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Thrombopoietin Rescues in Vitro Erythroid Colony Formation from Mouse Embryos Lacking the Erythropoietin Receptor MARK W

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Thrombopoietin Rescues in Vitro Erythroid Colony Formation from Mouse Embryos Lacking the Erythropoietin Receptor MARK W Proc. Natl. Acad. Sci. USA Vol. 93, pp. 9126-9131, August 1996 Medical Sciences Thrombopoietin rescues in vitro erythroid colony formation from mouse embryos lacking the erythropoietin receptor MARK W. KIERAN*, ANDREW C. PERKINS*, STUART H. ORKIN*t, AND LEONARD I. ZON*t# *Division of Hematology/Oncology, Department of Pediatrics, Children's Hospital and the Dana-Farber Cancer Institute, Boston, MA 02115; and tHarvard Medical School and the Howard Hughes Medical Institute, Boston, MA 02115 Contributed by Stuart H. Orkin, May 9, 1996 ABSTRACT The interaction of the hormone erythropoi- defect in both embryonic and liver erythropoiesis but normal etin and its receptor (EpoR) is thought to be required for megakaryopoiesis (refs. 21 and 22 and see below). normal hematopoiesis. To define the role of EpoR in this Recombinant Epo has been shown to increase human process, the murine EpoR was disrupted by homologous megakaryocytic colony formation (23), as well as murine recombination. Mice lacking the EpoR died in utero at em- platelet production (24, 25). On the other hand, thrombopoi- bryonic day 11-12.5 with severe anemia. Embryonic erythro- etin (Tpo) stimulation in syngeneic bone marrow transplant poiesis was markedly diminished, while fetal liver hematopoi- recipients resulted in increased numbers of erythroid blast- esis was blocked at the proerythroblast stage. Other cell types forming units (BFU-E), as well as higher erythrocyte nadir known to express EpoR, including megakaryocytes, mast, and counts and accelerated recovery of erythrocytes (17, 26). To neural cells were morphologically normal. Reverse transcrip- address the potential regulation of erythropoiesis by Tpo, we tion-coupled PCR analysis of RNA from embryonic yolk sac, have generated EpoR-/- mice and examined erythroid peripheral blood, and fetal liver demonstrated near normal progenitor proliferation and differentiation in the presence of transcripts levels for EKLF, thrombopoietin (Tpo), c-MPL, recombinant Tpo. Our results demonstrate that EpoR-/- GATA-1, GATA-2, and a- and embryonic PH1-globin but none erythroid progenitor cells express c-MPL and that exogenous for adult jmaj-globin. While colony-forming unit-erythroid Tpo in combination with either Kit ligand (KL) or interleukin (CFU-E) and burst-forming unit-erythroid (BFU-E) colonies (IL) 3 plus IL-11 rescues BFU-E proliferation and terminal were not present in cultures derived from EpoR-/- liver or differentiation. These findings indicate that a signal provided yolk sac cells, hemoglobin-containing BFU-E colonies were by Tpo-c-MPL interaction to cultured erythroid precursor detected in cultures treated with recombinant Tpo and Kit cells can substitute for that normally transmitted through the ligand or with Tpo and interleukin 3 and 11. Rescued BFU-E EpoR and raises the possibility that erythropoiesis might be colonies expressed adult 18-globin and c-MPL and appeared regulated in part in vivo by Tpo. morphologically normal. Thus, erythroid progenitors are formed in vivo in mice lacking the EpoR, and our studies demonstrate that a signal transmitted through the Tpo re- METHODS ceptor c-MPL stimulates proliferation and terminal differen- Generation of Targeted Embryonic Stem Cell Clones and tiation of these progenitors in vitro. EpoR Null Mice. The splice acceptor site of exon II and all of exon III of the EpoR (GenBank accession no. X53081) were The hematopoietic system in vertebrates is capable of gener- excised (XhoI to BamHI) and replaced by a neomycin cassette ating a diverse array of differentiated cell types, including red (Fig. 1A). Twenty-five micrograms of the targeting construct, blood cells, white blood cells, and platelets, from a few which also contained a thymidine kinase cassette for negative self-renewing stem cells. Interactions between specific cyto- selection, was linearalized by NotI digestion and electropo- kines and their receptors transmit signals for proliferation and rated into Jl embryonic stem cells, and recombinants were possibly differentiation within hematopoietic progenitors. The identified as described (27). Individual clones were selected erythropoietin receptor (EpoR) (1, 2) and its ligand erythro- and injected into C57BL/6 blastocysts, which were transferred poietin (Epo) (3) figure prominently in erythroid proliferation into C57BL/6 or BALB/c pseudopregnant foster mothers. and differentiation (4, 5). Chimeras were backcrossed to C57BL/6 mice and germ-line Several lines of evidence suggest that erythroid and transmission was detected by Southern blot analysis of tail megakaryocytic lineages arise from a common cell (6). Both DNA as described (27). Genotyping was performed both by lineages coexpress transcription factors (7, 8), as well as the Southern blot analysis usingAvrII digestion followed by prob- EpoR and the Tpo receptor (c-MPL) (9-12), which are ing with the 550-bp BamHI-AvrII fragment of exon VIII (Fig. structurally related members of the cytokine receptor super- 1B) or by PCR (data not shown). Staged embryos were family (13-15). Moreover, the ligands for these receptors removed on embryonic days (E) 10 to E15 (the morning of exhibit significant structural homology (16, 17). These struc- vaginal plug discovery was taken as EO) and disected free of tural similarities suggest common ancestry and raise the pos- maternal tissue without disrupting the placenta or visceral yolk sibility of vestigial overlap in downstream receptor-mediated sac (27). signaling cascades (18). Analysis ofPeripheral Blood, Yolk Sac, and Liver Cells. The Mice lacking either c-MPL (19, 20) or the EpoR (refs. 21 and embryos were rinsed in PBS to remove maternal red cells and 22 and see below) have been generated by homologous re- exsanginated via the umbilical and vitelline vessels in 1 ml of combination in embryonic stem (ES) cells. c-MPL-/- mice display a significant, albeit incomplete, reduction in platelet Abbreviations: EpoR, erythropoietin receptor; Epo, erythropoietin; production but apparently normal erythropoiesis (19, 20). By Tpo, thrombopoietin; IL, interleukin; KL, Kit ligand; E, embryonic contrast, both Epo and EpoR-/- mice demonstrate a major day(s); RT-PCR, reverse transcription-coupled PCR; CFU-E, ery- throid colony-forming unit(s); BFU-E, erythroid blast-forming unit(s). The publication costs of this article were defrayed in part by page charge ITo whom reprint requests should be addressed at: Division of payment. This article must therefore be hereby marked "advertisement" in Hematology, Children's Hospital, 300 Longwood Avenue, Boston, accordance with 18 U.S.C. §1734 solely to indicate this fact. MA 02115 9126 Downloaded by guest on September 29, 2021 Medical Sciences: Kieran et al. Proc. Natl. Acad. Sci. USA 93 (1996) 9127 Tail A 4.171 Kb B Embryo B H AE X B HB BH A B I + n dIA $ II IIIIV VI VIl VIII 2 I PROBE N B H AE X B HB BH A B II IV V VI VII VIII 5.60OKb PROBE C Gestational Total Number Number Number Age Number +/+, +/- -/- Live -/- Dead Embryos Embryos Embryos Embryos 13.0 12 6 0 6 12.5 46 35 7 4 12.0 21 17 4 0 11.5 21 13 6 2 11.0 14 11 3 0 :E G 0 H . y 0 _ y i:. n> .p I 0~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ _ FIG. 1. (A) Targeted disruption of the mouse EpoR gene. The EpoR locus and partial restriction map is shown (Upper) with the targeting construct (Lower). Tk, PGK-thymidine kinase cassette. Restriction enzymes are as follows: B, BamHI; H, HindIll; A, AvrII; E, EcoRI; X, XhoI; N, NotI. (B) Southern blot analysis of tail and embryo genomic DNA demonstrates the 4.2-kb wild-type and 5.6-kb mutant alleles. (C) Viability and genotypic analysis of embryos during gestation (days). (D) Phenotypic comparison of E13 wild-type (Left) and EpoR homozygous mutant (Right). (E and F) Wild-type day 12.5 peripheral blood (E) or fetal liver (F) May-Grunwald-Geimsa-stained cytospins. Mature primitive yolk sac-derived erythroid cells (y) as well as fetal liver-derived definitive erythroid cells, including proerythroblasts (p), normoblasts (n), and mature enucleated red cells (m) are present. (G and H) EpoR-/- peripheral blood (G) or fetal liver (H) May-Grunwald-Geimsa-stained cytospins. Mature primitive yolk sac-derived erythroid cells (y) as well as liver-derived definitive proerythroblasts (p) but no mature definitive liver-derived erythroid cells are present. PBS by surgical removal of the placenta. Single-cell suspen- Reactions were controlled by including amplification primers for sions of fetal liver were prepared by gentle passage through a ,B-actin or HPRT. Reactions were incubated in a Perkin-Elmer 26-gauge needle, and single-cell suspensions from yolk sacs 9600 thermal cycler at 94°C for 1 min, 65°C for 1 min, and 72°C were prepared by incubation for 1 h in 0.1% collagenase in for 2 min. Aliquots were removed after a variable number of 20% fetal calf serum/PBS at 37°C followed by passage through cycles and electropheresed through a 6% polyacrylamide gel. 25- and 26-gauge needles. Cell number and viability were Image analysis was performed using a Phospholmager and determined by trypan blue exclusion. Peripheral blood, yolk IMAGEQUANT software (Molecular Dynamics). All analyses were sac, and liver cytocentrifuge preparations were made by cen- performed on samples within the linear range of amplification. trifugation at 400 rpm in a Shandon Cytospin 3 rotor for 4 min The following primer sets were used: EKLF, 5'-TCGCCG- and stained in May-Grunwald-Geimsa (27). GAGACGCAGGCT-3' and 5'-CCCAGTCCTTGTGCAGGA- Preparation of RNA, DNA, and cDNA from Cells and 3'; c-MPL, 5 '-CCATCGATCTAGAGCTGCGCCCCCG- Tissue. Total RNA was prepared from tissues using a com- AGC-3' and 5' -CGGAATTCGTAGGAATGTATAG- mercial kit (RNAzol B, Tel-Test, Friendswood, TX). Genomic GTCTGC-3'; TPO, 5'-TCTGTCCAGCCCCGTAGGTC-3' and DNA was prepared from tails and carcasses by standard 5'-GTTCCATCCACAGTCCGTG-3' (28); EpoR, 5'-GGGAT- methods. cDNA was prepared using Moloney murine leuke- GGACTTCAACTACAG-3' and 5'-TGATGCGGTGATAGC- mia virus reverse transcriptase (GIBCO/BRL) by the manu- GAGGAGA-3'; GATA2, 5'-CGGAATTCGACACACCAC- factures recommendations.
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