Requirement for the TIE family of receptor tyrosine kinases in adult but not fetal hematopoiesis - PubMed (original) (raw)

Requirement for the TIE family of receptor tyrosine kinases in adult but not fetal hematopoiesis

Mira C Puri et al. Proc Natl Acad Sci U S A. 2003.

Abstract

In mammals, the continuous production of hematopoietic cells (HCs) is sustained by a small number of hematopoietic stem cells (HSCs) residing in the bone marrow. Early HSC activity arises in the aorta-gonad mesonephros region, within cells localized to the ventral floor of the major blood vessels, suggesting that the first HSCs may be derived from cells capable of giving rise to the hematopoietic system and to the endothelial cells of the vasculature. TIE1 (TIE) and TIE2 (TEK) are related receptor tyrosine kinases with an embryonic expression pattern in endothelial cells, their precursors, and HCs, suggestive of a role in the divergence and function of both lineages. Indeed, gene targeting approaches have shown that TIE1, TIE2, and ligands for TIE2, the angiopoietins, are essential for vascular development and maintenance. To explore possible roles for these receptors in HCs, we have examined the ability of embryonic cells lacking both TIE1 and TIE2 to contribute to developmental and adult hematopoiesis by generating chimeric animals between normal embryonic cells and cells lacking these receptors. We show here that TIE receptors are not required for differentiation and proliferation of definitive hematopoietic lineages in the embryo and fetus; surprisingly, however, these receptors are specifically required during postnatal bone marrow hematopoiesis.

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Figures

Fig. 1.

Fig. 1.

Analysis of Tie1/Tie2 deficiency in the hematopoietic system. (a) Tie1/Tie2 doubly deficient embryos display reduced HPC numbers in the yolk sac at E8.5. Reduced primitive erythroid (EryP) and macrophage (Mac) colony numbers in embryos doubly deficient for Tie1 and Tie2 (DM, n = 7) relative to control littermates (WT, n = 9, and DH, n = 18). No differences in colony numbers were observed for granulocyte–macrophage (GM) colonies, definitive erythrocytes (BFU-E), or mast cells (Mast), likely because of the low numbers of colonies produced. (b) Analysis of hematopoiesis in Tie1/Tie2 chimeras. Chimeras were generated by morula ↔ morula aggregation of donor embryos derived from intercross of recombinant Tie1/Tie2 DH parents and WT ICR or _Rag2_–/– host embryos. Donor cells carry the AA isoform of the GPI enzyme (GPIAA), whereas ICR host cells carry the BB isoform (GPIBB), allowing the fate of donor cells to be followed in chimeric tissue by colorimetric assay for GPI isoforms after electrophoresis. Donor cell fate in BM HCs was monitored by flow cytometry because donor animals express enhanced GFP (EGFP) in these cells. For quantitative analysis of donor-versus host-derived HPCs, a G418 selection strategy was implemented. Donor cells are resistant to G418 (G418R), whereas WT host cells are sensitive to G418 (G418S). (c) Validation of G418 HPC selection in chimeras. HC colonies were harvested from methylcellulose cultures of FL cells grown in the presence (+) or absence (–) of G418. Cells sensitive to G418 (derived from WT host, GPIBB) are selected against, but G418-resistant DH donor cells (lanes 1 and 2, GPIAA) carrying the _PGK_–neomycin-resistance gene survive selection.

Fig. 2.

Fig. 2.

Tie1/Tie2 doubly deficient cells contribute to fetal hematopoiesis. (a) E13.5 FL cells from WT (n = 9), DH (n = 15), and DM (n = 8) chimeras were cultured in methylcellulose containing cytokines for non-red myeloid (CFU-C) and erythroid (BFU-E) differentiation in the presence and absence of G418. Mutant and control colony numbers (adjusted for the overall extent of chimerism) show equivalent contribution. (b) Representative GPI analysis demonstrating equivalent donor (AA) versus host (BB) contribution to tail (lanes 1 and 4), FL (lanes 2 and 5), and erythro-myeloid cells (lanes 3 and 6) in DH and DM chimeric fetuses. Cells in lanes 3 and 6 were collected from methylcellulose cultures in a. (c) X-gal stained FL of E15.5 DM ↔ WT chimera reveals β-galactosidase-positive, mutant cell-derived megakaryocytes identified by their large size and multilobed nucleus. (d) Tie1/_Tie2_-deficient cells contribute to hemogenic endothelium in E10.5 chimeras. ECs and HCs of the ventral aorta contain equivalent β-galactosidase positive donor cells in DM and DH chimeras.

Fig. 3.

Fig. 3.

Tie1/_Tie2_-deficient cells are at a competitive disadvantage in the adult hematopoietic system. (a) Cells lacking Tie1 and Tie2 contribute poorly to adult hematopoietic tissues in chimeras. GPI analysis was performed on lysates from tissues as indicated, demonstrating a reduced presence of the donor-derived GPI-AA isoform in cells from hematopoietic organs of DM chimeras (DM ↔ WT) but not DH chimeras (DH ↔ WT). (b) Distribution of DM relative to DH cells in chimeras from three independent aggregation experiments. DH and DM chimeras with low donor cell contribution (<15% in nonhematopoietic tissues) were eliminated from the analysis. T, tail; H, heart; Lu, lung; Li, liver; K, kidney; B, blood; BMMC, BM mononuclear cells; Spl, spleen; Sc, splenocytes; Thy, thymus; Tc, thymocytes; LN, lymph node.

Fig. 4.

Fig. 4.

Tie1/_Tie2_-deficient HCs and progenitors are absent from adult chimeric BM. (a) Tie1/_Tie2_-deficient cells do not contribute to mature HC lineages in the BM. Flow cytometry was performed on BM cells isolated from DH and DM chimeras, a WT non-GFP-expressing control (ICR), and a control for the EGFP-expressing strain (B5/EGFP) by using anti-CD45 to mark all leukocytes, anti-B220 as a marker for B cells, anti-Gr-1 and anti-Mac1 for granulocytes and macrophages, respectively, and Ter119 to label erythroid cells. The hatched rectangles highlight the reduced frequency of donor-derived lymphocytes in DM versus DH BM. Results are representative of two DM ↔ WT and three DH ↔ WT chimeras. (b) BM-derived non-red myeloid (CFU-C) and erythroid (BFU-E) progenitors were enumerated from WT, DH, and DM chimeras after culture in cytokine-supplemented methylcellulose in the presence or absence of G418. DM ↔ WT, n = 8; DH, ↔ WT, n = 8; WT ↔ WT, n = 8.

Fig. 5.

Fig. 5.

Tie1/_Tie2_-deficient cells reconstitute lymphoid lineages in _Rag2_–/– host. (a) Flow cytometry was performed on HCs of DM ↔ _Rag2_–/–, control DH ↔ _Rag2_–/–, and WT ↔ _Rag2_–/– chimeras. Anti-B220 versus anti-IgM identified mature B cells in the BM and spleen (Spl) of mutant and control chimeras. Anti-CD4 and -CD-8 antibodies were used as T cell markers for thymus (Thy) and lymph node (LN) cells. Results are representative of DM (n = 8), DH (n = 11), and WT (n = 8). (b) DM cells contribute poorly to myelopoiesis in _Rag2_–/– host. Genomic DNA from tail (T), thymus (Thy), spleen (Spl), and myeloid (M) cells from a CFU-C assay were screened for the relative presence of the donor-specific _tie2_Δsp allele (19) in _Rag2_–/– chimeras reconstituted with DM or DH cells.

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References

    1. Sabin, F. R. (1920) Contrib. Embryol. 9 214–262.
    1. Kubo, H. & Alitalo, K. (2003) Genes Dev. 17 322–329. - PubMed
    1. de Bruijn, M. F., Ma, X., Robin, C., Ottersbach, K., Sanchez, M. J. & Dzierzak, E. (2002) Immunity 16 673–683. - PubMed
    1. Pardanaud, L., Luton, D., Prigent, M., Bourcheix, L. M., Catala, M. & Dieterlen-Lievre, F. (1996) Development (Cambridge, U.K.) 122 1363–1371. - PubMed
    1. Delassus, S. & Cumano, A. (1996) Immunity 4 97–106. - PubMed

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