Hematopoietic Commitment during Embryogenesis

9

Hematopoietic Commitment during Embryogenesis

SCOTT ROBERTSON,a MARION KENNEDY,a AND GORDON KELLER, a,b,c

aNational Jewish Medical and Research Center, 1400 Jackson Street, Denver, Colorado 80206, USAbThe Department of Immunology, University of Colorado Health Sciences Center,Denver, Colorado 80262, USA

ABSTRACT: Hematopoiesis develops initially as discrete blood islands in theextraembryonic yolk sac of the embryo. These blood islands consist of clustersof primitive erythrocytes surrounded by developing angioblasts that ultimatelyform the yolk sac vasculature. The close developmental association of theseearly hematopoietic and endothelial cells has led to the hypothesis that they de-velop from a common precursor, a cell known as the hemangioblast. Using adevelopmental model system based on the in vitro differentiation capacity ofembyronic stem (ES) cells, we have identified a precursor with the capacity togenerate endothelial as well as primitive and definitive hematopoietic progeny.The developmental potential of this precursor population suggests that it rep-resents the in vitro equivalent of the hemangioblast.

INTRODUCTION

The hematopoietic system undergoes dramatic changes throughout ontogenyboth with respect to the site of activity as well as to the lineages produced.1 Most ofour understanding of lineage relationships and regulation of growth and differentia-tion within the hematopoietic system has come from studies on adult bone marrowand fetal liver. While there are some notable differences between fetal and adult he-matopoiesis, in general they share many similarities including the simultaneous de-velopment of multiple lineages that derive from a common precursor known as themultipotential stem cell.2–4 Stem cells of both fetal and adult origin are able to pro-vide long-term hematopoietic repopulation following transplantation into adult re-cipient animals, a characteristic that distinguishes them from all other cells in thehematopoietic system. Prior to the development of the fetal liver, hematopoietic ac-tivity is found in the extraembryonic yolk sac, the first site of hematopoietic com-mitment.1,5 In contrast to the fetal and adult systems, yolk sac hematopoiesis showsunique developmental patterns which suggest the presence of novel precursorpopulations.1,5

cAddress for correspondence: Gordon Keller, Ph.D., National Jewish Medical and ResearchCenter, 1400 Jackson Street, Denver, Colorado 80206-2761. Phone, 303/398-1813; fax, 303/398-1396; e-mail, [email protected]

10 ANNALS NEW YORK ACADEMY OF SCIENCES

YOLK SAC HEMATOPOIESIS

The embryonic hematopoietic system initiates as discrete blood islands in theearly yolk sac at approximately day 7.5 of gestation.1,5 The potential of the firststage of the embryonic program is distinct from that of fetal liver and adult marrowhematopoiesis in that it appears to be restricted to the generation of two lineages:embryonic erythrocytes, which represent the major hematopoietic component of theblood islands, and macrophages that are dispersed throughout the yolk sac.1,5 Theseearly erythroid cells, known as primitive erythrocytes, are large, remain nucleatedand produce the embryonic forms of globlin.5–7 Given this potential, this stage ofyolk sac development is known as primitive hematopoiesis. Cells of the endotheliallineage represent the second component of the blood islands and are first detected asa layer of developing angioblasts which surround the inner clusters of primitiveerythrocytes. The observation that the hematopoietic and endothelial lineages devel-op simultaneously in close proximity in the blood islands provided the basis for thehypothesis that they share a common precursor, a cell called the hemangioblast (re-viewed in Refs. 8 and 9). Although experimental evidence supporting this notion hasaccumulated since the original hypothesis was put forward almost 100 years ago, acell with these characteristics has not yet been isolated from the developing mouseembryo.

While the initial stages of yolk sac hematopoiesis appear to be restricted, precur-sors for other hematopoietic lineages, including definitive erythroid, myeloid, andmast cells can be detected within 12–48 hours following the development of theblood islands.10–12 These cells, which are collectively referred to as definitive he-matopoietic precursors, appear in the yolk sac prior to the establishment of intraem-bryonic hematopoiesis, suggesting that they are produced at this site. However, incontrast to the primitive erythroid and macrophage precursors that mature in the yolksac, these precursors do not undergo significant differentiation in this environment.Although these precursors can be detected in the yolk sac by 8.0 to 9.0 days of ges-tation, transplantable stem cells capable of providing long-term repopulation in adultrecipients are not easily detected until day 10.5 to 11.13 Interestingly, these cells arefound within the embryo proper slightly earlier than in the yolk sac, leading to thesuggestion that they develop at some site within the embryo and then migrate to theyolk sac. Reports of lymphoid precursors within the yolk sac are somewhat variablewith respect to stage of development and have been detected between 8.5 and 9.5days of gestation.14–16 One of the most recent studies demonstrated that lymphoidpotential is present in both the yolk sac and embryo proper as early as day 8.5.17

However, these precursors appear to develop from multipotential rather than fromlymphoid committed cells and therefore the actual stage at which lymphoid-restrict-ed precursors develop may be later than originally reported.

The embryonic developmental sequence in which primitive erythroid cells appearbefore definitive erythroid/myeloid precursors and long-term repopulating stemcells (LTRSC) is reverse to that predicted by most models of fetal or adult hemat-poiesis. These models typically position the LTRSC as the most immature cell with-in the system. There are at least several explanations for these unusual observations.First, it is possible that yolk sac hematopoiesis is established by an embryonicmultipotential precursor that initially produces primitive erythroid progeny, then de-

11ROBERTSON et al.: HEMATOPOIETIC COMMITMENT

finitive hematopoietic cells, and finally LTRSC and lymphoid precursors. Theseconsecutive waves of differentiation including the late development of the repopu-lating stem cell would simply reflect maturation of the system. This putative multi-potential cell could be considered as the primordial hematopoietic precursor, the pre-LTRSC. A second possibility is that the yolk sac hematopoietic program is distinctfrom that of intraembryonic hematopoiesis and restricted to the development ofprimitive erythroid, definitive erythroid and myeloid precursors.

Yolk sac hematopoietic activity declines dramatically between days 10 and 12 ofgestation, concomitant with the initiation of intraembryonic hematopoiesis in the de-veloping fetal liver.1,12 The transition from yolk sac to fetal liver defines the switchfrom primitive to definitive hematopoiesis and the replacement of the primitiveerythroid program by multilineage hematopoiesis including definitive erythropoie-sis, myelopoiesis, and lymphopoiesis.1,12 Definitive erythroid cells generated in thefetal liver differ from primitive erythrocytes in the yolk sac in that they are small andthat they enucleate and produce adult globins.6,7

HEMATOPOIETIC DEVELOPMENT OF ES CELLS IN CULTURE RECAPITULATES YOLK SAC HEMATOPOIESIS

To define the developmental events involved in the establishment of the hemato-poietic and endothelial lineages, it is important to focus on the early yolk sac at astage prior to the appearance of the blood islands. Most attempts to study these earlydifferentiation steps have been severely hampered by the inaccessibility of the em-bryo at this stage of development as well as by the limited numbers of cells available.To overcome these problems a number of groups have utilized the ES in vitro differ-entiation system as a model for early hematopoietic and endothelial development.Under appropriate conditions, ES cells will differentiate and form colonies knownas embryoid bodies (EBs), which contain developing precursor populations frommultiple lineages, including those of the hematopoietic and vascular systems.18 Dur-ing the past 8 years cell culture systems and techniques have advanced significantlysuch that it is now possible to routinely generate both the primitive and definitiveerythroid lineages, most myeloid lineages, and endothelial cells in a predictable pat-tern from developing EBs.18–25

One concern with this model system is whether or not it reflects the developmen-tal program in the normal embryo. While this is difficult to determine in all aspects,several findings do indicate that at least the early events in hematopoietic and endo-thelial commitment in the EBs are similar to those found in utero. First, precursoranalysis of the EBs clearly demonstrated that the primitive erythroid and macro-phage lineages are the first to develop, followed by those of the definitive erythroidand other myeloid lineages, a pattern reminiscent of that of the early yolk sac.21

Moreover, as observed in the yolk sac, the primitive erythroid lineage within the EBappears to be transient, generated between days 4 and 10 of differentiation. Second,kinetic analysis of gene expression within the developing EBs indicates that markersexpressed in mesoderm appear earlier than those expressed in hematopoietic and en-dothelial precursors, which in turn precede those that define specific hematopoietic


lineages.21 These gene expression patterns are consistent with the well-establishedbiological data that demonstrate that the hematopoietic and endothelial lineages de-velop from mesoderm. Together, these findings suggest that, at least for the earlyevents, hematopoietic commitment in the ES/EB system is comparable to that of theearly embryo.

THE PRIMITIVE AND DEFINITIVE HEMATOPOIETIC LINEAGES DEVELOP FROM A COMMON PRECURSOR IN EBS

As indicated earlier, one model for the unusual pattern of lineage development inthe yolk sac could be the development of a multipotential cell with the potential togenerate both primitive and definitive hematopoietic progeny. The initial productionof the primitive erythroid lineage followed by the development of definitive precur-sors would reflect maturation of the system resulting from molecular changes withinthis precursor population and/or changes in the microenvironment. To determinewhether a multipotential cell with both primitive and definitive hematopoietic poten-tial does exist, we analyzed EBs prior to the establishment of the primitive erythroidlineage, specifically before day 4 of differentiation. Using this approach, we identi-fied a transient vascular endothelial growth factor (VEGF)-responsive precursor thatdeveloped in EBs within 2.5 days of differentiation and persisted for approximately24 hours.26 In methylcellulose cultures containing VEGF and conditioned mediumfrom an embryonic endothelial cell line (D4T), these precursors generate coloniesconsisting of undifferentiated blast cells. Replating studies demonstrated that theseblast cell colonies contain both primitive and definitive hematopoietic precursorsand, as such, documented for the first time that these lineages can develop from acommon precursor, the blast colony-forming cell (BL-CFC).26 To further character-ize the developmental status of the blast cell colonies, individual colonies were an-alyzed via RT–PCR for the expression of genes representing mesodermal,endothelial, and early and late hematopoietic precursors. This analysis includedBrachyury,27 flk-1,28,29 SCL,30–32 CD34,33,34 GATA-1,35 and βH1 and β majorglobins. None of the blast colonies analyzed expressed Brachyury, indicating thatthey represent a stage of development more advanced than mesoderm. All of the col-onies analyzed did, however, express flk-1, SCL/TAL-1, CD34 and GATA-1, sug-gesting that they contain hematopoietic precursors representing various stages ofdevelopment, a finding consistent with their replating potential. Many of the blastcolonies also expressed βH1 and β major globin, a finding that further supports thenotion that they have both primitive and definitive hematopoietic potential.

THE VEGF-RESPONSIVE BL-CFC HAS BOTHHEMATOPOIETIC AND ENDOTHELIAL POTENTIAL

The finding that the BL-CFC responded to VEGF and that the blast colonies ex-pressed flk-1 suggested that these colonies may have endothelial in addition to he-matopoietic potential. To determine whether this was true, individual blast colonies


were cultured in liquid in microtiter wells in the presence of cytokines that supportthe development of both the hematopoietic and endothelial lineages. Approximately30–40% of blast colonies from day-3.25 EBs were able to generate both hematopoi-etic and adherent cells under these conditions. The remainder gave rise to only he-matopoietic progeny. Analysis of the adherent population indicated that these cellsexpressed markers characteristic of endothelial cells, including PECAM-1, flk-1,tie-2, and flt-1. In addition, they displayed the capacity to take up acetylated low-density lipoprotein (LDL), also a characteristic of endothelial cells. The observationthat blast colonies contained both hematopoietic and endothelial precursors stronglysuggests that the BL-CFC they derive from has the properties of a hemangioblast.36

As indicated above, not all blast colonies displayed endothelial potential. Kineticanalysis revealed that the proportion with endothelial potential was highest (approx-imately 75%) in blast colonies generated from day-2.5 EBs. The proportion of bi-lineage blast colonies dropped dramatically as the age of the EBs from which theywere generated increased. Fewer than 25% of the blast colonies from day-3.5 EBsshowed both hematopoietic and endothelial potential. These findings suggest thatthe bi-potential BL-CFC represented a transient population that persists in EBs forapproximately 24 hours between day 2.5 and 3.5 of differentiation. Analysis of thehematopoietic potential of the blast colonies with endothelial potential indicated thatmost contain precursors for multiple lineages. However, a small number of these col-onies appeared to be more restricted and generated only primitive erythroid and ad-herent cell progeny. These findings suggest that populations of hemangioblasts withdifferent potentials may exist.

CONCLUSIONS

The identification of the VEGF-responsive BL-CFC provides the first demonstra-tion that the primitive and definitive hematopoietic and endothelial lineages can de-velop from a common precursor. That fact that these blast colonies develop inresponse to VEGF indicates that this precursor expresses Flk-1, and that this inter-action is required for the development of these early populations. This interpretationis consistent with in vivo gene-targeting studies which demonstrated that a function-al Flk-1 receptor is required for the development of these lineages in the embryo.37

Taken together, the characteristics of the BL-CFC are consistent with the interpreta-tion that it represents the in vitro equivalent of the hemangioblast and suggests thata comparable precursor should be present in the developing embryo. Current exper-iments are aimed at identifying the BL-CFC in early embryos. Utilizing the ES/EBsystem we have also been able to identify and characterize a novel colony that spansmesodem to hemangioblast commitment and as such likely develops from a precur-sor earlier than the BL-CFC. The majority of these colonies express Brachyury, flk-1 and SCL, indicating the presence of mesodermal and hemangioblastic cells. In ad-dition a subpopulation of these colonies also express GATA-1, βmajor and βH1,demonstrating further commitment to the hematopoietic lineages. Access to theseunique colonies will allow for a molecular analysis of the specification of mesodermto earliest stages of enothelial and hematopoietic development.


REFERENCES

1. METCALF, D. & M. MOORE. 1971. Haemopoietic Cells. Frontiers in Biology, Vol. 24.A. Neuberger and E.L. Tatum, Eds. North-Holland Publishing Co. London.

2. KELLER, G. 1992. Hematopoietic stem cells. Curr. Opin. Immunol. 4: 133–139.3. UCHIDA, N., W. FLEMING, E. ALPERN & I. WEISSMAN. 1993. Heterogeneity of hemato-

poietic stem cells. Curr. Opin. Immunol. 5: 177–184.4. MORRISON, S.J., N. UCHIDA & I.L. WEISSMAN. 1995. The biology of hematopoietic

stem cells. Annu. Rev. Cell. Dev. Biol. 11: 35–71.5. RUSSEL, E. 1979. Hereditary anemias of the mouse: a review for geneticists. Adv.

Genet. 20: 357–459.6. BARKER, J. 1968. Development of the mouse hematopoietic system. I. Types of hemo-

globin produced in embryonic yolk sac and liver. Dev. Biol. 18: 14-29.7. BROTHERTON, T., D. CHUI, J. GAULDIE & M. PATTERSON. 1979. Hemoglobin ontogeny

during normal mouse fetal development. Proc. Natl. Acad. Sci. USA 76: 2853–2857.8. WAGNER, R.C. 1980. Endothelial cell embryology and growth. Adv. Microcirc. 9: 45–

75.9. RISAU, W. & I. FLAMME. 1995. Vasculogenesis. Annu. Rev. Cell. Dev. Biol. 11: 73–91.

10. WONG, P., S. CHUNG, D. CHUI & C. EAVES. 1986. Properties of the earliest clono-genic hemopoietic precursor to appear in the developing murine yolk sac. Proc.Natl. Acad. Sci. USA 83: 3851–3854.

11. SONODA, T., C. HAYASHI & Y. KITAMURA. 1983. Presence of mast cell precursors inthe yolk sac of mice. Dev. Biol. 97: 89–94.

12. JOHNSON, G. & D. BARKER. 1985. Erythroid progenitor cells and stimulating factorsduring murine embryonic and fetal development. Exp. Hematol. 13: 200–208.

13. MULLER, A.M., A. MEDVINSKY, J. STROUBOULIS, F. GROSVELD & E. DZIERZAK. 1994.Development of hematopoietic stem cell activity in the mouse embryo. Immunity1: 291–301.

14. LIU, C. & R. AUERBACH. 1991. In vitro development of murine T cells from prethy-mic and preliver embryonic yolk sac hematopoietic stem cells. Development 113:1315–1323.

15. CUMANO, A., C. FURLONGER & C. PAIGE. 1993. Differentiation and characterizationof B cell precursor detected in the yolk sac and embryo body of embryos beginningat the 10- to 12-somite stage. Proc. Natl. Acad. Sci. USA 90: 6429–6433.

16. HUANG, H., L. ZETTERGREN & R. AUERBACH. 1994. In vitro differentiation of B cellsand myeloid cells from the early mouse embryo and its extraembryonic yolk sac.Exp. Hematol. 22: 19–25.

17. GODIN, I., F. DIETERLEN-LIEVRE & A. CUMANO. 1995. Emergence of multipotenthemopoietic cells in the yolk sac and para-aortic splanchnopleura in mouseembryos, beginning at 8.5 days postcoitus. Proc. Natl. Acad. Sci. USA 92: 773–777.

18. KELLER, G. 1995. In vitro differentiation of embryonic stem cells. Curr. Opin. CellBiol. 7: 862–869.

19. SCHMITT, R., E. BRUYNS & H. SNODGRASS. 1991. Hematopoietic development ofembryonic stem cells in vitro: cytokine and receptor gene expression. Genes Dev.5: 728–740.

20. BÜRKERT, U., T. VON RÜDEN & E. WAGNER. 1991. Early fetal hematopoietic develop-ment from in vitro differentiated embryonic stem cells. New Biol. 13: 698–708.

21. KELLER, G., M. KENNEDY, T. PAPAYANNOPOULOU & M.V. WILES. 1993. Hematopoie-tic commitment during embryonic stem cell differentiation in culture. Mol. Cell.Biol. 13: 473–486.

22. NAKANO, T., H. KODAMA & T. HONJO. 1994. Generation of lymphohematopoieticcells from embryonic stem cells in culture. Science 265: 1098–1101.


23. RISAU, W., H. SARIOLA, H.G. ZERWES, J. SASSE, P. EKBLOM, R. KEMLER & T. DOET-SCHMAN. 1988. Vasculogenesis and angiogenesis in embryonic-stem-cell-derivedembryoid bodies. Development 102: 471–478.

24. WANG, R., R. CLARK & V.L. BAUTCH. 1992. Embryonic stem cell-derived cysticembryoid bodies form vascular channels: an in vitro model of blood vessel devel-opment. Development 114: 303–316.

25. VITTET, D., M.H. PRANDINI, R. BERTHIER, A. SCHWEITZER, H. MARTIN-SISTERON, G.UZAN & E. DEJANA. 1996. Embryonic stem cells differentiate in vitro to endothe-lial cells through successive maturation steps. Blood 88: 3424–3431.

26. KENNEDY, M., M. FIRPO, K. CHOI, C. WALL, S. ROBERTSON, N. KABRUN & G.KELLER. 1997. A common precursor for primitive erythropoiesis and definitivehaematopoiesis. Nature 386: 488–493.

27. HERRMANN, B.G., S. LABEIT, A. POUSTKA, T.R. KING & H. LEHRACH. 1990. Cloningof the T gene required in mesoderm formation in the mouse. Nature 343: 617–622.

28. YAMAGUCHI, T.P., D.J. DUMONT, R.A. CONLON, M.L. BREITMAN & J. ROSSANT. 1993.flk-1, an flt-related receptor tyrosine kinase is an early marker for endothelial cellprecursors. Development 118: 489–498.

29. MILLAUER, B., S. WIZIGMANN-VOOS, H. SCHNURCH, R. MARTINEZ, N.P. MOLLER, W.RISAU & A. ULLRICH. 1993. High affinity VEGF binding and developmentalexpression suggest Flk-1 as a major regulator of vasculogenesis and angiogenesis.Cell 72: 835–846.

30. BEGLEY, C.G., P.D. APLAN, S. DENNING, B.F. HAYNES, T.A. WALDMANN & S.S. KIR-SCH. 1989. The gene SCL is expressed during early hematopoiesis and encodes adifferentiation-related DNA-binding motif. Proc. Natl. Acad. Sci. USA 86: 10128–10132.

31. ROBB, L., I. LYONS, R. LI, L. HARTLEY, F. KONTGEN, R.P. HARVEY, D. METCALF &C.G. BEGLEY. 1995. Absence of yolk sac hematopoiesis from mice with a targeteddisruption of the scl gene. Proc. Natl. Acad. Sci. USA 92: 7075–7079.

32. SHIVDASANI, R., E. MAYER & S.H. ORKIN. 1995. Absence of blood formation in micelacking the T-cell leukemia oncoprotein tal-1/SCL. Nature 373: 432–434.

33. CIVIN, C.I., L.C. STRAUSS, C. BROVALL, M.J. FACKLER, J.F. SCHWARTZ & J.H.SHAPER. 1984. Antigenic analysis of hematopoiesis. III. A hematopoietic progeni-tor cell surface antigen defined by a monoclonal antibody raised against KG-1acells. J. Immunol 133: 157–165.

34. KATZ, F.E., R. TINDLE, D.R. SUTHERLAND & M.F. GREAVES. 1985. Identification of amembrane glycoprotein associated with haemopoietic progenitor cells. LeukemiaRes. 9: 191–198.

35. ORKIN, S. 1992. GATA-binding transcription factors in hematopoietic cells. Blood80: 575–581.

36. CHOI, K., M. KENNEDY, A. KAZAROV, J.C. PAPADIMITRIOU & G. KELLER. 1998. Acommon precursor for hematopoietic and endothelial cells. Development 125:725–732.

37. SHALABY, F., J. ROSSANT, T.P. YAMAGUCHI, M. GERTSENSTEIN, X.F. WU, M.L.BREITMAN & A.C. SCHUH. 1995. Failure of blood-island formation and vasculogen-esis in Flk-1-deficient mice. Nature 376: 62–66.

DISCUSSION

S.J. SHARKIS (Johns Hopkins Oncology Center): I know that this is an in vitrosession, but have you taken any of these transitional colonies and transplanted theminto on-the-hoof mice?


KELLER: We are starting to look at that. We have decided to follow the model de-veloped by Merv Yoder and transplant conditioned newborn pups with both the blastcolonies and the transitional colonies. The other approach is to expand the blast col-onies and the transitional colonies in different combinations of cytokines or on dif-ferent stromal cell lines prior to transplantation. I think we also have to entertain thepossibility that we are looking at a developmental program that may never give riseto long-term repopulating stem cells.

R. MÖHLE (Eberhard Karls University): There are some data from partial Flk-1knockouts that there is a common mesodermal precursor which populates both yolksac and AGM (aorta-gonad-mesonehros) region. Migration of this cell depends onFlk-1. Is this cell identical with your blast colony cell?

KELLER: I would hope it is a comparable cell. We have not done much sortingfrom normal embryos. We have started to grow blast cell colonies from mid-streakstage embryos. Our preliminary studies indicate that the normal embryo does con-tain blast colony-forming cells. We are trying to determine whether they can gener-ate both hematopoietic and endothelial progeny.

D.A. WILLIAMS (Riley Hospital for Children): Two questions, both probablytrivial. When you talk about myeloid precursors in the yolk sac are those primitiveor definitive myeloid precursors?

KELLER: I don‘t know. WILLIAMS: The second question has to do with your genomics; based on what-

ever criteria you are going to use, what percentage of the 1,500 sequences fall intothe category that you are going to want to analyze in detail? In other words, how for-midable a task is it going to be to run these?

KELLER: It is not as bad as it sounds. Of the 1,500 sequences, we have eliminatedthe known genes and are now in the process of determining what proportion are dif-ferentially expressed between the driver and tracer. Using the slot blots with nine dif-ferent samples, we can probably analyze 700–800 clones in several months.

Documents

Hematopoietic Commitment during Embryogenesis