the AGM) or most posterior portion (the region that will later become the PBI) of the stripe of mesodermal-derived tissue marked by an lmo2:eGFP transgene in 13–15 somite stage (ss) embryos (Fig. 5A, B). Since EMPs peak in number at 30 hpf (Fig. 2B), we analyzed uncaged animals at this time point. 8/8 animals with medial lmo2 cells targeted showed marked progeny confined to the anterior trunk region (Table 1), mainly within presumptive vasculature (not shown). 0/8 animals showed marked progeny within the PBI (Fig. 5C). 5/8 animals with posterior lmo2+ cells targeted showed marked progeny within the PBI (Table 1). These cells were large and round and localized to the vascular plexus of the PBI (Fig. 5C), consistent with our localization of lmo2gata-1 EMPs at 30 hpf (Fig. 2A). None of these 8 animals showed labeled progeny in the trunk (Table 1).

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Erythromyeloid progenitors arise autonomously within the PBI. (A–D) Cells expressing GFP under control of the lmo2 promoter were lineage traced by uncaging a combination of caged rhodamine and FITC. GFP cells were targeted, either in the medial (bounded by somites 1–10; panel A) or most posterior (panel B) regions of the lmo2 expression domain between 13–15 somite stages. (C, D) Analysis of targeted progeny at 30 hpf showed no medially-derived cells in the PBI (C), whereas posterior-derived daughter cells were observed throughout the venous plexus of the PBI (D). Inset in (D) shows a close-up of the PBI in a second animal, with marked progeny found within the vascular plexus between the aorta (dashed red line) and caudal vein (dashed blue line). Primitive erythrocytes within each vessel are marked with asterisks, the daughters of posterior lmo2 cells by white arrowheads.

Together, these data suggest that lmo2gata-1 EMPs arise from the most posterior regions of the lmo2 (gata-1) hematopoietic/vasculogenic “stripes” that converge to form the posterior ICM/PBI. These results are consistent with previous studies demonstrating localized expression of “definitive” hematopoietic genes in this region such as scl (Liao et al., 2002) and gata-2 (Detrich et al., 1995).

The posterior blood island is a site of hematopoiesis

We wished to determine when and where the first definitive hematopoietic precursors arise in the zebrafish embryo. Early gene expression studies suggested that the most posterior portion of the ICM contained hematopoietic precursors distinct from the primitive erythroblasts that arise in the ICM (Thompson et al., 1998). To determine what types of blood cells are found in the PBI, we performed whole-mount in situ hybridization (WISH) with a panel of hematopoietic genes at 30 and 36 hpf. We first identified the zebrafish homologue of cd45, which in mammals is expressed in all hematopoietic cell lineages except for erythrocytes. Expression of the zebrafish cd45 gene was observed as early as 30 hpf, and positive cells increased in number by 36 hpf in the ventral part of the tail (Fig. 1A, G). Although some studies have treated l-plastin (also known as lcp1) as a macrophage-specific marker in the zebrafish, it has been shown to be expressed in all murine leukocytes, including hematopoietic progenitors (Arpin et al., 1994; Bertrand et al., 2005a) and in zebrafish granulocytes (Meijer et al., 2007). Expression of l-plastin was observed in a pattern similar to that of cd45 (Fig. 1B, H), suggesting that both genes similarly mark zebrafish leukocytes.

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Genes associated with multilineage hematopoiesis are expressed in the posterior blood island as early as 30 hpf. (A, B) By 30 hpf, expression of the pan-leukocyte markers cd45 and l-plastin are observed throughout the PBI. (G, H) By 36 hpf, the number of cells expressing each gene has increased in the PBI, and expressing cells begin to migrate throughout the embryo. Localized expression of lmo2 is observed in the PBI at 30 hpf (C) and 36 hpf (I). (D, J) Expression of gata-1 is also observed in cells within the vascular plexus of the PBI at both timepoints. (E, F, K, L) Expression of genes associated with myelopoiesis is also observed within the PBI, including pu.1 and mpx. Rare, pu.1 cells are observed at 30 hpf in the PBI (E) that increase in number by 36 hpf (K), whereas mpx expression is not observed until 36 hpf (F, L), consistent with it being a marker of relatively mature myelomonocytic cell types. Lower panels are 20X magnification views of the PBI from upper, 10X whole-embryo photographs.

To further assess the possible presence of hematopoietic progenitors, we analyzed expression of lmo2, gata-1 and pu.1. lmo2 is expressed by both vascular and hematopoietic precursors. In addition to widespread vascular staining in the tail, a small population of lmo2 cells was observed in the PBI (Fig. 1C, I). Similarly, expression of a canonical marker of erythroid potential, gata-1, was observed in small numbers of cells in the PBI at both time points (Fig. 1D, J). These cells did not appear to be within vessels, but rather within the vascular plexus of the PBI. pu.1 is a transcription factor that acts as a master regulator of myeloid commitment (Tenen et al., 1997). We first observed a small population of pu.1 cells in the PBI at 30 hpf that increased in number by 36 hpf (Fig. 1E, K). Myeloperoxidase (mpx) is expressed specifically in cells of the myelomonocytic lineages, and is transcriptionally controlled by Pu.1 (Tenen et al., 1997). Accordingly, pu.1 expression precedes transcription of mpx in the PBI by at least 6 hours, since pu.1 and mpx expression are first detected at 30 and 36 hpf, respectively (Fig. 1E, F, K, L). Expression of all genes appeared to localize to the region between the aorta and the caudal vein, just posterior to the yolk tube extension. Taken together, these data indicate that both erythroid and myeloid cells are present in the PBI at 30 hpf and are consistent with the hypothesis that the PBI is an early site of multilineage hematopoiesis.

We next took a cytological approach to determine if definitive hematopoietic cell types could be recognized morphologically from PBI preparations. Tails were dissected from embryos at time points ranging from 24–72 hpf and single cell suspensions prepared following enzymatic digestion. At 24 hpf, the only hematopoietic lineages observed were erythroblasts and rare monocyte/macrophages (sFig. 1), each likely originating from primitive hematopoiesis. At 30 hpf another cell subset appeared that showed characteristics of early hematopoietic precursors, including blastic morphology, open chromatin structure and basophilic cytoplasm. Eosinophilic and neutrophilic granulocytes were first observed by 36 and 48 hpf, respectively. Cells of the thrombocytic series were first observed at 48 hpf, with mature thrombocytes appearing only after 72 hpf. Finally, cells displaying a lymphoid-like morphology (small, round cells with scant cytoplasm) appeared at 48 hpf. Since circulating lymphocytes are not present at this time, these cells may be HSCs that have immigrated from the AGM region, consistent with the migration hypothesis of Murayama and colleagues (Murayama et al., 2006).

We performed similar analyses of hematopoietic cells present in dissected embryonic trunk and head segments. Before 48 hpf, we did not observe definitive cell types in regions other than the tail (not shown). Collectively, these hematological results demonstrate that multiple lineages of hematopoietic cells are present in the PBI, corroborating our WISH data.

Coexpression of lmo2 and gata-1 defines a novel hematopoietic population in the PBI

One possible explanation for the presence of definitive myeloid and erythroid cells uniquely in the PBI at 30 hpf is that they arise from local progenitor cells. In murine bone marrow, only hematopoietic stem or progenitor cells express a combination of both Lmo2 and Gata-1 (Miyamoto et al., 2002). Since both lmo2 and gata-1 are expressed in the PBI, we analyzed their coexpression in animals carrying lmo2:eGFP and gata-1:DsRed transgenes. As shown in Figure 2A, cells expressing high levels of both transgenes were observed in the PBI from 30–48 hpf. Flow cytometric analysis of double transgenic embryos showed a distinct population of lmo2gata-1 cells (Fig. 2B). This population peaks in number per embryo between 30–36 hpf, the time at which the first myeloid precursors were observed by morphology (sFig. 1).

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Coexpression of lmo2 and gata-1 reveals immature hematopoietic precursors in the PBI. (A) Fluorescence microscopy reveals cells within the vascular plexus of the PBI that expressed both gata-1:DsRed and lmo2:eGFP fluorochromes (arrowheads) in double transgenic animals. Blue boxes superimposed on embryonic photographs in upper panels denote the regions shown at 20× magnification in middle and 40× magnification in lower panels at 30, 36, and 48 hpf. Lower panels show a single, deconvolved Z slice, demonstrating coexpression of each transgene in single cells (arrowheads). (B) Cells coexpressing the gata-1:DsRed and lmo2:eGFP transgenes are prospectively isolated by flow cytometry. Double positive cells peak in number at 30 hpf, with approximately 160 cells per embryo (left panel). (C) Compared to purified primitive erythroblasts sorted by low levels of lmo2:eGFP and high levels of gata-1:DsRed (red gate in 30 hpf plot), cytological staining of purified 30 hpf lmo2gata-1 cells (black gate) showed immature morphologies indicative of early hematopoietic progenitors.

Before 30 hpf, a population of lmo2gata-1 cells was observed by flow cytometry that expressed approximately 15-fold lower levels of GFP than lmo2gata-1 cells (Fig. 2B). Cell sorting showed that the lmo2gata-1 fraction consisted entirely of primitive erythroblasts (Fig. 2C, left panel). Compared to purified primitive erythoblasts, purification of lmo2gata-1 cells showed morphologies characteristic of earlier hematopoietic progenitors (Fig. 2C, middle and right panels). At 30 hpf, two morphological subtypes were present in purified lmo2gata-1 cells. Approximately half of this population displayed morphologies characteristic of immature myelomonocytic progenitors, including asymmetric oval or bean-shaped nuclei with lightly stained cytoplasm containing darker granules (Fig. 2C, middle panel). The remaining half displayed morphologies characteristic of immature erythroid progenitors, including centered, round nuclei with stippled staining patterns and basophilic cytoplasm (Fig. 2C, right panel).

lmo2gata-1 cells express both erythroid and myeloid genes

Hematopoietic progenitor cells frequently express lineage markers for multiple mature cell types, reflecting their multilineage differentiation potential (Hu et al., 1997; Miyamoto et al., 2002). We therefore profiled the gene expression pattern of purified lmo2gata-1 cells. At 30 hpf, we isolated four populations of cells defined by differential expression of lmo2:eGFP and gata-1:dsRed transgenes in double transgenic embryos, including lmo2gata-1, lmo2gata-1, lmo2gata-1, and lmo2gata-1 fractions. In addition to expressing high levels of the erythroid-associated gata-1 reporter gene, RT-PCR analyses showed that purified lmo2gata-1 cells expressed the pan-leukocyte marker, l-plastin, and the myelomonocytic genes, mpx and pu.1 (“LG”, Fig. 3A). By contrast, expression of these genes in purified primitive erythoblasts (lmo2gata-1 cells) was undetectable (“G”, Fig. 3A). This expression pattern suggests that, despite expression of the gata-1:dsRed transgene, the lmo2gata-1 population is not committed to the erythroid lineage. Accordingly, these cells also express scl and runx1, genes associated with early hematopoietic progenitors. Expression of the mpx and runx1 genes was found in the lmo2gata-1 fraction (“L”, Fig. 3A). runx1 is known to be expressed in endothelial cells (Kalev-Zylinska et al., 2002), the most abundant cell type marked by the lmo2:eGFP transgene. The presence of mpx transcripts may be due to low-level expression of the lmo2:eGFP transgene in primitive macrophages (not shown).

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Gene expression profiling of hematopoietic precursors in the PBI suggest multipotency. (A) Cells were purified from 30 hpf embryos by flow cytometry based on expression of gata-1:DsRed and lmo2:eGFP transgenes (DN – Double Negative; L - lmo2:eGFP, gata-1:DsRed; LG - lmo2:eGFP, gata-1:DsRed; G – lmo2:eGFP, gata-1:DsRed+; WKM – Whole Kidney Marrow) and subjected to RT-PCR. (B) FACS analysis shows a population that coexpresses the gata-1:DsRed and mpx:eGFP transgenes at 30 hpf (black gate). (C) Two-color FISH demonstrates that cells within the PBI at 30 hpf coexpress gata-1 and pu.1 (C, upper panels) and gata-1 and mpx (lower panels).

We also analyzed cd41 in each isolated subset, since its expression appears to be one of the first markers of mesoderm commitment to definitive hematopoiesis in mammals (Ferkowicz et al., 2003; Mikkola et al., 2003; Mitjavila-Garcia et al., 2002). In 30 hpf embryos, cd41 expression was only detected within lmo2gata-1 cells. This result supports the hypothesis that lmo2gata-1 cells represent an early definitive hematopoietic progenitor population.

To determine whether individual lmo2gata-1 cells express multiple lineage markers, as would be expected for a hematopoietic progenitor cell, we performed single-cell expression studies by FACS and FISH. To assess myeloerythroid gene expression at the single cell level, we analyzed embryos carrying mpx:eGFP and gata-1:DsRed transgenes. mpx:eGFP transgenic zebrafish were generated recently and shown to express GFP in embryonic granulocytes (Renshaw et al., 2006). In double transgenic embryos, early expression of the mpx:eGFP transgene is largely localized to cells also expressing the gata-1:DsRed transgene (Fig. 3B). We have also observed coexpression of the mpx and gata-1 transcripts in single cells in the PBI using two-color FISH (Fig. 3C). In murine multipotent progenitors, myeloid versus erythroid fate decisions are largely dependent upon the Pu.1 and Gata-1 transcription factors, respectively (Huang et al., 2007; Nerlov et al., 2000; Zhang et al., 2000). We observed coexpression of pu.1 and gata-1 in single cells in the PBI (Fig. 3C). We did not observe coexpression of either pu.1 and gata-1 or mpx and gata-1 in any other anatomical site in the zebrafish embryo (not shown). Taken together, our gene expression analyses strongly support the existence of a definitive, multilineage precursor in the zebrafish PBI.

PBI hematopoietic progenitors lack key characteristics of HSCs

Because lmo2gata-1 cells displayed many of the features of hematopoietic progenitor cells, we wanted to test their homing, proliferative, and differentiation potentials in functional assays. lmo2gata-1 cells were purified by flow cytometry from 36 hpf embryos and transplanted into WT embryonic recipients, either stage-matched or at 48 hpf. Transplanted cells homed to the PBI within 24 hours post-transplantation (Fig. 4A). In contrast to transplanted adult whole kidney marrow (WKM) cells which populate the embryonic thymus and pronephros (Traver et al., 2003), embryonic lmo2gata-1 cells were never observed to seed these hematopoietic organs. Rather, donor-derived cells remained largely within the PBI and were observable for approximately one week following transplantation by fluorescent transgene expression.

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Functional studies demonstrate that gata-1lmo2cd41cells are committed erythromyeloid progenitors. (A) Dissociated gata-1:DsRedlmo2:eGFP cells were purified from 36 hpf embryos by flow cytometry and transplanted into wild-type embryonic recipients. Transplanted cells were observed to home back to the PBI in host animals (right panel, 20X magnification). (B) In vivo fate mapping studies were performed by laser activation of caged rhodamine in cd41cells in the 44 hpf AGM or 40 hpf PBI. Presumptive AGM HSCs were targeted as positive controls for thymus colonization (lower panels; outlined crescent-shaped structure). Boxed yellow and blue regions in upper panel denote close up areas shown in left panels for the AGM and right panels for the PBI, respectively. All animals are shown in lateral views, with heads oriented to the left and dorsal sides up. Dotted line at the left edge of lower boxes denotes outline of the eye for orientation. (C) Short-term culture of lmo2gata-1 cells atop kidney stromal cells demonstrates erythroid (E) and myeloid (M) differentiation potential.

Several reports have demonstrated that rare, definitive hematopoietic precursors arise in the mammalian yolk sac prior to circulation (Bertrand et al., 2005b; Ferkowicz et al., 2003; Palis et al., 1999; Yokomizo et al., 2007; Yokota et al., 2006). These precursors express Cd41 (Ferkowicz et al., 2003; Mikkola et al., 2003; Yokota et al., 2006), which appears to be the first marker of definitive hematopoietic commitment from mesoderm. Since cd41 was detected only within lmo2gata-1 cells in 30–36 hpf embryos, we analyzed expression of cd41:eGFP in the PBI of transgenic embryos. GFP expression was detected as early as 26 hpf, and increased in intensity and in number of cells labeled over time (not shown). The location, appearance, and proliferative kinetics of cd41 cells closely matched those of lmo2gata-1 cells, suggesting that cd41 expression represents another, independent method of detecting definitive hematopoietic precursors in the PBI.

Use of a single, GFP marker enabled lineage-tracing studies. We performed lineage tracing experiments in living embryos by laser targeting cd41:eGFP cells to activate caged rhodamine molecules. By 40 hpf, many GFP cells were found within the vascular plexus of the PBI (Fig. 4B). Rhodamine was uncaged in ten cd41:eGFP cells in the PBIs of 30 transgenic animals. 30/30 animals displayed robust proliferation of targeted cells and distribution of their progeny throughout the PBI when analyzed at 4 days post-uncaging (Fig. 4B). 0/30 animals, however, showed labeled progeny in the thymus (Fig. 4B, Table 1), suggesting that cd41:eGFP cells in the PBI are not HSCs.

Table 1

Fate Mapping of Hematopoietic Precursors by Laser Uncaging

Laser Activation of Caged Rhodamine/FITC in Transgenic cd41:eGFP Cells
TissueStagenMarked Progeny in PBIMarked Progeny in Thymus
PBI40 hpf30300
44 hpf40405
48 hpf771

AGM44 hpf865
48 hpf885
Laser Activation of Caged Rhodamine/FITC in Transgenic lmo2:eGFP Cells
TissueFixationnMarked Progeny in PBIMarked Progeny in Trunk

Posterior30 hpf850
Medial30 hpf808

In each experiment, 10 GFP cells were laser targeted to uncage a combination of Rhodamine and FITC. Marked progeny were followed in living animals by Rhodamine fluorescence and were detected at noted timepoints following fixation and antibody visualization. Cells targeted in lmo2:eGFP animals were either in medial GFP stripes (bounded by somites 1–10) or at the posterior most end of the GFP expression domain.

As a positive control for thymic immigration, we performed similar experiments targeting putative HSCs in the AGM region. We previously reported that cd41:eGFP cells are observed between the dorsal aorta and cardinal vein in the zebrafish equivalent of the AGM (Lin et al., 2005). Based on the similarities to other vertebrate AGMs, the regional fate-mapping results of Murayama et al. (Murayama et al., 2006), and the finding that Cd41 marks embryonic HSCs in the mouse (Bertrand et al., 2005a), we hypothesized that cd41:eGFP expression in the AGM may mark the first HSCs to arise in the zebrafish embryo. We therefore performed similar lineage tracing studies by laser targeting ten cd41 cells in the AGM. Unlike cd41 cells in the 40 hpf PBI which never populated the thymus, 10/16 embryos where cd41 cells were targeted in the AGM between 44–48 hpf showed thymic colonization (Fig. 4B). The progeny of AGM cells were also observed to colonize the PBI, but we never observed the progeny of PBI targeted cd41 cells to colonize the AGM, thymus or pronephros.

Our transplantation and fate mapping experiments demonstrated that hematopoietic progenitors in the PBI lack lymphoid potential, a hallmark of HSCs. PBI progenitors did, however, display transient but relatively robust proliferation potential. To assess the differentiation potential of these cells, we developed in vitro culture assays using a new zebrafish kidney stromal (ZKS) cell line. lmo2gata-1 cells were purified from 30 hpf embryos and deposited onto ZKS layers. Aliquots were removed daily and analyzed for cellular morphology. Compared to the immature morphology of uncultured cells (Fig. 2C), lmo2gata-1 cells rapidly differentiated into erythroid and myelomonocytic cell fates upon 2 days of culture (Fig. 4C). An approximately equivalent ratio of erythroid to myelomonocytic cells was observed on day 2 (sFig. 2). By day 4 of culture myelomonocytic cells predominated, with most erythroid cells presumably having differentiated and died (Fig. 4C, sFig. 2). Based on morphological analyses, cultured lmo2gata-1 cells generated multiple myeloid lineages, including neutrophilic granulocytes, monocytes and macrophages, whereas parallel experiments using purified lmo2gata-1 primitive erythroblasts showed only erythroid progeny (not shown).

Taken together, these functional assays demonstrate that lmo2gata-1cd41 cells in the PBI represent a population of cells with both erythroid and myeloid differentiation potential. Their proliferation capacity is limited both in transplantation experiments and in vitro, and their progeny remain largely within in the PBI. Furthermore, they lack lymphoid potential. We therefore term these cells erythromyeloid progenitors (EMPs).

EMPs arise independently of HSCs from caudal, lmo2 mesoderm

Collectively, our data suggest that lmo2gata-1, or cd41 cells in the PBI before 40 hpf are committed erythromyeloid progenitors that lack self-renewal and multilineage differentiation abilities. Our fate mapping experiments also suggest that cd41 cells in the nascent AGM region represent the first HSCs born in the embryo. Although EMPs appear to arise before presumptive HSCs are observed in the AGM, it is possible that EMPs derive from HSCs. To determine whether EMPs are born directly in the PBI or migrate from the AGM, we performed uncaging experiments in mid-somitogenesis embryos. Approximately 10 lmo2 cells were targeted in either the medial (bounded by somites 1–10; the region that will later contain the AGM) or most posterior portion (the region that will later become the PBI) of the stripe of mesodermal-derived tissue marked by an lmo2:eGFP transgene in 13–15 somite stage (ss) embryos (Fig. 5A, B). Since EMPs peak in number at 30 hpf (Fig. 2B), we analyzed uncaged animals at this time point. 8/8 animals with medial lmo2 cells targeted showed marked progeny confined to the anterior trunk region (Table 1), mainly within presumptive vasculature (not shown). 0/8 animals showed marked progeny within the PBI (Fig. 5C). 5/8 animals with posterior lmo2+ cells targeted showed marked progeny within the PBI (Table 1). These cells were large and round and localized to the vascular plexus of the PBI (Fig. 5C), consistent with our localization of lmo2gata-1 EMPs at 30 hpf (Fig. 2A). None of these 8 animals showed labeled progeny in the trunk (Table 1).

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Erythromyeloid progenitors arise autonomously within the PBI. (A–D) Cells expressing GFP under control of the lmo2 promoter were lineage traced by uncaging a combination of caged rhodamine and FITC. GFP cells were targeted, either in the medial (bounded by somites 1–10; panel A) or most posterior (panel B) regions of the lmo2 expression domain between 13–15 somite stages. (C, D) Analysis of targeted progeny at 30 hpf showed no medially-derived cells in the PBI (C), whereas posterior-derived daughter cells were observed throughout the venous plexus of the PBI (D). Inset in (D) shows a close-up of the PBI in a second animal, with marked progeny found within the vascular plexus between the aorta (dashed red line) and caudal vein (dashed blue line). Primitive erythrocytes within each vessel are marked with asterisks, the daughters of posterior lmo2 cells by white arrowheads.

Together, these data suggest that lmo2gata-1 EMPs arise from the most posterior regions of the lmo2 (gata-1) hematopoietic/vasculogenic “stripes” that converge to form the posterior ICM/PBI. These results are consistent with previous studies demonstrating localized expression of “definitive” hematopoietic genes in this region such as scl (Liao et al., 2002) and gata-2 (Detrich et al., 1995).

DISCUSSION

Our results demonstrate that definitive hematopoiesis initiates in the PBI through a committed progenitor that we have termed the erythromyeloid progenitor (EMP). These progenitors appear to exist transiently, and can be isolated prospectively by their coexpression of lmo2 and gata-1 from 24–48 hpf. Our description of a definitive progenitor in the PBI explains previous gene expression studies suggesting a distinct population of cells is present in the caudal most portion of the developing ICM. Based on the expression of gata-2 cells at the posterior tip of the gata-1 ICM, Detrich et al. postulated these cells to be undifferentiated, presumptive hematopoietic progenitors (Detrich et al., 1995). Additional early observations showed that cells in this region also expressed the lmo2 gene (Thompson et al., 1998). These authors hypothesized that these cells may be the founders of a distinct second wave of hematopoiesis. Our identification of the EMP confirms these hypotheses, and characterization of their molecular markers provides the means to isolate them for functional analyses, including lineage differentiation potentials.

The presence of the EMP in the PBI by 24 hpf may help clarify the ontogeny of the definitive myeloid lineages in teleosts. Histological and ultrastructural analyses have described granulocytic precursors in the PBI, both in the zebrafish (Willett et al., 1999; Zapata et al., 2006) and carp (Huttenhuis et al., 2006; Rombout et al., 2005). By morphology, we have observed eosinophilic granulocytes in zebrafish tail preparations by 36 hpf and neutrophilic granulocytes by 48 hpf. The first granulocytes produced in the zebrafish embryo may therefore be the daughters of EMPs, and not lineally related to the primitive macrophages that arise from cephalic mesoderm. This model is consistent with the observations of Herbomel and colleagues (Herbomel et al., 1999), and with the single lineage origin of embryonic macrophages produced in other organisms ranging from Drosophila to mammals (Evans et al., 2003; Lichanska and Hume, 2000). Based on the robust generation of mononuclear phagocytes by cultured EMPs, it appears that a second wave of macrophage production later occurs in the PBI through monocytic intermediates. The full differentiation potential of the EMP remains to be determined, since our in vitro conditions do not appear to support all myeloerythroid lineages, including eosinophils and thrombocytes. Isolation of autologous growth factors such as Interleukin-3 and Thrombopoietin will likely be necessary to reveal the complete fate potentials of zebrafish hematopoietic stem and progenitor cells.

The first blood cells born in the mammalian embryo are erythroid cells in the extraembryonic yolk sac. Based on their resemblance to the nucleated erythrocytes of avian and amphibian species, these cells were initially termed primitive, and their enucleated embryonic counterparts definitive (McGrath and Palis, 2005). More recently, these terms have become interchangeable with yolk sac and intraembryonic hematopoiesis, respectively. The correlation of each wave with anatomical sites has led to some confusion, since several reports have recently demonstrated the generation of definitive cell types in the extraembryonic yolk sac. Definitive hematopoietic progenitors have been identified in the precirculation yolk sac that generate granulocytes, macrophages, mast cells, and erythrocytes that express adult type globins (Bertrand et al., 2005b; Cumano, 1996; Palis et al., 1999; Wong et al., 1986; Yokomizo et al., 2007; Yokota et al., 2006). Subsequent studies showed that Cd41 expression marked early definitive precursors in the yolk sac (Ferkowicz et al., 2003; Mikkola et al., 2003). Before E9.5, Cd41 yolk sac cells generate multiple myeloid and erythroid lineages, but lack lymphoid potential in coculture assays (Yokota et al., 2006). Similar results were obtained at E10.5 using yolk sac cells purified by combined Cd41 c-Kit Cd45 expression (Bertrand et al., 2005a). In addition, studies utilizing a transgenic mouse expressing GFP under control of the Gata-1 promoter showed that Gata-1 cells purified from E7.5 yolk sacs could generate granulocytes, monocytes, macrophages and erythrocytes upon OP9 stromal cells (Yokomizo et al., 2007). Taken together, these studies, with our current report, highlight the initial development of definitive hematopoiesis through a committed progenitor that can be isolated prospectively based on the expression of Gata-1 and Cd41 in both the mouse and the zebrafish.

The nomenclature used for the hematopoietic waves in the zebrafish has often been inconsistent and confusing, with many publications referring to “primitive HSCs”, “primitive granulocytes”, and so forth. We propose that the same conventions applied to the murine hematopoietic waves (Keller et al., 1999; McGrath and Palis, 2005) be used to describe primitive versus definitive hematopoiesis in the zebrafish, namely that only ICM-derived erythrocytes and cephalic mesoderm-derived macrophages should be termed primitive. This nomenclature is consistent with observations in each species suggesting that the two primitive lineages appear to arise directly from mesoderm without transiting through a multipotent progenitor (Bertrand et al., 2005b; Detrich et al., 1995; Herbomel et al., 1999; Keller et al., 1999). Conversely, we propose that hematopoietic cells that derive from an oligopotent or multipotent progenitor be termed definitive.

In our current model, hematopoietic development in the zebrafish appears to occur in four independent waves, each through precursors that arise in different anatomical regions (Fig. 6). Two primitive waves produce macrophages from cephalic mesoderm and erythrocytes from the ICM. Likewise, two definitive waves occur, the first from EMP production in the PBI and the second from HSC generation in the AGM. It was previously proposed by Murayama and colleagues that all hematopoietic cells in the PBI derive from HSCs born in the AGM (Murayama et al., 2006). Our results do not support this hypothesis. Several lines of evidence show that hematopoietic precursors are present in the PBI before presumptive HSCs can be detected in the AGM region beginning at approximately 28 hpf. These include gene expression profiles suggestive of multilineage hematopoiesis in the PBI by 30 hpf, the appearance of definitive myelomonocytic cells within the PBI by 36 hpf, single cell coexpression of early hematopoietic genes such as lmo2/gata-1 or gata-1/pu.1 by 30 hpf, and cd41:eGFP cells appearing in the PBI before the AGM. Furthermore, our fate mapping experiments suggest that EMPs arise directly from the lmo2:eGFP descendents of posterior LPM that contribute to the formation of the PBI. The experiments of Murayama et al. were performed after 48 hpf. Our lineage tracing experiments show that cd41:eGFP cells in the PBI before 40 hpf lack thymus colonizing potential. After 40 hpf, we observed rare thymic immigrants from targeted cd41:eGFP cells in the PBI that increased in number over time. These results suggest that the first cd41:eGFP cells in the PBI are EMPs and that cd41:eGFP HSCs immigrate into the PBI from the AGM after 40 hpf. It appears that the EMP disappears gradually after this time, with definitive hematopoiesis in the PBI increasingly deriving from incoming HSCs. As previously suggested in the mouse, the generation of committed EMPs in the embryo before HSCs arise has likely evolved to provide a rapid burst of definitive cell types to meet the needs of the growing embryo (Keller et al., 1999; McGrath and Palis, 2005).

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Model of hematopoietic ontogeny in the zebrafish embryo. (A) Different regions of lateral plate mesoderm (LPM) give rise to anatomically distinct regions of blood cell precursors. Cartoon depicts a dorsal view of a 5 somite stage embryo. (B) Embryonic hematopoiesis appears to occur through four, independent waves of precursor production. Each wave is numbered based on the temporal appearance of functional cells from each subset.

In conclusion, we have demonstrated that definitive hematopoiesis initiates through a committed progenitor that is generated independently of HSCs in the posterior blood island of the zebrafish embryo. These findings provide a better understanding of hematopoietic ontogeny in the zebrafish and help clarify the beginnings of the definitive blood cell lineages in teleosts. The development of prospective isolation approaches and a variety of functional assays to both purify and distinguish EMPs and HSCs now enable the means to genetically dissect the cues that pattern each population from mesoderm. Our results also highlight the close conservation between zebrafish and mammals in the temporal development of each hematopoietic wave during development of the blood-forming system.

Supplementary Material

Supplemental Figure 1. Temporal appearance of definitive blood cell types in the embryonic tail.Hematopoietic cells identified from tail preparations at noted timepoints were assigned to presumptivelineage affiliations by morphological criteria.

Supplemental Figure 2. Lineage differentials of cell types produced from cultured EMPs. n = numberof cells counted from each timepoint.

Supplemental Figure 1. Temporal appearance of definitive blood cell types in the embryonic tail.Hematopoietic cells identified from tail preparations at noted timepoints were assigned to presumptivelineage affiliations by morphological criteria.

Supplemental Figure 2. Lineage differentials of cell types produced from cultured EMPs. n = numberof cells counted from each timepoint.

Click here to view.(330K, pdf)

Acknowledgments

We thank Amy Kiger, Patricia Ernst, and Wilson Clements for critical evaluation of the manuscript, Steve Renshaw for providing mpx:eGFP animals, David Kosman, Bill McGinnis, Samantha Zeitlin and Scott Holley for assistance with FISH techniques and confocal microscopy, Shutian Teng for performing initial CD45 WISH experiments, Shrey Purohit and Jason Reyes for assistance with progenitor cultures, Colin Jamora for microscope use, Adam O’Connor and Karen Ong for assistance with flow cytometry, and Roger Rainville, Evie Wright, and Lisa Phelps for excellent animal care. Supported by the Irvington Institute for Immunological Research (J.Y.B.), the Cancer Research Prevention Foundation (D.L.S.), National Institutes of Health grant # DK074482, the March of Dimes Foundation, and the Arnold and Mabel Beckman Foundation (D.T.).

Section of Cell and Developmental Biology, Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093-0380
These authors contributed equally to this work

SUMMARY

Shifting sites of blood cell production during development is common across widely divergent phyla. In zebrafish, like other vertebrates, hematopoietic development has been roughly divided into two waves, termed primitive and definitive. Primitive hematopoiesis is characterized by the generation of embryonic erythrocytes in the intermediate cell mass and a distinct population of macrophages that arises from cephalic mesoderm. Based on previous gene expression studies, definitive hematopoiesis has been suggested to begin with the generation of presumptive hematopoietic stem cells (HSCs) along the dorsal aorta that express cmyb and runx1. Here we show, using a combination of gene expression analyses, prospective isolation approaches, transplantation, and in vivo lineage tracing experiments, that definitive hematopoiesis initiates through committed erythromyeloid progenitors (EMPs) in the posterior blood island (PBI) that arise independently of HSCs. EMPs isolated by coexpression of fluorescent transgenes driven by the lmo2 and gata1 promoters exhibit an immature, blastic morphology and express only erythroid and myeloid genes. Transplanted EMPs home to the PBI, show limited proliferative potential, and do not seed subsequent hematopoietic sites such as the thymus or pronephros. In vivo fate mapping studies similarly demonstrate that EMPs possess only transient proliferative potential, with differentiated progeny remaining largely within caudal hematopoietic tissue. Additional fate mapping of mesodermal derivatives in mid-somitogenesis embryos suggests that EMPs are born directly in the PBI. These studies provide phenotypic and functional analyses of the first hematopoietic progenitors in the zebrafish embryo and demonstrate that definitive hematopoiesis proceeds through two distinct waves during embryonic development.

SUMMARY
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