Both primitive and definitive blood cells are derived ...

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Prepublished online October 28, 2008; doi:10.1182/blood-2008-06-162750

Both primitive and definitive blood cells are derived from Flk-1+ mesoderm Jesse J Lugus, Changwon Park, Yunglin D. Ma and Kyunghee Choi

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Blood First Edition Paper, prepublished online October 28, 2008; DOI 10.1182/blood-2008-06-162750

Both primitive and definitive blood cells are derived from Flk-1+ mesoderm

Jesse J. Lugus1, 2*, Changwon Park1*#, Yunglin D. Ma1, 3, and Kyunghee Choi1, 2,3, §

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Department of Pathology and Immunology, 2Molecular Cell Biology Program, 3

Developmental Biology Program

Washington University School of Medicine, St. Louis, MO,

*These authors contributed equally #Current Address: Emory University, Department of Cardiology, Atlanta, GA §Corresponding author, Washington University School of Medicine, Department of Pathology and Immunology, 660 S. Euclid Ave. Campus Box 8118, St. Louis, MO 63110 [email protected]

Running title: Flk-1+ mesodermal origin for blood Key words: Embryo, Blood, Endothelial, Fate mapping, Blood Island, Flk-1,

1 Copyright © 2008 American Society of Hematology


Abstract Emerging evidence suggests that all hematopoietic and endothelial cells originate from Flk-1+ mesoderm in the mouse. However, this concept has not been completely proven, especially for the origin of blood cells. Utilizing either Flk1+/Cre;Rosa26R-EYFP or Flk1+/Cre;Rosa26R-LacZ mice, we permanently marked Flk-1+ cells and their progenies to determine the relationship between hematopoietic tissues and cells that express Flk-1. In embryos, all blood cells within the yolk sac and aorta were of Flk-1+ origin. Additionally, nearly all CD45+ cells in bone marrow and circulating blood in adults were of Flk-1+ origin. These results provide clear evidence that all blood cells, primitive and definitive, in mice are derived from Flk-1+ mesodermal cells.

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Introduction Flk-1 is a receptor tyrosine kinase that binds vascular endothelial growth factor (VEGF). In primitive streak stage embryos, Flk1 expression is first detectable in cells within and exiting the primitive streak as well as the extraembryonic mesoderm 1,2. Subsequently, it can be detected in the endothelial cells lining the blood islands of the extraembryonic yolk sac, the first site of embryonic hematopoiesis by e7.5. Genetic studies indicate that Flk1 is required for blood and vessel formation as Flk1-/mice fail to develop blood or vessels and die by e9.53. By making chimeric mice with Flk1-/- ES cells, it was revealed that Flk1 is also required for adult blood, as no Flk1-/cells contributed to adult hematopoietic tissues4. Flk-1+ cells within the primitive streak are capable of giving rise to hematopoietic cells whereas Flk-1- cells of the primitive streak do not possess hematopoietic potential5. Utilizing the ES-EB system to generate Flk-1+ cells in vitro, it is clear that hematopoietic and endothelial progenitors are contained within Flk-1+ cells6-8 . Additionally, Flk-1+ cells, but not Flk-1- cells derived from ES cells could generate T lymphocytes when co-cultured with lymphocyte-depleted thymic lobes9 and Flk-1+ cells differentiated from ES cells in vitro were able to reconstitute the hematopoietic systems of SCID mice upon transplantation10.

Though these studies show a functional requirement for Flk1 in hematopoietic development, they do not explain the origin of embryonic or adult blood. The complicated issue in hematopoietic ontogeny is that Flk1 is downregulated within cells of the hematopoietic system, such that any given hematopoietic cell is Flk-1-11. To circumvent this issue, it is necessary to utilize a lineage tracing system where cells that express Flk1, or are the progeny of Flk-1+ cells, will be permanently marked. An ideal means to perform this tracing is by utilizing a Flk1+/Cre mouse, which was generated by knocking-in Cre recombinase into the Flk1 locus by homologous recombination12. By

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comparing the endogenous Flk-1 and LacZ expression in Flk1+/LacZ and Cre expression in Flk1+/Cre mice, it was established that Cre expression patterns recapitulate endogenous Flk-1 protein and mRNA. Previous studies utilizing this strategy to permanently mark cells expressing Flk1 suggest blood cells within the yolk sac blood islands originate from Flk-1+ mesoderm12. Conversely, lineage tracing utilizing chimaeric mice suggested that not all primitive blood cells are derived from Flk-1+ mesoderm13. In this report, we examined the origin of primitive and definitive blood cells by lineage tracing and demonstrate that all blood cells are the progeny of Flk-1+ mesoderm.

Materials and methods Flk1+/Cre mice 12 were crossed with Rosa26R-LacZ 14,15 or Rosa26R-EYFP 16 mice to generate Flk1+/Cre; Rosa26R-LacZ and Flk1+/Cre; Rosa26R-EYFP reporter mice, respectively. The use of mouse models in these experiments received IACUC approval (approval number 20070074) from all participating institutions.

FACS analyses were performed as previously described 7,8. E9.5 yolk sacs were dissected. Embryos were subjected to genotyping and yolk sacs were incubated for 90 minutes at 37oC in 0.1% collagenase (Sigma) with 20% fetal bovine serum in PBS. After incubation, the yolk sacs were separated into single-cell suspension by passing through 20-gauge syringes. The cells were stained with Ter119 and Mac1 antibodies (eBioscience), and analyzed by FACS. BM cells were obtained by flushing the femur and peripheral blood (PB) samples were taken retroorbitally. Both BM and PB were treated with Red blood cell lysis buffer. After centrifugation, cells were stained with CD45, CD4, CD8, B220, Gr1, and Mac1 antibodies (eBioscience) and analyzed by FACS.

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Whole mount LacZ staining was performed as previously described 4. After staining, embryos were cryosectioned at 5-6 microns.

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Results and Discussion To trace the lineage of Flk-1+ cells, Flk1+/Cre mice12, in which Cre recombinase is knocked into the Flk1 locus, were crossed to flox-STOP-flox-LacZ (Rosa26R-LacZ)14,15 or flox-STOP-flox- EYFP (Rosa26R-EYFP)16 Rosa reporter mice, whereby cells that express Flk1 will express Cre recombinase and delete the floxed-STOP sequence. Due to the constitutively active nature of the Rosa26 locus, the cells and their progeny will permanently express LacZ or EYFP. We first examined embryonic hematopoiesis in the yolk sac blood islands of e8.5 Flk1+/Cre;Rosa26R-LacZ mice. As shown in Figure 1A, all blood island endothelial and blood cells are LacZ+. Whole mount examination revealed the obvious presence of LacZ+ cells in the extraembryonic yolk sac (asterisks in Fig 1A) and in the dorsal aorta (arrowheads in Fig 1A’). In control Rosa26R-LacZ littermates, no LacZ+ cells were found throughout the embryos or yolk sacs (Fig 1B, E, and E’). Upon sectioning embryos, it could be seen that all blood cells present in the yolk sacs are LacZ+, surrounded by endothelial cells which are also LacZ+ (Fig 1 C, C’, D, D’). We found there was both strong (entire cell stains blue) and weak (staining confined to cytoplasm) LacZ staining in nearly every type of cell that stained positively. It is likely that the processes underlying X-gal staining, including fixation, tissue permeabilization and stain penetration, could affect the uniformity of staining within these cells. At the single cell level, about 97% (average 96.73 ± 3.27, n=8) of the erythroid cells (Ter119+) and 97% (average 97.12 ± 2.22, n=8) of the macrophage (Mac1+) of the e9.5 Flk1+/Cre;Rosa26R-EYFP yolk sac cells were EYFP+ (Fig 1F, G). About ~2.7% of the e9.5 yolk sac cells were CD45+, which were also EYFP+ (not shown). Within the developing embryo proper, the paired dorsal aortas (DA) were present and visible in the e9.5 Flk1+/Cre;Rosa26R-LacZ mice and the endothelial cells lining the DA were clearly LacZ+

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(Fig 1H and H’). All blood cells visible in the dorsal aorta were LacZ+ when e9.5 Flk1+/Cre;Rosa26R-LacZ mice were examined (Fig 1H and H’).

To examine the hematopoietic tissues of adult mice, we analyzed the bone marrow (BM) of Flk1+/Cre;Rosa26R-EYFP mice. Over 98% (Average 98.6 ± 0.098, n=3) of CD45+ BM cells were EYFP+, while less than 2% of CD45+ cells were EYFP- (Fig 2A, B). More importantly, over 98% (average 98.8 ± 0.528, n=4) of CD45+ peripheral blood cells were EYFP+ (Fig 2C, D). B220+, Gr1+ and Mac1+ peripheral blood cells were all EYFP+. With reasons unclear, a small fraction of the peripheral T cells (CD4+ or CD8+) were EYFP(Fig 2C). Potentially, inactivation of the Rosa locus in some T cells could contribute to this minor CD45+EYFP- cell population. The data strongly argue that all blood cells found in embryos and in adult mice originate from Flk-1+ mesodermal cells.

The Cre/loxP system is an ideal way to trace cell lineage in vivo without complicated manipulations 17. Using Flk1+/Cre and Rosa26 reporter mice, we have demonstrated for the first time that adult blood cells are of an Flk-1+ origin. Although our findings that primitive blood cells are of an Flk-1+ origin are consistent with current literature, recent work reported that blood cells present in the yolk sac have a heterogeneous cellular origin, Flk-1+ or Flk-1- 13. In this report, the authors transfected Flk1+/Cre ES cells with floxed-STOP reporter vectors driving expression of fluorescent proteins. The transfected cells were then injected into blastocysts, and the blood islands of the chimeric embryos were analyzed. Based on the presence of fluorescent cells, they argued that the majority of blood cells have an Flk-1- rather than Flk-1+ origin. This study falls short for a number of reasons.

By transfecting Flk1+/Cre ES cells and relying on the excision of the

floxed STOP sequences in the fluorescent reporter vector, the authors ignore both the

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inherent difficulty in transfecting ES cells as well as the confounding issue of integration site effect, the latter compounded by the failure to select for stable clones allowing for non-transfected ES cell to be further analyzed. Secondly, the composition of ES cells in a chimaeric embryo may be extremely low, varied or result in an embryo that will fail to develop properly to parturition18. Thirdly, the authors compare the presence of fluorescent endothelial cells to that of hematopoietic cells with no regard for possible differences in the rates of proliferation between the two cell types. Lastly, as the blood island is a three dimensional structure, the proper way to look for populations of clonal cells would be via 3-D reconstructions, not a two dimensional plane of section. In short, these complications could account for large numbers of non-fluorescent Flk-1+ cells in the embryos that could lead to the spurious conclusion that some blood cells arise from a Flk-1- origin. In contrast, our study used a ‘genuine’ cell lineage tracing system and examined the hematopoietic cells of embryos and adult mice with minimal technical complications such as transfection efficiency or ES cell clonality. We clearly demonstrate that from the first hematopoietic cells in the yolk sac blood islands to the resident hematopoietic cells in the adult bone marrow, all blood cells are derived from Flk-1+ mesodermal precursors.

Acknowledgements We thank Tom Sato (Cornell) and Frank Costantini (Columbia) for the Flk1+/Cre and Rosa26 reporter mice, respectively. J.J.L was supported by the American Heart Association predoctoral fellowship. This work was supported by grants from the National Institutes of Health, NHLBI, HL63736 and HL55337 (K.C.).

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Author Contributions J.J.L., C.P. and Y.D.M. designed and performed experiments and wrote the manuscript. K.C. designed experiments and wrote the manuscript. None of the authors declare a conflict of interest.

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References 1. Yamaguchi TP, Dumont DJ, Conlon RA, Breitman ML, Rossant J. flk-1, an fltrelated receptor tyrosine kinase is an early marker for endothelial cell precursors. Development. 1993;118:489-498. 2. Ema M, Takahashi S, Rossant J. Deletion of the selection cassette, but not cisacting elements, in targeted Flk1-lacZ allele reveals Flk1 expression in multipotent mesodermal progenitors. Blood. 2006;107:111-117. 3. Shalaby F, Rossant J, Yamaguchi TP, et al. Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice. Nature. 1995;376:62-66. 4. Shalaby F, Ho J, Stanford WL, et al. A requirement for Flk1 in primitive and definitive hematopoiesis and vasculogenesis. Cell. 1997;89:981-990. 5. Huber TL, Kouskoff V, Fehling HJ, Palis J, Keller G. Haemangioblast commitment is initiated in the primitive streak of the mouse embryo. Nature. 2004;432:625-630. 6. Choi K, Kennedy M, Kazarov A, Papadimitriou JC, Keller G. A common precursor for hematopoietic and endothelial cells. Development. 1998;125:725-732. 7. Faloon P, Arentson E, Kazarov A, et al. Basic fibroblast growth factor positively regulates hematopoietic development. Development. 2000;127:1931-1941. 8. Chung YS, Zhang WJ, Arentson E, Kingsley PD, Palis J, Choi K. Lineage analysis of the hemangioblast as defined by FLK1 and SCL expression. Development. 2002;129:5511-5520. 9. de Pooter RF, Cho SK, Carlyle JR, Zuniga-Pflucker JC. In vitro generation of T lymphocytes from embryonic stem cell-derived prehematopoietic progenitors. Blood. 2003;102:1649-1653. 10. Miyagi T, Takeno M, Nagafuchi H, Takahashi M, Suzuki N. Flk1+ cells derived from mouse embryonic stem cells reconstitute hematopoiesis in vivo in SCID mice. Exp Hematol. 2002;30:1444-1453. 11. Kabrun N, Buhring HJ, Choi K, Ullrich A, Risau W, Keller G. Flk-1 expression defines a population of early embryonic hematopoietic precursors. Development. 1997;124:2039-2048. 12. Motoike T, Markham DW, Rossant J, Sato TN. Evidence for novel fate of Flk1+ progenitor: contribution to muscle lineage. Genesis. 2003;35:153-159. 13. Ueno H, Weissman IL. Clonal analysis of mouse development reveals a polyclonal origin for yolk sac blood islands. Dev Cell. 2006;11:519-533. 14. Zambrowicz BP, Imamoto A, Fiering S, Herzenberg LA, Kerr WG, Soriano P. Disruption of overlapping transcripts in the ROSA beta geo 26 gene trap strain leads to

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widespread expression of beta-galactosidase in mouse embryos and hematopoietic cells. Proc Natl Acad Sci U S A. 1997;94:3789-3794. 15. Soriano P. Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet. 1999;21:70-71. 16. Srinivas S, Watanabe T, Lin CS, et al. Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. BMC Dev Biol. 2001;1:4. 17. Branda CS, Dymecki SM. Talking about a revolution: The impact of site-specific recombinases on genetic analyses in mice. Dev Cell. 2004;6:7-28. 18. Eggan K, Akutsu H, Loring J, et al. Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation. Proc Natl Acad Sci U S A. 2001;98:6209-6214.

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Figure legends Figure 1. A Flk-1+ origin for embryonic blood cells of Flk1+/Cre;Rosa26R-LacZ embryos. Images A-E’ are e8.5 embryos and images F and F’ are an e9.5 embryo. A) Whole mount image of 3 representative Flk1+/Cre;Rosa26R-LacZ e8.5 embryos. Asterisks indicate plane of major blood islands in yolk sacs. A’) Whole mount high-power magnification of an embryo showing stained dorsal aorta (arrowheads). B) Control e8.5 embryos display no LacZ+ cells in their yolk sacs or embryo proper. Asterisks as in A). C) and D) Five-micron sections through Flk1+/Cre;Rosa26R-LacZ e8.5 embryos. C’) and D’) High-power views of blood islands from embryos in C) and D), respectively. E) and E’) Sections of a control embryo showing an absence of LacZ+ cells in the embryo, yolk sac, or blood islands. F) and G) Representative FACS plots of e9.5 R26R-EYFP and e9.5 Flk1+/Cre;Rosa26REYFP yolk sacs staining for the erythroid cell marker Ter119 and macrophage marker Mac1 respectively (Y-axis) and EYFP (X-axis) to assess the origin of these lineage cells within the e9.5 YS. On the right panel, summary data of multiple experiments (n=8) analyzing YS of Flk1+Cre;Rosa26R-EYFP mice. Numbers indicate the percentage of Ter119+ and Mac1+ cells either EYFP- or EYFP+, respectively. H) Transverse section through an e9.5 embryo. DA- Dorsal Aorta, LPC- Left Pericardialperitoneal Cavity, LVV- Left Vitelline Vein, NT- Neural Tube, RPC- Right Pericardialperitoneal Cavity, RVV- Right Viteline Vein. H’) Magnification of region containing paired dorsal aorta demonstrating uniformity of LacZ+ blood cells in the embryonic circulation.

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Figure 2. A Flk-1+ origin for blood cells of adult Flk1+/Cre;Rosa26R-EYFP mice. A) Representative FACS plots of bone marrow (BM) staining for the hematopoietic marker CD45 (Y-axis) and EYFP (X-axis) to assess the origin of hematopoietic cells within the BM of 3 month-old Flk1+/Cre;Rosa26R-EYFP mice. B) Summary data of multiple experiments analyzing BM of Flk1+/Cre;Rosa26R-EYFP mice. Numbers above bars indicate percentage of CD45+ cells either EYFP+ or EYFPC) Representative FACS plots of peripheral blood (PB) staining for the hematopoietic marker CD45 (Y-axis) and EYFP (X-axis) to assess the origin of hematopoietic cells within the PB. Individual lineage markers were also assessed to examine if all mature blood lineages are indeed Flk-1+ origin. Hematopoietic lineages analyzed include CD4 and CD8, T cells; B220, B cells; Gr1, granulocytes and Mac1, macrophages. D) Summary data of multiple experiments (n=4) analyzing PB of Flk1+Cre;Rosa26R-EYFP mice. Numbers above bars indicate percentage of CD45+ cells either EYFP- or EYFP+, respectively.

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*

G 20 µm

100 µm

Flk1+/Cre;R26R-LacZ

C

C’

100 µm

Flk1+/Cre;R26R-LacZ

R26R-LacZ

D

E

D’

1.32

0.08

0.02

0.73

79.13

0.09

1.54

Mac1

*

B

A’

*

84.68

Ter119

A

R26R-LacZ

100 80 60 40 20 0

% of total Mac1+ cells

Flk1+/Cre;R26R-LacZ

Flk1+/Cre;R26R-EYFP

R26R-EYFP

F

E’ 200 µm

50 µm

200 µm

50 µm

e9.5 Flk1+/Cre;R26R-LacZ

H

NT DA LPC RPC

50 µm

RVV 200 µm

Lugus et al., Figure 1

LVV

3.27% EYFP-

H’ 10 µm

EYFP+ 97.12%

2.88% EYFP-

EYFP 200 µm

100 80 60 40 20 0

96.73%

EYFP+


e8.5

% of total Ter119+ cells

e9.5

CD45

C Flk1+/Cre;R26R-EYFP

1.52

98.53

B220

CD45

0.44

21.77

CD4/8

1.31

Flk1+/Cre;R26R-EYFP

Flk1+/Cre;R26R-EYFP

EYFP

EYFP

EYFP

Flk1+/Cre;R26R-EYFP

Gr1

EYFP

Lugus et al., Figure 2

Flk1+/Cre;R26R-EYFP 0.05

11.20

13.95

Mac1

0.17

62.90

EYFP

98.6%

100 80 60 40 20

1.34%

0 EYFP-

D

EYFP+

120

98.8%

100 80 60 40 20

1.19%

0 EYFP-

EYFP+


EYFP

EYFP

120

% of total CD45+ cells (PB)

R26R-EYFP

Flk1+/Cre;R26R-EYFP

% of total CD45+ cells (BM)

B

A