Characterization of prethymic progenitors within the chicken embryo
Miia Lampisuo,
Jussi Liippo,
Olli Vainio,
Kelly M. McNagny1,3,
Jarmo Kulmala2 and
Olli Lassila
Turku Immunology Centre, Department of Medical Microbiology, University of Turku, Kiinamyllynkatu 13, 20520 Turku, Finland
1 European Molecular Biology Laboratory, 69117 Heidelberg, Germany
2 Department of Oncology, Turku University Central Hospital, 20520 Turku, Finland
Correspondence to:
J. Liippo
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Abstract
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The thymic primordium in both birds and mammals is first colonized by cells emerging from the intra-embryonic mesenchyme but the nature of these precursors is poorly understood. We demonstrate here an early embryonic day 7 prethymic population with T lymphoid potential. Our work is a phenotypic analysis of, to date, the earliest embryonic prethymic progenitors arising in the avian para-aortic area during ontogeny. The phenotype of these cells, expressing the cell surface molecules
2ß1 integrin, c-kit, thrombomucin/MEP21, HEMCAM and chL12, reflects functional properties required for cell adhesion, migration and growth factor responsiveness. Importantly, the presence of these antigens was found to correlate with the recolonization of the recipient thymus following intrathymic cell transfers. These intra-embryonic cells were also found to express the Ikaros transcription factor, the molecular function of which is considered to be prerequisite for embryonic lymphoid development.
Keywords: cell surface molecules, hematopoiesis, T lymphocytes, thymus, transcription factors
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Introduction
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The intra-embryonic source for lymphoid cells has originally been described in the avian embryos (13). The splanchnopleural mesoderm and its derivatives, para- and intra-aortic hematopoietic clusters, have been shown to contain precursors for lymphopoiesis. Recently, analogous intraembryonic hematopoietic areas, the para-aortic splanchnopleura and aortagonadmesonephros (AGM) region, have been demonstrated in mammalian embryos (47). The characteristics of the early embryonic lymphocyte progenitors that first colonize the primary lymphoid organs are still largely unknown. It has been demonstrated that Ig gene recombination precedes the colonization of the bursa of Fabricius, suggesting prebursal B cell commitment of intra-embryonic progenitors (810). In contrast, the question of whether the thymic rudiment is colonized by committed progenitors or by multipotent precursors is still unresolved. Chicken embryos provide an easily accessible model to study the embryonic lymphoid progenitors arising in the para-aortic region before the colonization of the thymus and the bursa. The thymic rudiment is seeded by para-aortic progenitors in the first wave of colonization between embryonic day (E) 6.5 and 8. During later embryonic development, the thymus receives T cell progenitors in the second and third wave, and these cells are derived from the bone marrow, blood and spleen. Recently, new cell surface antigens have been identified that are expressed by the hematopoietic precursors of avian embryos. Expression of
2ß1 Integrin (VLA-2) (11,12) has been detected very early during avian embryogenesis, e.g. in E4 intra-aortic clusters of hematopoietic cells (K. M. McNagny et al., unpublished data).
2ß1 integrin is present on multipotent Myb-Ets-transformed hematopoietic progenitors (MEP) which also express a newly characterized antigen, thrombomucin (13). This protein is expressed on hematopoietic cells and has a similar distribution but is distinct from c-kit or CD34, which are generally used as markers for hematopoietic stem cells in mammals. Thrombomucin is distantly related to the CD34 molecule and is similarly expressed on both vascular endothelium and hematopoietic cells. Moreover, thrombomucin and CD34 are expressed on early hematopoietic cells in the intra-aortic clusters of respectively avian and human embryos (13,14). As other mucins, thrombomucin has been suggested to have both adhesive and anti-adhesive functions, but its exact role as an adhesion molecule is not yet resolved. During early ontogeny the avian chL12 antigen is also expressed on the intra-embryonic cells from the third day of development onwards (15,16). In addition, most thymocytes and peripheral T cells express this 3840 kDa molecule (9). Avian embryonic bone marrow cells, which have high thymus recolonizing capacity after intrathymic (i.t.) injections, are positive for c-kit tyrosine kinase receptor and co-express a novel adhesion molecule HEMCAM (17). In mammals c-kit is also present on various subsets including hematopoietic stem cells and T cell progenitors (1821).
Studies on the regulation of lymphoid gene expression have identified a number of transcription factors involved in T and B lymphocyte differentiation (22). Gene inactivation studies in mouse have revealed Ikaros, PU.1 and GATA-3 as transcription factors required for the generation of the earliest embryonic T and B cell progenitors (2325). These regulatory proteins establish the developmental expression of lymphoid genes (26). However, the molecular complexes that govern the first decisive events in lymphoid cell fate determination are currently unresolved. The Ikaros proteins are known to be essential regulators in early lymphocyte development, and embryonic expression of Ikaros transcripts is regarded to be prerequisite for lymphoid commitment as well as T and B lineage determination (23,2730). However, Ikaros has also been shown to be present on bone marrow cells which have capacity to generate various hematopoietic lineages (31). The target genes for Ikaros transcription factors are poorly understood and other factors such as Aiolos and Helios, known to act in concert with Ikaros, are also involved in lineage progression (3133). The recently demonstrated avian Ikaros homolog is evolutionarily highly conserved and is expressed during early embryonic development (34).
Given the high degree of conservation as well as early embryonic expression and, thus, the important role of Ikaros also in early avian lymphopoiesis, we wanted to study its presence in early prethymic progenitors. In the present study, we demonstrate by i.t. cell transfers that these progenitor cells expressing
2ß1 integrin (11), c-kit (17), thrombomucin/MEP21 (13), HEMCAM (17) and chL12 (9) are capable of recolonizing the irradiated thymus. Furthermore, we show that the prethymic intra-embryonic cells with T cell differentiation potential express the Ikaros transcription factor.
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Methods
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Cells
The chL12 surface molecule was originally described as an antigen expressed by thymocytes and peripheral T cells (9). It is also an alloantigen that is present only on cells of a H.B2+ chicken strain. The H.B2 strain of chickens is, in turn, totally negative for this antigen. chL12 can therefore be used to trace T lineage cells in H.B2+/H.B2 adoptive cell transfers. Positive recolonization can be demonstrated as the presence of chL12+ thymocytes and T cells in a H.B2 host. The chL12 antigen is recognized by the mAb 11A9 (9). Between 30 and 70 E47 embryos of the chL12+ strain were dissected free from the yolk sac, and the tissue surrounding the dorsal aorta and heart (which was excluded) was prepared as described (35). In order to increase the number of sorted cells the whole E7 embryonic body, excluding the head, was dissociated in some experiments (i.t. transfer of E7 MEP21 and c-kit-sorted cells). The spleens of E1314 embryos were dissected, and single-cell suspensions from these embryonic tissues were prepared as described (35) and resuspended in PBS with 2% FCS.
Flow cytometry and cell sorting
Cells were stained with purified mAb 11A9 (IgM isotype) (9), MEP17 against
2 chain of ß1 integrins (IgG1) (11), MEP21 against thrombomucin (11,13), kit2c75 against chicken c-kit (IgG2a) (17) and c264 against HEMCAM (IgG2b) (17). Culture supernatant of Sp 2/0 myeloma cells served as a negative control. FITC- or phycoerythrin-conjugated isotype-specific goat anti-mouse Ig reagents (Southern Biotechnology Associates, Birmingham, AL) were used as secondary antibodies. Flow cytometry was carried out using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA) and data were processed by using the CellQuest program (Becton Dickinson). Cell sortings were carried out using a FACStar Plus instrument (Becton Dickinson) and Lysys II software. For sorted E67 intra-embryonic
2ß1+ cells the purity was 7593%, 6394% for chL12+ cells, 8590% for MEP21+ cells and 4084% for c-kit+ cells (the frequency of c-kit expression was 0.53% on cells prepared from E7 intra-embryonic mesenchyme). The purity of surface marker-negative cells was >96% (except for c-kit cells 8097%). The purities of E13 splenocytes were >98% for chL12+ cells and >99% for chL12 cells. The expression data of these antigens has previously been described (16). For RT-PCR reactions E7 cells were sorted into chL12 and c-kit positive and negative fractions. Sorted cells were 85% pure for c-kit+ and 95% for chL12+, and for the corresponding negative cells the purity was >96%. The expression of T lineage markers by donor-derived (chL12+) cells on the recipient thymic lobes was analyzed with two-color immunofluorescence staining. mAb 11A9 was used together with mAb TCR1 (anti-TCR 
, IgG1) (36), TCR2 (anti-TCR Vß1, IgG1) (both purchased from Southern Biotechnology) (36), 2-6 (anti-CD4, IgG1) (15) or 11-39 (anti-CD8
, IgG1) (15) followed by FITC- or phycoerythrin-conjugated isotype-specific secondary antibody.
i.t. transfer
Recipients were 2-week-old chickens of the chL12 strain which were irradiated 1 h before the cell transfer with a radiation dose of 6 Gy from a radiotherapy unit (linear accelerator, Clinac 4/100; Varian, Palo Alto, CA). Donor cell suspensions (chL12+ strain) were injected into individual thymic lobes in a volume of 10 or 20 µl per lobe. As controls, non-sorted embryonic cells were injected. After 1219 days (exact durations are given in Table 1
) the injected thymic lobes of the recipients were individually dissected and the frequency of donor-derived chL12+ thymocytes was analyzed by immunofluorescence staining and flow cytometry.
RT-PCR
E7 intra-embryonic cells were sorted into chL12 and c-kit positive and negative fractions. The Ultraspec-II RNA kit (Biotecx, Houston, TX) was used to isolate the total RNA which was subjected to reverse transcription (by AMV RT-enzyme; reaction volume 20 µl) according to the supplier's instructions, to obtain oligo-p(dT)-primed cDNAs (1st Strand cDNA Synthesis Kit for RT-PCR; Boehringer Mannheim, Indianapolis, IN). Reverse transcriptase was replaced by water in control reverse transcription reactions. Also, the primers were prepared from different exons to obtain products that are of expected molecular size. Following reverse transcription, 2 µl of cDNA was used in a 100 µl PCR amplification reaction containing 0.2 mM of each dNTP, 1 U of DynaZyme DNA polymerase supplied with 10xPCR reaction buffer (Finnzymes, Espoo, Finland) and 15 pmol/µl of each primer set. Ikaros cDNA-specific primers were as described (34). To account for possible variability in the cDNA level following reverse transcription from total RNA, the constitutively expressed ß-actin gene was amplified with specific primers (34) from the same cDNA. PCR amplifications were performed using a DNA thermal cycler (Perkin Elmer, Norwalk, CT) for a total of 30 cycles. Reaction conditions were 94°C for 30 s, 55°C for 30 s and 72°C for 60 s. Finally, RT-PCR products were electrophoretically separated on a 1.5% SeaKem Agarose gel (FMC Bioproducts, Rockland, ME), transferred to a Hybond-N+ nylon membrane (Amersham, Amersham, UK) and hybridized with an internal 32P-labeled oligonucleotide probe (34,37).
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Results
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Prethymic T cell progenitors express
2ß1 integrin, c-kit, thrombomucin/MEP21, HEMCAM and chL12
The T cell differentiation potential and the phenotype of cells from the intra-embryonic mesenchyme before the first thymic colonization were studied. We performed adoptive i.t. transfer of FACS-sorted cells from E67 embryos and used a congenic chL12 marker to recognize donor-derived cells. Cells positive for
2ß1 integrin and c-kit were able to recolonize the recipient thymic lobes, whereas negative cells lacked this potential (Table 1
). The functional T cell differentiation (36) of donor-derived embryonic cells was verified by the expression of CD4, CD8, TCR 
and TCR Vß1 of the chL12+ thymocytes (Fig. 1
). Similarly, the intra-embryonic cell population expressing MEP21/thrombomucin antigen was capable of recolonization, whereas the negative population was not.
Although the frequencies of c-kit-,
2ß1 integrin- and thrombomucin-expressing early embryonic cells are low, two-color immunofluorescence analysis suggested that already at this early prethymic stage most or all c-kit+,
2ß1 integrin+ and thrombomucin+ cells express HEMCAM. In addition, HEMCAM+ E7 cells, in contrast to negative cells, were able to recolonize thymus. Our results confirm and extend the previous observations (17) demonstrating that already the progenitors for the first thymic colonization express both c-kit and HEMCAM.
In a similar way as
2ß1 integrin-, c-kit-, thrombomucin/MEP21- and HEMCAM-expressing cells, chL12+ cells from E67 intra-embryonic mesenchyme also gave rise to T cells, whereas the negative population failed to reconstitute thymic lobes (Table 1
). As embryonic spleen is known to contain T cell progenitors for the second wave of thymic colonization, we next transferred E13 spleen cells sorted according to their expression of the chL12 marker. In a similar way as the intra-embryonic progenitors, chL12+ embryonic splenocytes colonized the thymi after i.t. injection, whereas negative cells never did (data not shown).
To study whether even earlier intra-embryonic cells have potential towards the T lineage we carried out i.t. transfers with E4 progenitors (Table 1
). After transfer of 12x105 intra-embryonic cells the frequencies of donor-specific cells in recolonized thymic lobes were 36%. This finding further confirms the T cell capacity of the very early E4 intra-embryonic cells.
Ikaros transcripts are expressed by prethymic progenitors able to recolonize the thymus
The avian Ikaros gene is expressed early in embryogenesis from the second day of incubation onwards (34). Moreover, all the major alternatively spliced transcripts can be detected from very early on, suggesting Ikaros activity already in the early hematolymphoid development (34). Since the recolonization-associated cell surface molecules are also expressed early in the sites generating cells with T cell progenitor capacity, we studied Ikaros expression in the E7 intra-embryonic cells both bearing these surface antigens and capable of thymus repopulation. Cells were FACS sorted according to the expression of chL12 and c-kit antigens. The fractions containing thymus-recolonizing chL12+ and c-kit+ cells showed enriched Ikaros expression (Fig. 2
). Therefore, demonstration of thymus-recolonization capacity in concomitance with Ikaros suggests that early embryonic Ikaros expression involves also the prethymic hematopoietic progenitors that colonize the thymus.

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Fig. 2. RT-PCR analysis of Ikaros expression in sorted E7 intra-embryonic cells and E14 thymic and bursal cells. Cells were sorted into chL12+ and chL12- and into c-kit+ and c-kit- fractions as described in Methods. Total RNA was isolated and subjected to oligo-dT-primed reverse transcription to produce cDNA. Thymus recolonizing sorted chL12+ as well as c-kit+ E7 cells clearly express Ikaros. Cell numbers in chL12+/- fractions were 1.6 x 105 cells and c-kit+/- populations had 0.2 x 105 cells. Non-sorted fractions had the same numbers of cells. To normalize for the amount and integrity of the cDNA, constitutively expressed ß-actin was amplified from the same pools of cDNA. ns, non-sorted cells; thy, thymus; bu, bursa; c-, control lacking RT-enzyme; c0, no cDNA template.
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Discussion
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The sites of origin for the first embryonic precursors generating definitive lymphopoiesis have been under extensive studies (1,3,6,7,38). The migratory routes, degree of developmental restriction and, most importantly, phenotype of progenitor cells derived from intra-embryonic regions have still remained largely unclarified. Therefore, in the present study we have further characterized the phenotype and functional properties of the avian prethymic para-aortic cells (Fig. 3
).
Lymphoid progenitors require adhesion molecules to home and migrate into the thymus. These include VLA-4 (39), CD34 and CD44 (40). Interestingly, hematopoietic progenitors fail to colonize fetal liver in ß1 integrin-deficient mice, thus suggesting an essential role for these integrins in cell migration (41). To date, in particular the expression of VLA-2 has not been demonstrated on T cell progenitors in any species. Our results, however, indicate that
2ß1 integrin (VLA-2) is present on prethymic progenitors and might be important for thymus homing. Thrombomucin/MEP21 is present on thrombocytes and mono- and multipotent hemopoietic progenitors of yolk sac and bone marrow (13) but like VLA-2, its presence on prethymic progenitors has not been evaluated before. As we obtained thymus-recolonizing cells exclusively from the MEP21+ fraction of sorted intra-embryonic cells, it is now evident that already the prethymic T cell progenitors express thrombomucin. The adhesion molecule HEMCAM is co-expressed with c-kit both on bone marrow (17) and intra-embryonic progenitors. HEMCAM expression may thus further facilitate the migration and homing of early progenitors into the thymus. The function of the avian chL12 antigen is currently unknown. However, since both thymocytes and peripheral T cells express this antigen, it is likely to have a role also in the later development and function of T lymphoid cells.
Similarly to mammalian T cell progenitors which express c-kit (18,20), avian prethymic T cell progenitors are also positive for c-kit. Mouse fetal blood has been demonstrated to contain T lineage-restricted progenitor cells which are low-positive for c-kit (19). Furthermore, it seems that c-kit could be more important for T than B lymphocyte development since it has been shown that murine B cells differentiate normally without the interaction between c-kit and the c-kit ligand (42). Recently, the murine AGM region (K. Ohmura et al., personal communication) and fetal liver have also been shown to harbor c-kit+ T cell progenitors (21). Thus c-kit seems to have a role in the early differentiation of the intra-embryonic T cell progenitors in both avians and mammals.
Definitive conclusions about the efficiency of the recolonization capacity of intra-embryonic progenitors are difficult to draw since usually not all of the recipients or thymic lobes were repopulated. Intra-embryonic cells are not as effective in recolonization as, for example, embryonic bone marrow cells (17), partly because they are relatively vulnerable during ex vivo management and sorting. It is not known either how many of the transferred cells actually manage to give rise to T cell progeny and how many die in situ either immediately or upon negative selection. The estimated absolute numbers of surface marker-positive cells used in i.t. transfers of non-sorted intra-embryonic cells correspond to the number of positive cells in most sorted populations (16). For example, 8100x104 transferred non-sorted E67 cells contain ~1% c-kit+ cells, indicating that 0.081x104 c-kit+ progenitors were injected per lobe. Between 2.5 and 5x104 HEMCAM+ intra-embryonic cells recolonized lobes with relatively low frequency probably due to the fact that most (~80%) E7 cells express this molecule and thus the sorted subset is diluted with cells other than progenitor cells. In general, the lowest cell number needed for detectable recolonization was 0.20.3x104. However, under physiological conditions much less progenitors are required due to the significantly smaller size of the E67 thymic rudiment compared to the 2-week-old recipient thymus. An average of 10x104 intra-embryonic cells were obtained from a single whole embryo body and ~1% of these (the frequency of c-kit+ cells), 0.1x104 cells/embryo, can represent potential prethymic progenitors. The in vivo number of progenitors is presumably higher because some progenitors are lost during the cell preparation.
In addition to the cell surface molecules characteristic for early cells with T cell differentiation potential, we also studied the expression of the Ikaros transcription factor in the intra-embryonic cells. Our data show already at the prethymic stage functional association of the Ikaros expression and the potential to colonize the thymus. Although Ikaros is essential for early lymphopoiesis, its target genes and exact regulatory role in lymphocyte development have not yet been determined (23,27). In contrast to previous findings showing Ikaros as a lymphoid gene activator, a recent report demonstrates that Ikaros associates with transcriptionally inert genes at centromeric heterochromatin (43). Accordingly, early prethymic Ikaros expression in thymus-recolonizing cells may be involved in the first signs of lineage restriction that is later characterized by specific genes and regulatory factors, like GATA-3, Aiolos and Tcf-1 (25,31,44). On one hand our data are in line with studies suggesting early determination of lymphoid progenitors, but our results can also be interpreted by the fact that we deal with more clearly defined avian hematopoietic stem cells. Thus the observed prethymic Ikaros expression would agree with recent findings suggesting that mammalian multipotential hematopoietic stem and progenitor cells start multilineage gene expression and locus activation prior to lineage commitment (34,45). In addition, we could show that E4 intra-embryonic cells were capable of thymus reconstitution following i.t. transfers. Although these cells were not fractionated by sorting for surface antigen expression, they bear a repertoire of surface molecules largely similar to that of E7 precursors. Importantly, it has been demonstrated that these E4 embryonic cells also clearly express Ikaros transcripts (34). Thus, cells competent for thymus reconstitution arise earlier in ontogeny than has previously been demonstrated and well before the thymic primordium receives the first progenitors.
In conclusion, we have defined intra-embryonic prethymic progenitors with T lymphoid potential and identified markers which can be used to purify them. Thrombomucin and
2ß1 integrin have not yet been found on prethymic mouse progenitors from later hematopoietic tissues like fetal liver or bone marrow. Neither have the counterparts of these antigens (nor HEMCAM and chL12) been shown on progenitors of early para-aortic splanchnopleura or the AGM region. Future studies are likely to reveal the role of these markers in mammalian lymphopoiesis also.
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Acknowledgments
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We thank Marjo Hakkarainen, Raija Raulimo and Marianne Laine for their expert technical assistance. Dr Petteri Arstila, now at Institut Pasteur, Paris, is acknowledged for his comments on the manuscript. This work was financially supported by the Academy of Finland, the Turku University Foundation and EU program BIOTECH (BIO4-CT97-2706). K. M. M. was supported by NRSA scholarship F32 HL0736 from the National Heart, Lung and Blood Institute, NIH.
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Abbreviations
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AGM | aortagonadmesonephros |
E | embryonic day |
i.t. | intrathymic |
MEP | Myb-Ets-transformed hematopoietic progenitors |
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Notes
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3 Present address: Biomedical Research Centre, Department of Medical Genetics, University of British Columbia, Vancouver, BC V6T 1Z3, Canada 
The first two authors contributed equally to this work
Transmitting editor: S. Nishikawa
Received 15 June 1998,
accepted 29 September 1998.
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