The fibrinogen receptor GPIIb-IIIa integrin is known to be expressed on cells of the megakaryocytic lineage, but its presence on hematopoietic progenitors has been a controversial issue. To resolve this ambiguity unequivocally, we performed clonogenic assays and intrathymic cell-transfer experiments in congenic animals. As the ontogeny of the avian hematopoietic system is well documented, we used this experimental model to trace GPIIb-IIIa expression during embryogenesis. Consequently, we now report that the GPIIb-IIIa integrin is expressed as early as embryonic day 3.5 (E3.5) to 4 in intraaortic hematopoietic clusters, the first site of intraembryonic hematopoietic progenitor emergence, and later in E6 paraaortic foci. Myeloid and erythroid progenitors were also detected within the GPIIb-IIIa+ CD45+ population isolated from the E3.5 to 4 aortic area, while in embryonic and adult bone marrow, myeloid, erythroid, and T-cell progenitors were present in the GPIIb-IIIa+ c-kit+ population. Furthermore, we also provide the first evidence, that GPIIb-IIIa+ bone marrow cells can differentiate into T cells. Hence, GPIIb-IIIa can be used as a marker for multilineage hematopoietic progenitors, permitting identification of early intraembryonic sites of hematopoiesis, as well as the isolation of embryonic and adult hematopoietic progenitors.

HEMATOPOIETIC STEM CELLS (HSC) from adult mouse bone marrow express the markers Sca-1 and Thy-1, but lack expression of lineage-specific markers.1 At late embryonic stages, HSC are present in the Thy-1lo, c-kit+ population of blood,2 whereas at earlier embryonic stages HSC are defined as CD34+c-kit+ cells.3 Such findings demonstrate the need for a combination of markers for accurate identification of hematopoietic progenitors.

The platelet integrin GPIIb-IIIa has been extensively studied in mammals as it plays a fundamental role in the function of megakaryocytes (MK).4 It is an important molecule in cell-substratum adhesion and platelet aggregation, and mutations in the gene for this receptor are responsible for pathologic diseases such as Glanzmann’s thrombasthenia in humans.5 

Evidence that GPIIb-IIIa is also expressed on hematopoietic progenitors has been provided by several laboratories. Based on morphologic and in vitro culture data, Debili et al6 found GPIIb-IIIa expressed on MK progenitors. In addition, Berridge et al7showed that anti–GPIIb-IIIa antibodies could reduce spleen colony-forming units (CFU-S), granulomonocytic colony-forming units (CFU-GM), and CFU-MK from bone marrow cells. The same group demonstrated that treatment of cord blood cells with polyclonal antiserum against GPIIb-IIIa inhibited the growth of early progenitors, which had the potential to differentiate into mixed colonies (CFU-Mix), while CFU-GM and erythroid burst-forming units (BFU-E) were unaffected.8 Recently, conditional knock-out mice were generated, in which a thymidine kinase gene was placed under the control of the αIIb promoter. As a result, all thymidine kinase–expressing cells were eradicated upon ganciclovir administration. These animals suffered from thrombocytopenia, and the growth of bone marrow CFU-Mix, myeloid, and erythroid progenitors was dramatically reduced.9,10 Finally, Murray et al11 reported that the GPIIb-IIIa+ cell population, which contains CFU-MK and CFU-Mix, is also positive for CD34, a known marker for HSC in humans. Thus, although GPIIb-IIIa is a marker expressed throughout the megakaryocytic differentiation pathway, there is some evidence suggesting that it is also expressed by other hematopoietic progenitor cells, at a commitment stage as yet undefined. However, there has been no substantial evidence to date to indicate that GPIIb-IIIa+ progenitors can differentiate into lymphocytes.

In an attempt to address these issues and characterize the differentiation potential of GPIIb-IIIa+ HSC further, we used the chicken as an experimental model. This animal offers an easy access to embryonic and adult thymuses and also to accurately staged embryos, allowing precise localization and labeling of emerging HSC, which can be detected as early as embryonic day 3.5 (E3.5) to 4, in intraaortic foci and at E6 in the paraaortic mensenchyme.12 Furthermore, T-cell differentiation can be readily followed by intrathymic injection of progenitors, using two congenic strains of chickens.

Intraembryonic sites of hematopoiesis were originally described in birds, and only recently have homologous sites been identified in mammals. In mice, intraembryonic hemopoiesis is initiated in the region of the paraaortic splanchnopleura13 and later in the aorta-gonad-mesonephros region (AGM).14 In human embryos, CD34+ hematopoietic cells have been identified in the ventral endothelium of the aorta at the fifth week of gestation.15 

In this study, we used a monoclonal antibody (MoAb) against GPIIb-IIIa to investigate the expression of this integrin on hematopoietic progenitors during chicken ontogeny. Using flow cytometry, we sorted GPIIb-IIIa+ cells isolated from intraaortic clusters, and embryonic and adult bone marrow. Clonogenic assays for the multilineage potential of HSC showed that progenitors for myeloid, erythroid, and thrombocytic (the avian homolog for megakaryocytic) cells all express GPIIb-IIIa. In addition, in vivo adoptive transfer experiments showed that T-cell progenitors could also express GPIIb-IIIa. GPIIb-IIIa+ progenitors in the bone marrow were found in the c-kit+ population. In the intraaortic and paraaortic foci, GPIIb-IIIa+ progenitors were found in the CD45+ population.

In summary, we show that antibodies against GPIIb-IIIa integrin could be a useful tool for the characterization and localization of hematopoietic progenitors in embryos, and that this integrin is no longer an exquisite marker for the megakaryocytic lineage.

Animals

Outbred JA57 chick embryos were obtained from a local commercial source. Embryonated eggs from the H.B19 strain were produced at the facility of the Basel Institute for Immunology in Oberfrick, Switzerland. The H.B19 strain was subdivided into two congenic lines: H.B19 ov+ and ov, distinguished by the presence or absence of the ov antigen on T-lineage cells.

Immunohistology on Sections

Embryos were fixed in 4% (wt/vol) paraformaldehyde, embedded in gelatin-sucrose, and frozen in isopentane at −70°C. These were then sliced into 20-μm sections. Antibody staining and immunoperoxidase tissue analysis on the cryostat sections were performed as previously described.16 Immunofluorescence staining on tissue sections was performed with the tyramide-based detection method that increases the fluorescent signal (Renaissance Tyramide Signal Amplification kit; NEN Life Science Products, Les Ulis, France).

Bone Marrow and Aortic Region Suspensions

Bone marrow cells from 14-day-old embryos (E14) and 4-week-old chicks were removed with 25- and 21-gauge needles, respectively. Aortic regions from E3.5 to 4 chick embryos, stage 19 to 22 of Hamburger and Hamilton (HH)17 were retrieved as described previously.18 They were treated with 0.1% collagenase at 37°C for 1 hour, after which the cells were washed and resuspended in alpha-medium (GIBCO-BRL, France) with 3% fetal calf serum (FCS). Approximately 106 cells were usually obtained from 40 embryos.

Immunocytologic Labeling, Fluorescence-Activated Cell Sorting Analysis, and Sorting

Cells were incubated for 30 minutes with primary MoAbs, washed in medium with FCS, and incubated for 30 minutes with secondary goat anti-mouse IgM, IgG1, or IgG2a conjugated with phycoerythrin (PE) or fluorescein isothiocyanate (FITC) (Southern Biotechnology, Clinisciences, Montrouge, France).

Cells were then washed twice and resuspended at 5 × 106/mL and filtered through a nylon sieve before analysis and sorting by florescence-activated cell sorting (FACS) (FACS Star+; Becton Dickinson, France). The purity of the sorted population was found to be approximately 97%. After sorting, cells were collected in tubes containing 5% bovine serum albumin (BSA) or FCS. Regular two-color FACS analyses were performed on a FACSCalibur (Becton Dickinson) using the FL1, FL2, or FL4 channels with appropriate compensations.

Antibodies

The following mouse MoAbs were used: 11C3, which detects the GPIIb-IIIa chicken molecule, expressed by cells of the thrombocytic lineage19; HISC7, which recognizes CD45, present on all leukocytes20; MYL 51/2 specific for myelomonocytic cells21; c75, anti-chicken c-kit MoAb against the tyrosine kinase receptor expressed on hematopoietic progenitors22; AP2, which is specific for the human GPIIb-IIIa complex and crossreacts with GPIIb-IIIa on chicken thrombocytes23,24; 11A9, which recognizes the ov epitope expressed by T-lymphoid lineage cells of H.B19+.22 Anti-chicken CD4 (2-6) and CD8 (11-39) MoAbs were directly conjugated to FITC or PE.25 26Anti-human αvβ3 (MAB 1976; Chemicon International, Temecula, CA) was directly coupled to Cy5 using the FluoroLink Cy5 reactive dye from Amersham Life Science (Arlington Heights, IL). This antibody crossreacts with αvβ3 integrin of several species, including the chicken.

Assay for Hematopoietic Progenitor Cells

Progenitor cells were detected by their colony-forming ability in semisolid cultures as previously described.27 28 Cells were seeded in 0.5 mL medium and clotted by the addition of citrated bovine plasma (GIBCO-BRL) and thrombin (1 IU/mL; Miles Inc, Kankakee, IL).

Myeloid differentiation medium.

Myeloid colony-forming cells developed in the presence of 3% fibroblast-conditioned medium and gave rise to macrophage (M), granulocytic (G), and macrophage/granulocytic (M/G) colonies. Macrophage colonies were identified by the specific MYL 51/2 MoAb.

Thrombocytic differentiation medium.

In mammals, MK terminal differentiation is characterized by cytoplasmic fragmentation, which gives rise to platelets. Avians do not have platelets, and mononucleated thrombocytes are the terminal form of differentiation for this lineage in birds.29 

Thromboblastic (Tb) and thrombocytic (Tc) colonies both developed after the addition of 15% E10 kidney conditioned medium (in serum-free Dulbecco’s modified Eagles’ medium [DMEM] supplemented with 1 μg/mL bovine insulin, 15 μg/mL conalbumin, 20 μmol/L ethanolamine, 2.5 nmol/L Na-selenite, glutamine, 5 × 10−4 mol/L 2-β mercaptoethanol (ME), and nonessential amino acid30). Few granulocytic clusters developed under these conditions.

Thromboblast/erythroblast and granulocyte differentiation medium.

Chicken recombinant soluble stem-cell factor (SCF) (Amgen, Thousand Oaks, CA) was used in serum-free cultures at 100 ng/mL, a concentration that allows the differentiation of avian erythroid progenitors.31 

Erythroid differentiation medium.

Erythroid progenitor cells and erythroid burst-forming units (BFU-E) differentiate in the presence of 1 ng/mL transforming growth factor-α (TGFα; Biomedical Technologies, Stoughton, MA), 10 ng/mL bovine insulin (Sigma, France), and 0.5 U/mL mouse recombinant erythropoietin (provided by Dr E. Goldwasser, Chicago, IL), and both chicken serum (5%,) and FCS (20%) as described by Pain et al.32 Erythroblastic (Eb) and erythrocytic (Ec) colonies will readily grow under these conditions. Benzidine dihydrochloride (Sigma) staining was used to identify hemoglobin-containing cells as described by Palis et al.33 Five microliters of 30% hydrogen peroxide (Sigma) was added, immediately before use, to 100 μL of benzidine (0.1%) and mixed 1:1 with phosphate-buffered saline (PBS). The reaction was performed for 10 to 15 minutes at room temperature. Hemoglobin-containing cells stained dark blue.

In the presence of serum, myeloid colonies (M, G, M/G) and Tb colonies can also develop. Since Tb and Eb have similar morphology after May-Grünwald-Giemsa (MGG) staining, these colonies were referred to as Tb/Eb. The cultures were dried and MGG-stained for morphologic and quantitative examination using a Nikon Microphot-FXA microscope (Nikon, France). In some cases, the dried cultures were immunostained with different antibodies.

In Vivo T-Cell Progenitor Assay by Intrathymic Injection

The potential of progenitor cells to differentiate into T lymphocytes was studied according to a previously described method.22H.B19 8-day-old ov chicks were irradiated with 600 rad from a 137Cs source (110 rad/min) 6 hours before receiving sorted bone marrow cells from congenic H.B19 ov+E14 donor animals. Each thymic lobe was injected with a 10 μL cell suspension in PBS-BSA, using a Tridak Stepper syringe (Tridak, Brookfield, CT). Two weeks later, the chickens were killed and cells from the injected thymic lobes were isolated. The level of chimerism of the recipient thymus by ov+ donor cells was determined by immunofluorescence with the MoAb 11A9 directed against the ov+ antigen. The T-cell identity of these cells was determined by CD4/CD8 staining.

Limiting Dilution Assay for CFU

Bone marrow cells were seeded in 96-well tissue culture plates in 100 μL semisolid medium, at concentrations of 1 to 100 cells per well in the presence of kidney-conditioned medium. After 3 days, the cell clusters were harvested, stained with MGG and the number of colonies counted. The frequencies were estimated according to Poisson’s analysis.

Hematopoietic Progenitor Activity in Embryonic and Adult Bone Marrow

We previously showed that thromboblasts and thrombocytes are exclusively present in the GPIIb-IIIa+ cell population from embryonic day 14 (E14) bone marrow.19 To analyze whether the GPIIb-IIIa+ bone marrow cells also contained progenitors for the thrombocytic lineage, we performed in vitro colony-forming assays in semisolid medium. Accordingly, sorted E14 GPIIb-IIIa+ bone marrow cells (Fig1A) cultured in thrombocytic differentiation medium were able to develop into thromboblastic (Tb, Fig 1B) and thrombocytic (Tc, Fig 1C) colonies. Their numbers were sevenfold higher as compared with numbers obtained from similarly cultured unfractionated bone marrow cells. The approximate frequency after limiting dilution was calculated according to Poisson’s analysis. As determined from three independent experiments, this was found to be one in 10 (data not shown). In contrast, almost no colonies developed from the GPIIb-IIIa cell population. Thus, the GPIIb-IIIa integrin is expressed by thrombocytic progenitors in embryonic bone marrow.

Fig. 1.

Distribution of thromboblastic progenitors in embryonic bone marrow. (A) Thromboblastic colonies were scored from sorted GPIIb-IIIa positive (+) and negative (−) populations, and from total E14 bone marrow cells (unsorted) in thromboblastic differentiation medium. Data were normalized to 1,000 cultured cells. Mean ± SD is from eight experiments. (B) Morphology of colonies of thromboblasts (Tb), and (C) thrombocytes (Tc) (MGG staining). These cells were GPIIb-IIIa+ (not shown). Thromboblasts colonies are composed of several clusters. Bar = 12 μm.

Fig. 1.

Distribution of thromboblastic progenitors in embryonic bone marrow. (A) Thromboblastic colonies were scored from sorted GPIIb-IIIa positive (+) and negative (−) populations, and from total E14 bone marrow cells (unsorted) in thromboblastic differentiation medium. Data were normalized to 1,000 cultured cells. Mean ± SD is from eight experiments. (B) Morphology of colonies of thromboblasts (Tb), and (C) thrombocytes (Tc) (MGG staining). These cells were GPIIb-IIIa+ (not shown). Thromboblasts colonies are composed of several clusters. Bar = 12 μm.

Close modal

We then wondered whether, in addition to thrombocytic progenitors, the positive bone marrow population that was stained with the anti–GPIIb-IIIa MoAb 11C3 also contained progenitors for other hematopoietic lineages that would develop if the cells were cultured with the appropriate growth factors. Subsequent experiments then demonstrated that under myeloid differentiation conditions, cells from the 11C3+ population could predominantly develop into M, G, or M/G colonies as compared with cells from the 11C3population. Furthermore, erythroid differentiation medium allowed the differentiation of GPIIb-IIIa+ cells into macrophages (M, Fig 2C), granulocytes (G, Fig 2D), macrophages/granulocytes (M/G, Fig 2E), erythroblasts/thromboblasts (Eb/Tb, Fig 1B), and erythrocytes (Ec, Fig 2F). Again, no colonies developed from the GPIIb-IIIacells under similar conditions. The same bone marrow cell preparations were stained and sorted with the cross-reacting anti-human GPIIb-IIIa antibody AP2.19 24 The percentage of AP2+ cells was found to be identical to that obtained using the anti-chicken GPIIb-IIIa antibody 11C3. Furthermore, in both cases, the myeloid and erythroid differentiation potential of the sorted cells was comparable (Fig 2A and B). These results clearly indicated that colonies of different hematopoietic lineages can develop from GPIIb-IIIa+ embryonic bone marrow cells, and that cells selected by these two antibodies have identical differentiation potentials.

Fig. 2.

Distribution of myeloid and erythroid progenitors in GPIIb-IIIa+–sorted embryonic bone marrow cells. Embryonic bone marrow cells were sorted with the anti-chicken GPIIb-IIIa MoAb 11C3 or with the anti-human chicken cross-reacting GPIIb-IIIa MoAb AP2. Positively (+) or negatively (−) sorted cells were then cultured in erythroid or myeloid differentiation conditions in semisolid medium. Colonies were MGG stained and scored. (A) Colonies from E14 bone marrow sorted with MoAb 11C3. (B) Colonies from E14 bone marrow sorted with MoAb AP2. (a) Erythroid differentiation medium, (b) myeloid differentiation medium. Mean number of colonies developed from 1,000 cells, in duplicate cultures. Morphology of colonies after MGG staining. (C) Macrophages (M). These colonies were large and dispersed. Bar = 31 μm. (D) Granulocytes (G). Bar = 62 μm. (E) Macrophages/granulocytes (M/G). A tight granulocytic center is surrounded with dispersed macrophages. Bar = 62 μm. (F) Erythrocytes (Ec). Hemoglobinized cells developed in erythroid differentiation medium. Bar = 31 μm.

Fig. 2.

Distribution of myeloid and erythroid progenitors in GPIIb-IIIa+–sorted embryonic bone marrow cells. Embryonic bone marrow cells were sorted with the anti-chicken GPIIb-IIIa MoAb 11C3 or with the anti-human chicken cross-reacting GPIIb-IIIa MoAb AP2. Positively (+) or negatively (−) sorted cells were then cultured in erythroid or myeloid differentiation conditions in semisolid medium. Colonies were MGG stained and scored. (A) Colonies from E14 bone marrow sorted with MoAb 11C3. (B) Colonies from E14 bone marrow sorted with MoAb AP2. (a) Erythroid differentiation medium, (b) myeloid differentiation medium. Mean number of colonies developed from 1,000 cells, in duplicate cultures. Morphology of colonies after MGG staining. (C) Macrophages (M). These colonies were large and dispersed. Bar = 31 μm. (D) Granulocytes (G). Bar = 62 μm. (E) Macrophages/granulocytes (M/G). A tight granulocytic center is surrounded with dispersed macrophages. Bar = 62 μm. (F) Erythrocytes (Ec). Hemoglobinized cells developed in erythroid differentiation medium. Bar = 31 μm.

Close modal

We next determined whether GPIIb-IIIa expression was restricted to hematopoietic progenitor cells of the embryonic bone marrow. Subsequently, we detected GPIIb-IIIa high (hi) and low (lo) cells in adult bone marrow (Fig 3A). Interestingly however, while the GPIIb-IIIahi cells had the morphology of mature thrombocytes (Fig 3B) and gave rise to no colonies, the GPIIb-IIIalo cells were enriched for thromboblasts (Fig 3C) and also contained myeloid and erythroid colony-forming progenitors (Fig 3D). Thus the GPIIb-IIIa+ populations, in both embryonic and adult bone marrow, contain other hematopoietic progenitors in addition to thrombocytic progenitors.

Fig. 3.

FACS analysis of adult bone marrow cells stained for GPIIb-IIIa expression. Four-week-old bone marrow cells were immunostained with anti–GPIIb-IIIa MoAb. (A) Two populations with high and low fluorescence intensity were sorted. The percentages of cells in the GPIIb-IIIahi (high) and GPIIb-IIIalo (low) groups are indicated in the windows. The cell morphology of each fraction is illustrated in B and C, respectively. (B) Morphology of GPIIb-IIIa highly fluorescent cells (thrombocytes). (C) Thromboblasts in the GPIIb-IIIa weakly fluorescent cell population. (D) GPIIb-IIIalo (+low) and GPIIb-IIIahi(+high) cells, as well as the negative (−) and unsorted populations were cultured in erythroid differentiation medium. Each column represents the number of colonies arising from 1,000 cells in duplicate cultures.

Fig. 3.

FACS analysis of adult bone marrow cells stained for GPIIb-IIIa expression. Four-week-old bone marrow cells were immunostained with anti–GPIIb-IIIa MoAb. (A) Two populations with high and low fluorescence intensity were sorted. The percentages of cells in the GPIIb-IIIahi (high) and GPIIb-IIIalo (low) groups are indicated in the windows. The cell morphology of each fraction is illustrated in B and C, respectively. (B) Morphology of GPIIb-IIIa highly fluorescent cells (thrombocytes). (C) Thromboblasts in the GPIIb-IIIa weakly fluorescent cell population. (D) GPIIb-IIIalo (+low) and GPIIb-IIIahi(+high) cells, as well as the negative (−) and unsorted populations were cultured in erythroid differentiation medium. Each column represents the number of colonies arising from 1,000 cells in duplicate cultures.

Close modal

It has been previously shown that bone marrow hematopoietic progenitors can be enriched on the basis of the expression of the receptor tyrosine kinase, c-kit. Therefore, we performed double staining on E14 bone marrow cells with anti–GPIIb-IIIa and anti–c-kit MoAbs (Fig 4). Within the c-kit+ population, GPIIb-IIIa was expressed on a population of cells containing myeloid, erythroid, and thrombocytic progenitors. The c-kit+GPIIb-IIIa population had small progenitor activity, while the c-kit GPIIb-IIIa+cells contained mainly thrombocytic and erythroid progenitors, which were probably already lineage-committed (Table1). We concluded from these experiments that anti–GPIIb-IIIa antibodies can select multilineage progenitors within the c-kit+ bone marrow cell population.

Fig. 4.

c-kit and GPIIb-IIIa expression on E14 bone marrow cells. E14 bone marrow cells staining with anti c-kitand antiGPIIb-IIIa MoAbs. Three populations, GPIIb-IIIac-kit+ (1), GPIIb-IIIa+c-kit+ (2), and GPIIb-IIIa+c-kit (3), were sorted by FACS, for functional analysis (see Table 1).

Fig. 4.

c-kit and GPIIb-IIIa expression on E14 bone marrow cells. E14 bone marrow cells staining with anti c-kitand antiGPIIb-IIIa MoAbs. Three populations, GPIIb-IIIac-kit+ (1), GPIIb-IIIa+c-kit+ (2), and GPIIb-IIIa+c-kit (3), were sorted by FACS, for functional analysis (see Table 1).

Close modal

Since our 11C3 antibody detected GPIIb-IIIa integrin, we wanted to exclude the possibility of a crossreaction of our antibody with αvβ3. Dual-labeling experiments on E14 bone marrow cells with anti–GPIIb-IIIa and anti–αvβ3-specific antibody LM 609 showed that 12% of the cells expressed αvβ3, 7% expresssed GPIIb-IIIa, and less than 3% of the cells were double-labeled (data not shown). This finding suggests that 11C3 MoAb does not crossreact with αvβ3 integrin.

GPIIb-IIIa Expression by T-Cell Progenitors

Since the GPIIb-IIIa integrin was expressed on hematopoietic progenitors able to give rise to myeloid and erythroid lineages, we wondered if GPIIb-IIIa+ cells could also differentiate into lymphocytes. We previously showed that pro-T cells were contained within the c-kit+ embryonic bone marrow cell population.22 Thus, we tested whether these c-kit+ cells also expressed the GPIIb-IIIa integrin. E14 bone marrow cells from H.B19 ov+ animals were sorted using anti–GPIIb-IIIa and anti–c-kit antibodies, and injected into thymic lobes of 8-day-old H.B19 ovcongenic animals.34 The chimerism of the recipient thymuses was measured after 14 days by flow cytometry using the anti-ov MoAb 11A9. Injection of 1,000 GPIIb-IIIa+c-kit+ double-positive cells resulted in a 20.9% chimerism, while the same number of GPIIb-IIIac-kit+ cells led to a 3.7% chimerism (Table2). This was still inferior to the 5.8% chimerism obtained by injecting only 100 double-positive cells. Therefore, these experiments demonstrated that a majority of T-cell progenitors were present in the GPIIb-IIIa+c-kit+ population. As in age-matched control thymuses, most of the ov+ T cells recovered from the recipient thymus were immature CD4+ CD8+double-positive cells (data not shown).

In adult bone marrow, the GPIIb-IIIahi thrombocytes seen in Fig 3B were found to be c-kit, whereas the GPIIb-IIIalo cells were c-kit+ (data not shown). This latter population was subsequently tested for its T-cell potential. Similar to data obtained with embryonic bone marrow, adult bone marrow were also found to harbor T-cell progenitors in the GPIIb-IIIa+ c-kit+ population (Table2).

GPIIb-IIIa Expression by Intraembryonic Hematopoietic Progenitors

In the avian embryo, intraembryonic hematopoietic cells emerge at E3.5 to 4 in the wall of the aorta.12 The GPIIb-IIIa integrin is typically expressed, albeit at low levels, by intraaortic clusters on the luminal side of the ventral aortic wall, as well as on a few cells scattered in the dorsal mesenchyme beneath the aorta (Fig 5A and B [see page 2901]). It should be noted that the anti–GPIIb-IIIa antibody 11C3 does not stain vascular aortic endothelial cells. In addition to the GPIIb-IIIalo intraembryonic cells, some GPIIb-IIIahi cells are found in the blood following the establishment of the circulation. Double labeling of these cells with the pan-leukocyte marker anti-CD45 (HISC7) showed that the GPIIb-IIIalo cells were also CD45+ (Fig 5C and D).

Fig. 5.

GPIIb-IIIa expression in intraembryonic hematopoietic sites. (A) Transverse section of an E3.5 to 4 embryo: immunoperoxidase staining with the anti–GPIIb-IIIa MoAb. In the embryo proper, GPIIb-IIIa immunoreactivity is concentrated on cells located ventral to the aorta. (B) Higher magnification from the field boxed in A. Intraaortic clusters are GPIIb-IIIa+ (arrow) and a few positive cells are scattered in the mesenchyme beneath the aortic endothelium (arrowhead). (C) The same section, double stained with anti-CD45MoAb, HISC7, and shown by indirect immunofluorescence. Numerous isolated CD45+ cells were distributed throughout the embryo, in the vessels and in the mesenchyme. Bar = 119 μm. (D) Higher magnification from the field boxed in C, showing the same aortic area as in B. The staining patterns of the two antibodies are similar at the level of the intraaortic clusters, showing coexpression of GPIIb-IIIa and CD45 antigens at this site. Bar = 59 μm. (E) Transverse section of an E6 embryo. GPIIb-IIIa immunoperoxidase staining. Bar = 297 μm. (F) Higher magnification from the field boxed in E, showing a paraortic foci containing GPIIb-IIIa+ cells. Bar = 119 μm. Ao, aorta; PCV, posterior cardinal vein; PA, pulmonary artery; NC, notochord; NT, neural tube; O, esophagus.

Fig. 5.

GPIIb-IIIa expression in intraembryonic hematopoietic sites. (A) Transverse section of an E3.5 to 4 embryo: immunoperoxidase staining with the anti–GPIIb-IIIa MoAb. In the embryo proper, GPIIb-IIIa immunoreactivity is concentrated on cells located ventral to the aorta. (B) Higher magnification from the field boxed in A. Intraaortic clusters are GPIIb-IIIa+ (arrow) and a few positive cells are scattered in the mesenchyme beneath the aortic endothelium (arrowhead). (C) The same section, double stained with anti-CD45MoAb, HISC7, and shown by indirect immunofluorescence. Numerous isolated CD45+ cells were distributed throughout the embryo, in the vessels and in the mesenchyme. Bar = 119 μm. (D) Higher magnification from the field boxed in C, showing the same aortic area as in B. The staining patterns of the two antibodies are similar at the level of the intraaortic clusters, showing coexpression of GPIIb-IIIa and CD45 antigens at this site. Bar = 59 μm. (E) Transverse section of an E6 embryo. GPIIb-IIIa immunoperoxidase staining. Bar = 297 μm. (F) Higher magnification from the field boxed in E, showing a paraortic foci containing GPIIb-IIIa+ cells. Bar = 119 μm. Ao, aorta; PCV, posterior cardinal vein; PA, pulmonary artery; NC, notochord; NT, neural tube; O, esophagus.

Close modal

Later on in development, GPIIb-IIIalo cells are also detected in E6 paraaortic foci (Fig 5E and F), which are diffuse intramesodermal hematopoietic cells, previously designated as paraaortic foci by Dieterlen-Lièvre and Martin.12 

Hematopoietic Progenitor Activity in the E3.5 to 4 Aortic Area

Since myeloid and erythroid progenitors have been described to develop from the chick embryonic aortic region,18,27 35 we investigated whether the GPIIb-IIIa+ E3.5 to 4 intraaortic cell population also contained multilineage hematopoietic progenitors. The precision of staging of the embryo for these experiments was improved using the HH criteria. Progenitor cells from HH 21 to 22 chick embryos were routinely used for these experiments, since twofold to threefold more colonies were obtainable at this stage than with those prepared from HH 19 to 20 staged embryos.

FACS analysis showed that 7% ± 2% (mean ± SD from nine experiments) of the E3.5 to 4 paraaortic cells were GPIIb-IIIa+, and most of the colonies obtained under myeloid and erythroid conditions grew from this GPIIb-IIIa+cell population (Table 3).

To further characterize the intraaortic progenitors, cells were double stained with anti–GPIIb-IIIa and anti-CD45 MoAbs and sorted (Fig6). GPIIb-IIIa+CD45 cells were present at a frequency of 2.5%, GPIIb-IIIa+ CD45+ cells at 4%, and GPIIb-IIIa CD45+ cells at 0.8% (Fig 6). No colonies developed from the GPIIb-IIIa+CD45 or GPIIb-IIIaCD45 sorted cells when cultured under myeloid or erythroid conditions. In contrast, when GPIIb-IIIa+CD45+ cells were cultured under the same conditions, all types of progenitors (myeloid, thrombocytic, and erythroid) developed (Table 4). Furthermore, in comparison to progenitor development from the unfractionated population of the dissected aortic region, cells selected by GPIIb-IIIa expression led to a 20-fold enrichment in hematopoietic progenitors.

Fig. 6.

Flow cytometric analysis of E3.5 to 4 intraaortic cells double-stained with anti-CD45 (HISC7) and anti–GPIIb-IIIa (11C3) MoAbs. Populations 1, 2, and 3 defined as GPIIb-IIIa+CD45, GPIIb-IIIa+ CD45+, and GPIIb-IIIa CD45 cells, respectively, were sorted for functional analysis (see Table 4).

Fig. 6.

Flow cytometric analysis of E3.5 to 4 intraaortic cells double-stained with anti-CD45 (HISC7) and anti–GPIIb-IIIa (11C3) MoAbs. Populations 1, 2, and 3 defined as GPIIb-IIIa+CD45, GPIIb-IIIa+ CD45+, and GPIIb-IIIa CD45 cells, respectively, were sorted for functional analysis (see Table 4).

Close modal

The platelet-specific integrin GPIIb-IIIa (αIIbβ3 or CD41/CD61) is expressed by all cells of the thrombocytic lineage,36 and mature thrombocytes express it at a high level. However, the bone marrow of embryonic and adult animals also contained a cell population expressing this integrin at a low level. This population contains c-kit+ hematopoietic progenitors, which have the potential to differentiate into myeloid, erythroid, and lymphoid lineages.

During early embryogenesis, intraembryonic GPIIb-IIIalocells are found as clusters on the wall of the aorta, and later in paraaortic foci. Cells from this region, positive for both GPIIb-IIIa and the leukocyte marker CD45, are also able to differentiate into myeloid and erythroid lineages.

The GPIIb-IIIa integrin has long been considered a lineage marker for MK and platelets. Now we clearly demonstrate that myeloid, erythroid and lymphoid progenitors also express this adhesion molecule, although expression is lost upon cell differentiation. Thus, the expression of the GPIIb-IIIa integrin correlates with the hematopoietic differentiation status along the pathway between stem-cell and lineage-committed cells. Previously, the expression of GPIIb-IIIa by hematopoietic progenitors has been indirectly demonstrated, using an elegant gene knock-out system. In mice, expressing a GPIIb-IIIa transgene linked to a conditional toxigene, GPIIb-IIIa expression was eradicated at 5 weeks of age. The bone marrow cells of these mice then showed severe reduction in the potential to generate mixed colonies in CFU assays. Furthermore, these mice suffered subsequently from thrombocytopenia.9,10 Together with our findings, this demonstrates that hematopoietic progenitors of most hematopoietic lineages need to express the GPIIb-IIIa integrin, albeit at a low level, before undergoing differentiation. Furthermore, the level of GPIIb-IIIa expression can serve as an indicator of whether the cell is a progenitor or a differentiated thrombocyte. The correlation between expression levels of cell-surface molecules and the differentiation stage of progenitor cells is not unusual, and has been formerly used to separate hematopoietic progenitors from committed cells. For example, mature T cells express Thy-1 at high density, whereas HSCs have low Thy-1 expression.37 Mastocytes are c-kithi cells, while hematopoietic progenitors express c-kit at lower levels.38 Thus, GPIIb-IIIa can be considered another example of a marker for hematopoietic progenitors, that exhibits a regulated expression linked to differentiation. The reason for such fine tuning of expression is presently unknown, but presents an important avenue for future studies.

A subpopulation of the GPIIb-IIIa hematopoietic progenitors of adult and embryonic bone marrow cells also express the receptor tyrosine kinase c-kit. It has been previously shown that c-kitis coexpressed with several other surface molecules on HSC in the bone marrow and embryonic blood of mice.1 2 Interestingly, we found that GPIIb-IIIa+ c-kit+ bone marrow cells can develop into T lymphocytes, myeloid cells, thrombocytes, and erythrocytes. In contrast, c-kitsingle-positive cells, although still able to differentiate into myeloid, thrombocytic, and erythroid cells, did not develop into T cells in the thymic environment. GPIIb-IIIa+c-kit+ bone marrow cells have thus definitive multipotential differentiation capacities.

Blastoderm-derived target cells for the avian myb-ets retrovirus E26 express GPIIb-IIIa along with thrombomucin, a molecule belonging to the same family as CD34. c-kit+thrombomucin+ bone marrow cells, can only differentiate into erythroid lineage cells.39 40 Although, the lymphoid potential of these cells has not been evaluated, we would assume that GPIIb-IIIa+ c-kit+ bone marrow cells are more primitive than these cells.

The c-kit+ HSC are found in the intraembryonic mesodermal region of mouse embryos, including the region of the paraaortic splanchnopleura41 and AGM.3 Using immunohistochemistry, we found that c-kit is not expressed by hematopoietic progenitors from the paraaortic region in chick embryos at E3.5 to 4 or E5 to 8, although it is present in E14 embryonic bone marrow. These data suggest that the first differentiation steps of early embryonic hematopoietic progenitors do not depend on tyrosine kinase activation through the c-kit receptor. This is in agreement with Ogawa et al,42 who showed that c-kitis not functionally required for the establishment of the hematopoietic system. They suggested that the first hematopoietic wave in the embryo was c-kit-independent, whereas the second was c-kit-dependent.

Expression of GPIIb-IIIa integrin by hematopoietic progenitors may be functionally significant: progenitor cells in the bone marrow or on aortic endothelium may use GPIIb-IIIa integrin to adhere to extracellular matrix molecules such as fibronectin or vitronectin.43 In addition, since occupancy of the GPIIb-IIIa integrin leads to outside-in signals mediated by phosphatidyl inositol 3-kinase, this activation could thus allow differentiation of progenitors by costimulation with other molecules.4 Such events of facilitated activation have been described with various integrins expressed by immature or mature leukocytes.44 For instance, pro B lymphocytes use α4β1 integrin for interactions with the VCAM-1 ligand, expressed by bone marrow stromal cells. Blocking this interaction can partially block B-cell differentiation in vitro, although this effect is less pronounced in vivo.45 In platelets, GPIIb-IIIa occupancy induces blood coagulation, and mutations in GPIIb-IIIa lead to severe diseases such as Glanzmann’s thrombasthenia. The role for GPIIb-IIIa–mediated adhesion in the differentiation process of hematopoietic progenitors, is a subject to be addressed in future studies.

In conclusion, we show that the presence of GPIIb-IIIa on early progenitors along with CD45 could be a useful tool to trace hematopoiesis during embryogenesis. In bone marrow, it should be used in conjunction with c-kit to distinguish cells already irreversibly engaged in the megakaryocytic differentiation pathway. The presence of GPIIb-IIIa on early precursors and its coexpression with other markers might also be useful to elucidate precisely the status of cells in the hematopoietic maturation pathway towards terminally differentiated cells. This may be of interest in selecting cells at a particular maturation step for gene therapy or autologous bone marrow transplantation.

We thank Drs F. Dieterlen-Lièvre for critical reading of the manuscript; A. Lehmann and D. Wohlwend for technical assistance; Drs D. Pidard (Institut Pasteur, France) and S. Jeurissen (Lelystad, the Nederlands) for providing us with the MoAbs AP2 and HISC7, respectively; F. Viala for the illustrations; and C. Guilloteau for help with the preparation of the manuscript.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact.

1
Aguila
HL
Akashi
K
Domen
J
Gandy
KL
Lagasse
E
Mebius
RE
Morrison
SJ
Shizuru
J
Strober
S
Uchida
N
Wright
DE
Weissman
IL
From stem cells to lymphocytes: Biology and transplantation.
Immunol Rev
157
1997
13
2
Rodewald
HR
Pathways from hematopoietic stem cells to thymocytes.
Curr Opin Immunol
7
1995
176
3
Sanchez
M-J
Holmes
A
Miles
C
Dzierzak
E
Characterization of the first definitive hematopoietic stem cells in the AGM and liver of the mouse embryo.
Immunity
5
1996
513
4
Naik
UP
Parise
LV
Structure and function of platelet αIIbβ3.
Curr Opin Hematol
4
1997
317
5
Rosenberg
N
Yatuv
R
Orion
Y
Zivelin
A
Dardik
R
Peretz
H
Seligsohn
U
Glanzmann thrombasthenia caused by an 11.2-kb deletion in the glycoprotein IIIa (β3) is a second mutation in Iraqi Jews that stemmed from a distinct founder.
Blood
89
1997
3654
6
Debili
N
Issaad
C
Massé
J-M
Guichard
J
Katz
A
Breton-Gorius
J
Vainchenker
W
Expression of CD34 and platelet glycoproteins during human megakaryocytic differentiation.
Blood
80
1992
3022
7
Berridge
MV
Ralph
SJ
Tan
AS
Cell-lineage antigens of the stem cell-megakaryocyte-platelet lineage are associated with the platelet IIb-IIIa glycoprotein complex.
Blood
66
1985
76
8
Fraser
JK
Leahy
MF
Berridge
MV
Expression of antigens of the platelet glycoprotein IIb/IIIa complex on human hematopoietic stem cells.
Blood
68
1986
762
9
Tropel
P
Roullot
V
Vernet
M
Poujol
C
Pointu
H
Nurden
P
Marguerie
G
Tronik-Le Roux
D
A 2.7-kb portion of the 5′ flanking region of the murine glycoprotein αIIb gene is transcriptionally active in primitive hematopoietic progenitor cells.
Blood
90
1997
2995
10
Tronik-Le Roux
D
Roullot
V
Schweitzer
A
Berthier
R
Marguerie
G
Suppression of erythro-megakaryocytopoiesis and the induction of reversible thrombocytopenia in mice transgenic for the thymidine kinase gene targeted by the platelet glycoprotein αIIb promoter.
J Exp Med
181
1995
2141
11
Murray
LJ
Mandich
D
Bruno
E
DiGiusto
RK
Fu
W-C
Sutherland
DR
Hoffman
R
Tsukamoto
A
Fetal bone marrow CD34+ CD41+ cells are enriched for multipotent hematopoietic progenitors, but not for pluripotent stem cells.
Exp Hematol
24
1996
236
12
Dieterlen-Lièvre
F
Martin
C
Diffuse intraembryonic hemopoiesis in normal and chimeric avian development.
Dev Biol
88
1981
180
13
Cumano
A
Dieterlen-Lièvre
F
Godin
I
Lymphoid potential, probed before circulation in mouse, is restricted to caudal intraembryonic splanchnopleura.
Cell
86
1996
907
14
Medvinsky
A
Dzierzak
E
Definitive hematopoiesis is autonomously initiated by the AGM region.
Cell
86
1996
897
15
Tavian
M
Coulombel
L
Luton
D
San Clémente
H
Dieterlen-Lièvre
F
Péault
B
Aorta-associated CD34+ hematopoietic cells in the early human embryo.
Blood
87
1996
67
16
Corbel
C
Martin
C
Ohki
H
Coltey
M
Hlozanek
I
Le Douarin
N
Evidence for peripheral mechanisms inducing tissue tolerance during ontogeny.
Int Immunol
2
1990
33
17
Hamburger
V
Hamilton
HL
A series of normal stages in the development of the chick embryo.
J Morphol
88
1951
49
18
Cormier
F
de Paz
P
Dieterlen-Lièvre
F
In vitro detection of cells with monocytic potentiality in the wall of the chick embryo aorta.
Dev Biol
118
1986
167
19
Lacoste-Eleaume
A-S
Bleux
C
Quéré
P
Coudert
F
Corbel
C
Kanellopoulos-Langevin
C
Biochemical and functional characterization of an avian homolog of the integrin GPIIb-IIIa present on chicken thrombocytes.
Exp Cell Res
213
1994
198
20
Jeurissen
SHM
Janse
EM
Ekino
S
Nieuwenhuis
P
Koch
G
de Boer
GF
Monoclonal antibodies as probes for defining cellular subsets in the bone marrow, thymus, bursa of Fabricius, and spleen of the chicken.
Vet Immunol Immunopathol
19
1988
225
21
Kornfeld
S
Beug
H
Döderlein
G
Graf
T
Detection of avian hematopoietic cell surface antigens with monoclonal antibodies to myeloid cells: Their distribution on normal and leukemic cells of various lineages.
Exp Cell Res
143
1983
383
22
Vainio
O
Dunon
D
Aı̈ssi
F
Dangy
JP
McNagny
K
Imhof
B
HEMCAM, an adhesion molecule expressed by c-kit+ hemopoietic progenitors.
J Cell Biol
135
1996
1655
23
Pidard
D
Montgomery
RR
Bennett
JS
Kunicki
TJ
Interaction of AP-2, a monoclonal antibody specific for the human platelet glycoprotein IIb-IIIa complex, with intact platelets.
J Biol Chem
258
1983
12582
24
Kunicki
TJ
Newman
PJ
Synthesis of analogs of human platelet membrane glycoprotein IIb-IIIa complex by chicken peripheral blood thrombocytes.
Proc Natl Acad Sci USA
82
1985
7319
25
Luhtala
M
Salomonsen
J
Hirota
Y
Onodera
T
Toivanen
P
Vainio
O
Analysis of chicken CD4 by monoclonal antibodies indicates evolutionary conservation between avian and mammalian species.
Hybridoma
12
1993
633
26
Luhtala
M
Koskinen
R
Toivanen
P
Vainio
O
Characterization of chicken CD8-specific monoclonal antibodies recognizing novel epitopes.
Scand J Immunol
42
1995
171
27
Cormier
F
Dieterlen-Lièvre
F
The wall of the chick embryo aorta harbours M-CFC, G-CFC, GM-CFC and BFU-E.
Development
102
1988
279
28
Corbel
C
Cormier
F
Pourquié
O
Bluestein
HG
BEN, a novel surface molecule of the immunoglobulin superfamily on avian hemopoietic progenitor cells shared with neural cells.
Exp Cell Res
203
1992
91
29
Lucas
AM
Jamroz
BS
Atlas of Avian Hematology.
1961
181
US Deaprtment of Agriculture
Washington, DC
30
Corbel
C
Pourquié
O
Cormier
F
Vaigot
P
Le Douarin
NM
BEN/SC1/DM-GRASP, a homophilic adhesion molecule, is required for in vitro myeloid colony formation by avian hemopoietic progenitors.
Proc Natl Acad Sci USA
93
1996
2844
31
Hayman
M
Meyer
S
Martin
F
Steinlein
P
Beug
H
Self-renewal and differentiation of normal avian erythroid progenitor cells: Regulatory roles of the TGFα/c-ErbB and SCF/c-kit receptors.
Cell
74
1993
157
32
Pain
B
Woods
CM
Saez
J
Flickinger
T
Raines
M
Peyrol
S
Moscovici
C
Moscovici
MG
Kung
HJ
Jurdic
P
Lazarides
E
Samarut
J
EGF-R as a hemopoietic growth factor receptor: The c-erbB product is present in chicken erythrocytic progenitors and controls their self-renewal.
Cell
65
1991
37
33
Palis
J
McGrath
K
Kingsley
P
Initiation of hematopoiesis and vasculogenesis in murine yolk sac explants.
Blood
86
1995
156
34
Corbel
C
Imhof
BA
Vainio
O
Development of somatic and lymphoid chimeras in avian species
Immunology Methods Manual: The Comprehensive Sourcebook of Techniques.
Lefkovits
I
1996
2171
Academic
San Diego, CA
35
Cormier
F
Avian pluripotent haemopoietic progenitor cells: Detection and enrichment from the para-aortic region of the early embryo.
J Cell Sci
105
1993
661
36
Block
KL
Poncz
M
Platelet glycoprotein IIb gene expression as a model of megakaryocyte-specific expression.
Stem Cells
13
1995
135
37
Ikuta
K
Uchida
N
Friedman
J
Weissman
IL
Lymphocyte development from stem cells.
Ann Rev Immunol
10
1992
759
38
Galli
SI
Hammel
I
Mast cell and basophil development.
Curr Opin Hematol
1
1994
33
39
Frampton
J
McNagny
K
Sieweke
M
Philip
A
Smith
G
Graf
T
v-Myb DNA binding is required to block thrombocytic differentiation of Myb-Ets-transformed multipotent haematopoietic progenitors.
EMBO J
14
1995
2866
40
McNagny
KM
Petterson
I
Rossi
F
Flamme
I
Shevchenko
A
Mann
M
Graf
T
Thrombomucin, a novel cell surface protein that defines thrombocytes and multipotent hematopoietic progenitors.
J Cell Biol
138
1997
1395
41
Marcos
MAR
Morales-Alcelay
S
Godin
IE
Dieterlen-Lièvre
F
Copin
SG
Gaspar
M-L
Antigenic phenotype and gene expression pattern of lymphohemopoietic progenitors during early mouse ontogeny.
J Immunol
158
1997
2627
42
Ogawa
M
Nishikawa
S
Yoshinaga
K
Hayashi
SI
Kunisada
T
Nakao
J
Kina
T
Sudo
T
Kodama
H
Nishikawa
SI
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
1993
1089
43
Pelletier
AJ
Kunicki
T
Ruggeri
ZM
Quaranta
V
The activation state of the integrin αIIbβ3 affects outside-in signals leading to cell spreading and focal adhesion kinase phosphorylation.
J Biol Chem
270
1995
18133
44
Miyake
K
Weissman
IL
Greengerger
JS
Kincade
PW
Evidence for a role of the integrin VLA-4 in lympho-hemopoiesis.
J Exp Med
173
1990
599
45
Friedrich
C
Cybulsky
MI
Gutierrez-Ramos
J-C
Vascular cell adhesion molecule-1 expression by hematopoiesis-supporting stromal cells is not essential for lymphoid or myeloid differentiation in vivo or in vitro.
Eur J Immunol
26
1996
2773

Author notes

Address reprint requests to Catherine Corbel, PhD, Institut d’Embryologie Cellulaire et Moléculaire du CNRS et du Collège de France, 49 bis, avenue de la Belle Gabrielle, 94736 Nogent/Marne cedex, France; e-mail: ccorbel@infobiogen.fr.

Sign in via your Institution