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Blood, Vol. 96 No. 3 (August 1), 2000:
pp. 950-957
HEMATOPOIESIS
From the Department of Medicine, Mount Sinai School of
Medicine, New York, NY; the Department of Pathology, University of
Washington, and the Seattle Biomedical Research Institute, Seattle, WA.
To study the regulation of the early stages of hematopoiesis, cDNA
representational difference analysis was used to isolate genes that
were differentially expressed in primitive hematopoietic progenitors.
The reasoning was that such genes were more likely to provide functions
important to hematopoietic stem cells and progenitors. One of the genes
identified through this approach encodes mouse Jagged2
(mJagged2). Using quantitative reverse
transcription-polymerase chain reaction, it was shown that
mJagged2 was differentially expressed in
c-kit+ hematopoietic progenitors, including those with
the phenotypes of Lin
Although much progress has been made in understanding
the regulation of the intermediate to late stages of hematopoiesis, relatively little is known about the control of hematopoietic stem
cells (HSC). Part of the difficulty is the scarcity of HSC. To identify
genes that might be important in the regulation of HSC, we capitalized
on the availability of 2 complementary cell lines, EML C1 and MPRO. EML
C1 is a stem cell factor (SCF)-dependent, multipotent hematopoietic
cell line with erythroid, myeloid, and B-lymphoid
potential.1 MPRO is a granulocyte
macrophage-colony-stimulating factor (GM-CSF)-dependent neutrophilic
promyelocyte cell line with a reversible block in terminal
differentiation.2 Developmentally, EML C1 is relatively
close to HSC, whereas MPRO is already committed to the neutrophil
lineage. A subtraction of EML C1 cDNA by MPRO cDNA should yield genes
that are differentially expressed in HSC or in progenitors committed to
various hematopoietic lineages except the neutrophil lineage. The
approach we used was cDNA representational difference analysis
(RDA),3 which produces fewer false-positive results than
most methods of subtraction.
Here we report the isolation and functional characterization of one
such gene, E11-5, with the main focus on its roles in hematopoiesis.
The cDNA represented by E11-5 encodes the mouse homologue of rat and
human Jagged2 (rJagged2 and
hJagged2)4,5 and is designated as mJagged2.
mJagged2 belongs to the so-called DSL ligand
family.6-8 All members of this ligand family are
transmembrane proteins that serve as the ligands for Notch and related
receptors.9 They are characterized by a conserved DSL motif
in their extracellular domains. This motif was first noted in
Drosophila Delta,10,11 Serrate,12,13
and Caenorhabditis Lag27 and is essential for
interaction with Notch and related receptors. In the embryos of
Drosophila and C. elegans, Notch and related receptors
play important roles in cell fate decisions through the process of
lateral inhibition or inductive signaling.8,9 Available
evidence indicates that on binding of DSL ligands, the Notch receptor
is cleaved at a site external to the transmembrane domain. The
intermediate is further cleaved by presenilin or a presenilin-dependent Compared with Delta and Serrate of Drosophila, less is known
about the functions of mammalian DSL ligands. To date, at least 4 mammalian DSL ligands have been described: Delta-like 1 (Dll-1),16 Delta-like 3 (Dll-3),17
Jagged1,18,19 and Jagged2 (4,5 and this
report). Four mammalian Notch receptors have been identified: Notch-1,20,21 Notch-2,22 Notch-3,23
and Notch-4.24,25 The functions and the receptor usage of
mammalian DSL ligands are largely unknown. Among the mammalian DSL
ligands, rJagged14 was cloned first and has been studied
more extensively. Earlier studies of mammalian DSL ligands focused on
their involvement in differentiation.18,26,27 More recent
studies indicate that the DSL-Notch signal transduction pathway may
also regulate apoptosis28,29 and cellular
proliferation.30 In mouse embryos, Jagged1 and Jagged2 show both overlapping and unique patterns of
expression. For example, Jagged2 is highly expressed in thymus,
whereas Jagged1 is not.4,5 Jagged2 is
expressed in the basal layer of epidermis, whereas Jagged1 is
expressed in the suprabasal layer (4,5 and our unpublished
data). Thus, Jagged1 and Jagged2 may have nonredundant roles. Human
CD34+ cells cocultured with hJagged2-expressing NIH3T3
cells in the presence of Flt3 ligand and PIXY 321, a hybrid of human
IL-3 and GM-CSF, showed a slower decline of the CD34+
cells.31 In the current study, we found that
membrane-bound, full-length mJagged2 promoted the survival and
proliferation of mouse hematopoietic progenitors in the absence of
added hematopoietic growth factors. This effect required direct
cell-to-cell contact. Furthermore, we found that endothelial cells
expressed high levels of Jagged2 but that stromal fibroblasts did not.
These new observations further implicate mJagged2 as a regulator of hematopoiesis.
cDNA RDA
cDNA library screening and 5' RACE
RT-PCR and Northern blot analyses All primers chosen for reverse transcription (RT)-PCR span the intron-exon junctions to prevent amplification of genomic DNA. The correct PCR product was 477 bp in length. To prepare total RNA for RT-PCR, cells were pelleted and lysed on ice in diethylpyrocarbonate-treated water containing RNasin (0.5 U/µL; Stratagene) at a ratio of 10 µL/1000 cells. Serial 5-fold dilutions (up to 625-fold) were made from this master lysate and used as a set in RT-PCR. The first-strand synthesis and the primary PCR were performed in a volume of 50 µL in the same tube containing 10 µL cell lysate, 25 pmol each of antisense primer (5'-CCCATGCTGACACTGCCCAT) and sense primer (5'-GTGTGGAGCCCTGGCACTGT), 1.5 mmol/L MgCl2, 50 mmol/L KCl, 10 mmol/L Tris-HCl (pH 9 at 25°C), 0.1% Triton X-100, 200 µmol/L dNTP, 1.25 U Taq polymerase (Qiagen, Valencia, CA), 10 U RNasin, and 5 U SuperScript II (Boehringer Mannheim, Indianapolis, IN). The mixture was incubated for 30 minutes at 42°C (RT reaction), heated to 95°C for 5 minutes, and amplified using 25 cycles at 94°C for 30 seconds, 65°C for 30 seconds, and 72°C for 1 minute, followed by a final elongation of 4 minutes at 72°C. For secondary PCR, 5 µL of the primary PCR was added to a new tube containing 25 pmol each of nested antisense primer (5'-GGCAATCACAGTAATAGCCGCCAAT) and the nested sense primer (5'-GGCACTGTGACTGTGAGACCAACT), 1.5 mmol/L Mg Cl2, 50 mmol/L KCl, 10 mmol/L Tris-HCl (pH 9 at 25°C), 0.1% Triton X-100, 1 mmol/L dNTP, and 1.25 U Taq polymerase in a volume of 50 µL. Samples were denatured at 94°C for 30 seconds and amplified using 25 cycles at 94°C for 30 seconds, 65°C for 30 seconds, and 72°C for 1 minute, followed by a final extension at 72°C for 4 minutes. Five microliters of the secondary PCR products was resolved on a 2% agarose gel and stained with ethidium bromide. In all mJagged2 RT-PCR, only one band was observed. The sequence of the single PCR product was verified by direct cycle sequencing. Primers used in mouse -actin RT-PCR were antisense primer for cDNA synthesis
and primary PCR (5'-GCCACGCTCGGTCAGGATCTT), sense primer
(5'-GCCCAGAGCAAGAGAGGTATCCT), nested antisense primer
(5'-TTTGATGTCACGCACGATTTCC), and nested sense primer
(5'-TGTGATGGTGGGAATGGGTCAG). For Northern blot analyses,
RNA was resolved on 1% agarose gels and blotted onto HyBond-N
(Amersham, Buckinghamshire, UK) and hybridized with random-primed,
P32-labeled probes at 65°C in Rapid Hyb buffer (Amersham).
Retroviral vectors and transduction of Rab-9 cells The entire coding region of mJagged2 was cloned into the LXSN vector.32 The sequence preceding the first ATG was modified to conform to the Kozak consensus sequence. This vector was named LMJSN. Similarly, a 3.3-kb DNA encoding the entire extracellular domain (ECD) of mJagged2 (aa 1-1083) plus the Kozak consensus sequence and a stop codon was cloned into the LXSN vector. The resultant vector was named LECDSN. Amphotropic producer cell lines (PA317/LXSN, PA317/LMJSN, and PA317/LECDSN) were established as described.33 Rab-9 cell strain was obtained from American Type Culture Collection and maintained in Dulbecco's modified Eagle's medium (DMEM)/10% fetal calf serum (FCS). This diploid cell strain was established from the skin of a rabbit and was not transformed or immortalized. A clonal strain, Rab-9 C.14, was established and infected with 1 mL supernatant from each viral producer line for 24 hours and was selected with G418 (0.75 mg/mL) for 10 days. The resultant cell strains, designated as Rab-9/LXSN, Rab-9/LMJSN, and Rab-9/LECDSN, were subcultured at a 1:4 ratio every 3 to 5 days.Immunofluorescence staining and Western blot analyses The entire intracellular domain (ICD) (aa 1109-1247) of mJagged2 was expressed as GST fusion proteins per standard protocol (Gibco BRL) and was used to immunize a New Zealand White rabbit. Indirect immunofluorescence staining of Rab-9 cells grown on glass coverslips was performed using rabbit preimmune and immune sera (1:200 dilution) as the first antibody and fluorescein isothiocyanate-conjugated donkey antirabbit IgG (H+L) (1:400 dilution; PharMingen, San Diego, CA) as the second antibody. For Western blot analysis, cells were lysed in lysis buffer (50 mmol/L Tris, pH 7.5, 100 mmol/L NaCl, 5 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride, 1% Triton X-100, 0.5% sodium dodecyl sulfate [SDS]). From each cell line, 3 to 10 µg total protein was run on 8% SDS-polyacrylamide gel electrophoresis (PAGE) and blotted onto nitrocellulose (HyBond-ECL; Amersham). Western blot was probed with preimmune or immune rabbit sera at 1:2000 dilution, followed by horseradish peroxidase-conjugated donkey antirabbit IgG (Amersham), and visualized by the enhanced chemiluminescence method (Amersham).Fractionation and enrichment of bone marrow Fractionation and enrichment of bone marrow were performed according to published methods with modification.34,35 Briefly, low-density mononuclear bone marrow cells from B6SJL mice were separated by Nycodenz (1.077 mg/mL; Nycomed) density centrifugation. For further purification, mononuclear cells (MNC) were stained with a cocktail of monoclonal antibodies (anti-7/4, anti-B220, anti-YW25.12.7, Mac-1, Gr-1, Ter-119) (PharMingen) spun over an FCS cushion and rosetted twice with magnetic beads coated with sheep antirat IgG (Dynabeads M-450; Dynal, Oslo, Norway) at a bead:cell ratio of 10:1. Unstained cells were separated by the Dynal magnet and collected as the Lin (lineage marker-negative) fraction. The
Lin cells were stained with Hoechst 33342 (Ho)
(Sigma, St Louis, MO) and rhodamine (Rh) (Sigma), then with
phycoerythrin-labeled antic-kit antibody (PharMingen) and propidium
iodide (Sigma) and sorted with a FACStar Plus flow cytometer
(Becton Dickinson, Franklin Lakes, NJ) equipped with dual
argon lasers. The long-term repopulating potential of the
Lin c-kit+ Rhlo
Holo cells was verified by single-cell transplantation
(together with supporting cells).
Coculture of bone marrow cells with Rab-9 cells Monolayers of Rab-9/LXSN, LMJSN, and LECDSN were established in 12-well plates 1 week before coculture experiments. Bone marrow MNC or Lin c-kit+ cells were cocultured with
Rab-9 cells in DMEM/10%FCS (4 mL/well). Half the medium was
replaced every 2 to 4 days. Subculturing was performed at each colony
assay and at additional time points.
Hematopoietic colony assays Lin c-kit+ Rhlo
Holo cells were cultured in Iscove's modified Dulbecco's
medium (IMDM) supplemented with FCS (12.5% vol/vol), horse serum
(12.5% vol/vol), SCF (100 ng/mL), mouse IL-3 (100 ng/mL), mouse IL-6
(20 ng/mL), and mouse GM-CSF (5 ng/mL). For colony assays, cells were
plated in 1% methylcellulose medium supplemented with 20% horse serum
and the 4 growth factors specified above at the same concentrations.
Colony assays of nonadherent cells harvested from Rab-9 cocultures were
carried out in 1% methylcellulose medium supplemented with 30% FCS,
10% bovine serum albumin (wt/vol), SCF (100 ng/mL), IL-3 (10 ng/mL),
and GM-CSF (5 ng/mL).
Stromal cell lines and human umbilical vein endothelial cells STW, STV, STS, and STD are spontaneously immortalized fibroblast cell lines established from long-term bone marrow cultures of W(+/+), W/Wv, Sl (+/+), and Sl/Sld mice, respectively.36 The S17 cell line37 was a gift from Robert Rottapel (University of Toronto, Toronto, Canada). Human umbilical vein endothelial cells (HUVEC) were kindly provided by Nasserine Haque and Peter Harpel (Mount Sinai School of Medicine, New York, NY).
Cloning of mJagged2 from a multipotent hematopoietic cell line cDNA RDA was performed as described3 with some modifications. The EML C1 cDNA representation appeared as a smear of DNA fragments ranging from 0.2 to 1.2 kb on the gel (Figure 1A) and was used as the "tester" in RDA. The MPRO cDNA representation also appeared as a smear (not shown) and served as the "driver" in RDA. After 3 rounds of RDA, the products were resolved on an agarose gel (Figure 1B). Individual DNA was cloned and sequenced. Among the genes identified by EML C1-MPRO cDNA RDA were CD34 and c-kit (both are expressed by HSC and progenitors), Flt-1, mFrizzled-2, and Pax-6. Another gene, designated E11-5, exhibited EML C1-specific expression among a panel of hematopoietic cell lines and was further characterized (Figure 1C).
Normal hematopoietic progenitors express mJagged2 Although mJagged2 was initially cloned from the multipotent hematopoietic cell line EML C1, it was important to demonstrate that normal HSC and progenitors also expressed mJagged2. To determine whether normal mouse HSC expressed mJagged2, we performed RT-PCR using mJagged2-specific primers on Lin c-kit+ Rhlo
Holo (lineage markers-negative, c-kit [SCF
receptor]-positive, Rh 123-low uptake, and Ho-low uptake) mouse bone
marrow cells. The Lin c-kit+
Rhlo Holo fraction is highly enriched for
LTR-HSC.34,35 For comparison, RT-PCR was also performed on
other fractions of mouse marrow enriched or depleted of HSC and
progenitors. The fractionation of bone marrow is outlined in Figure
2A. To allow comparison of expression levels, RT-PCR was performed on undiluted and serial 5-fold diluted lysates. Results are shown in Figure 2B. EML C1 lysate served as a
positive control for RT-PCR (Figure 2Bi). The unfractionated bone
marrow showed no detectable mJagged2 expression (Figure 2Bii). When bone marrow was directly separated into c-kit
and c-kit+ fractions, only the c-kit+ fraction
(enriched for progenitors) showed mJagged2 expression (Figure
2Biii-iv). When marrow was first fractionated by density-gradient centrifugation over Nycodenz to remove mature cells, low-level expression of mJagged2 was detected in MNC (Figure 2Bv).
Purification of the MNC by antibody depletion of lineage
markers-positive cells yielded the Lin fraction,
which showed slightly increased levels of mJagged2 expression
(Figure 2Bvi). Further enrichment for HSC and progenitors by positive
selection for c-kit expression yielded the Lin
c-kit+ fraction that showed markedly increased
mJagged2 expression (Figure 2Bviii). The Lin
c-kit fraction, devoid of HSC and progenitors, did
not express mJagged2 (Figure 2Bvii). The Lin
c-kit+ Rhhi Holo fraction contained
spleen colony-forming units (CFU-S8-12) capable of
short-term repopulation of lethally irradiated mice
(STR-HSC).34,38,39 This fraction also expressed high levels
of mJagged2 (Figure 2Bix). The Lin
c-kit+ Rhlo Holo fraction
(containing LTR-HSC) showed relatively high levels of mJagged2
(Figure 2Bx). Overall, the expression of mJagged2 appears to be
restricted to the c-kit+ hematopoietic progenitors. In
all mJagged2 RT-PCR experiments (except Figure 2Bvi), there was
only one visible PCR product (477 bp) whose identity was verified by
direct sequencing of the gel-purified DNA. (The faint, 1.4-kb PCR
product in panel vi of Figure 2B resulted from amplification of the
genomic mJagged2 DNA.)
Expression of mJagged2 is down-regulated on differentiation To examine the modulation of mJagged2 during hematopoiesis, we cultured Lin c-kit+ Rhlo
Holo cells in a medium containing SCF, IL-3, IL-6, and
GM-CSF and performed mJagged2 RT-PCR on cells sequentially
harvested from the liquid culture. The results showed that the level of
mJagged2 expression was high in the starting population and was
down-regulated during subsequent proliferation and differentiation
(Figure 3A-C) and was almost undetectable
after 11 days (Figure 3D). Wright-stained cytospin preparations of
cells harvested on days 8 and 11 showed increasing and predominantly
mature neutrophils, macrophages, and mast cells (not shown).
Endothelial cells express high levels of Jagged2 whereas fibroblasts do not Preliminary in situ hybridization studies revealed that the endothelial cells lining the lumen of the dorsal aortae of day 8.5 and day 12.5 mouse embryos expressed mJagged2, whereas most of the mesenchymal cells did not (Queva C, Tsai S, unpublished data). Furthermore, the hJagged2 cDNA was initially cloned from a HUVEC cDNA library.5 These 2 observations suggested that endothelial cells could be an important source of mJagged2 in the hematopoietic microenvironment. Therefore, we examined the expression of mJagged2 protein in primary HUVEC and in 5 independently derived stromal fibroblast cell lines (S17, STW, STV, STS, STD)36,37 by Western blot analysis using the anti-ICD antibodies (recognizing the intracellular domain of both human and mouse Jagged2). The results are shown in Figure 4. Although HUVEC expressed high levels of Jagged2 (Figure 4A), none of the stromal fibroblast cell lines examined showed detectable levels of Jagged2 expression (Figure 4B). These findings were corroborated by the results of Northern blot analysis (Figure 4C). Stimulation of stromal fibroblast cell lines with tumor necrosis factor did not result in the induction of Jagged2 (not shown). Western and Northern blot analyses of additional fibroblast cell lines or strains such as mouse NIH3T3 (Figure 1C, lane 1), primary human foreskin fibroblasts (not shown), and primary rabbit skin fibroblasts (Figure 4A, lane 1) also failed to detect Jagged2 expression.
Full-length mJagged2 promotes the survival and proliferation of mouse bone marrow progenitors To determine the role of mJagged2 in the survival, proliferation, or differentiation of hematopoietic progenitors, we first expressed mJagged2 in a rabbit-skin fibroblast cell strain (Rab-9) through retroviral vector-mediated gene transfer. Mouse hematopoietic progenitors were then cocultured with Rab-9 cells expressing ectopic mJagged2. Mouse fibroblasts or mouse stromal cell lines were not used for expressing mJagged2 because they produce multiple mouse hematopoietic growth factors that could overshadow or antagonize the action of mJagged2 on mouse hematopoietic progenitors. For the same reason, no exogenous hematopoietic growth factors were added to the cocultures. The Rab-9 fibroblast cell strain differs from fibroblast cell lines in that it is not transformed or immortalized and behaves like normal fibroblasts. To ensure homogeneity, we established a clonal strain, Rab-9 C.14, which was then transduced with retroviral vectors LXSN (empty vector control), LMJSN (expressing full-length mouse Jagged2; a.a 1-1247), and LECDSN (expressing the entire extracellular domain of mJagged2; a.a. 1-1083) and selected with G418. Northern blot analyses showed the expression of mJagged2 RNA of predicted sizes in Rab-9/LMJSN and Rab-9/LECDSN but not in Rab-9/LXSN. The level of the full-length mJagged2 message was less than one tenth that of truncated mJagged2 (Figure 5A), indicating that the sequence downstream from the extracellular domain might be involved in transcriptional or post-transcriptional regulation of the gene. Western blot analyses using anti-ICD antibodies verified the production of the approximately 150-kd mJagged2 protein in the multipotent hematopoietic cell line EML C1 (positive control) and Rab-9/LMJSN but not in Rab-9/LXSN or Rab-9/LECDSN (Figure 5B). Immunofluorescence staining with anti-ICD antibodies also verified the expression of mJagged2 in Rab-9/LMJSN but not in Rab-9/LXSN or Rab-9/LECDSN (Figure 5C-E).
Survival effect of membrane-bound mJagged2 requires direct
cell-to-cell contact
In this report, we describe the cloning of mJagged2 by cDNA
RDA from EML C1, an SCF-dependent, multipotent hematopoietic cell line.1 mJagged2 encodes a single-pass membrane
protein characterized by the DSL motif in its extracellular domain.
Using quantitative RT-PCR, we showed that mJagged2 was
expressed in freshly isolated c-kit+ mouse hematopoietic
progenitors, including those in the Lin We thank Peter Harpel, Nasserine Haque, Ewa Sitnicka, Raphael Kopan,
and Robert Rottapel for sharing research materials, Gordon Keller for
critical reading of the manuscript, Carl Storey and Lily Kiang for
expert technical assistance, and Doris Damian for advice on statistical analysis.
Submitted December 23, 1999; accepted April 5, 2000.
Supported by Public Health Service grants DK48622 (S.T.) and
DK 48708 (S.B.).
Reprints: Schickwann Tsai, The Mount Sinai School of Medicine,
Box 1496, 1425 Madison Avenue, Room 13-23B, New York, NY 10029; e-mail:
tsais01{at}doc.mssm.edu.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
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Top
Abstract
Introduction
-secretases within the transmembrane domain to
release the intracellular domain.
gt11 was
screened with radiolabeled E11-5 DNA. Eleven positive clones
representing 2 overlapping fragments were picked. After secondary
screening, phage DNA was purified for cycle sequencing. For 5'
RACE (rapid amplification of cDNA ends), 1 µg EML C1 mRNA was primed
with a gene-specific primer (5'-GCTGAAGTGCAGGCTCTT) using
sequence data from the 
174/HaeIII markers. (B) The result of EML
C1-MPRO cDNA RDA was resolved on 2% agarose and stained with ethidium
bromide. Some bands contained more than one DNA species. (C) Northern
blot analysis of the expression of E11-5 in cell lines representing
different hematopoietic lineages. Each lane contains 5 µg total RNA
from NIH3T3 (fibroblast), MPRO (neutrophil), J774 (macrophage), EML C1
(stem cell-like), MC/9 (mast cell), FLDS-19 (erythroblast), BaF3
(B-cell), and EL-4 (T-cell). The blot was hybridized with a
radiolabeled, nick-translated E11-5 (mJagged2) probe.
a Serrate-like gene expressed during rat embryogenesis.
Dev Biol.
1996;180:370-376
/
T cell lineage decision.
Cell.
1997;88:833-843
