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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 11 (June 1), 1998:
pp. 4084-4091
The Notch Ligand, Jagged-1, Influences the Development of Primitive
Hematopoietic Precursor Cells
By
Barbara Varnum-Finney,
Louise E. Purton,
Monica Yu,
Carolyn Brashem-Stein,
David Flowers,
Steven Staats,
Kateri A. Moore,
Isabelle Le Roux,
Robert Mann,
Grace Gray,
Spyros Artavanis-Tsakonas, and
Irwin
D. Bernstein
From the Clinical Research Division, Fred Hutchinson Cancer Research
Center, Seattle, WA; the Department of Pediatrics, The University of
Washington, Seattle; the Department of Molecular Biology, Lewis Thomas
Laboratory, Princeton University, Princeton, NJ; Developmental Genetics
Laboratory, Imperial Cancer Research Fund, London, UK; and the
Departments of Biology and Cell Biology and Howard Hughes Medical
Institute, Yale University, New Haven, CT.
 |
ABSTRACT |
We examined the expression of two members of the
Notch family, Notch-1 and Notch-2, and one
Notch ligand, Jagged-1, in hematopoietic cells. Both Notch-1
and Notch-2 were detected in murine marrow precursors
(Lin Sca-1+c-kit+).
The Notch ligand, Jagged-1, was not detected in whole
marrow or in precursors. However, Jagged-1 was seen in cultured primary murine fetal liver stroma, cultured primary murine bone marrow stroma,
and in stromal cell lines. These results indicate a potential role for
Notch-Notch ligand interactions in hematopoiesis. To further test this
possibility, the effect of Jagged-1 on murine marrow precursor cells
was assessed by coculturing sorted precursor cells
(LinSca-1+c-kit+)
with a 3T3 cell layer that expressed human Jagged-1 or by
incubating sorted precursors with beads coated with the purified
extracellular domain of human Jagged-1 (Jagged-1ext). We
found that Jagged-1, presented both on the cell surface and on beads,
promoted a twofold to threefold increase in the formation of primitive
precursor cell populations. These results suggest a potential use for
Notch ligands in expanding precursor cell populations in vitro.
| |
INTRODUCTION |
HEMATOPOIETIC STEM CELLS either
self-renew, thereby maintaining stem cell properties, or alternatively,
give rise to cells increasingly committed to differentiate into the
various hematopoietic lineages. It is unclear if this cell fate
decision is controlled by a purely stochastic mechanism1 or
is the result of environmental cues mediated through specific receptor
ligand interactions. In several invertebrate and vertebrate
developmental systems, cell fate is influenced both by soluble
molecules and by molecules acting via cell-cell interactions, including
those mediated by the Notch receptor family.2,3
The Notch superfamily encodes cell-surface receptors that
influence numerous cell-fate decisions in both invertebrates and vertebrates, including decisions made during central and peripheral nervous system development, wing, eye, bristle, and ovary development in Drosophila melanogaster,4-8 the germinal
vesicle and nervous system development in Caenorhabditis
elegans,9 and eye development in
Xenopus.10 Moreover, Notch
mRNA and protein have been found in hematopoietic precursors,
indicating a potential role for Notch interactions in
hematopoiesis11 (and Flowers et al,
submitted).
Both Notch and its ligands are transmembrane proteins that consist of
several epidermal growth factor (EGF) repeats in the extracellular domain. At present, four Notch homologs have been identified in vertebrates: Notch-1, -2, -3, and
-4.12-16 Also, candidate ligands for Notch have
been cloned from vertebrates, including two homologs for Drosophila
Serrate. The first Serrate homolog has been referred to as
Jagged-1 in rat17 and C-Serrate-1 in
chicken,18 and the second Serrate homolog as
Jagged-2 in rat19 and in human.20 Also,
two homologs for D Delta have been cloned. The first
Delta homologue has been referred to as C-Delta-1 in
chicken21 and Delta-like-1 in mouse,22
and the second as Delta-like-3 in mouse.23 At the
present time very little is known about the specificity of the various
Notch receptors for each ligand. Notch ligands consist of a highly
homologous DSL (the first letters of Delta,
Serrate, and Lag-2)24 domain at the
amino terminus followed by EGF repeats (see Fig 1). In Jagged-1 and -2, as in their Drosophila homolog Serrate, there is a
cysteine-rich region between the EGF repeats and the transmembrane domain. Delta is missing this domain in both vertebrates
and in Drosophila. The function of the cytoplasmic domain,
whose structure varies among the different ligands, is unknown but
deletion analyses have shown it to be essential for wild-type
function.10,25-28
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| Fig 1.
A schematic diagram of Jagged-1 and
Jagged-1ext. The top diagram depicts the protein product of
Jagged-1 expressed in 3T3 cells and the bottom diagram the
truncated product of Jagged-1ext. Indicated are the
signal peptide (SP), the DSL domain, the EGF repeats, the cysteine rich
region (Cys), the transmembrane domain (TM), the myc tag
epitope recognized by the 9E10 antibody (myc tag), and the
poly-histidines (poly-his).
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To identify a potential role for Notch in hematopoiesis, we first
assessed the expression of two members of the Notch family, Notch-1 and Notch-2, in murine whole marrow and in
purified precursors. We also determined the expression of the Notch
ligand, Jagged-1, in murine cells including whole marrow,
purified precursors, primary cultured fetal liver stroma, primary
cultured bone marrow (BM) stroma, and in stromal cell
lines.29,30 To assess the effect of Jagged-1 on early
precursors, we cultured purified primitive mouse marrow cells
(LinSca-1+c-kit+)
either with a 3T3 layer expressing full-length Jagged-1 or in wells containing beads coated with truncated Jagged-1, consisting of
the extracellular domain of Jagged-1 (Jagged-1ext, see Fig
1). The results presented in this report show the potential for Notch
and Jagged-1 interactions within the marrow and further demonstrate
that Jagged-1 affects the in vitro development of a primitive precursor
cell population.
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MATERIALS AND METHODS |
Transfections, retroviral infections, and full-length and mutant
Jagged-1 generation.
Full-length human Jagged-1 cDNA in pBluescript (KS+;
Stratagene, San Diego, CA; Gray and Artavanis, in
preparation; GenBank accession no. U61276) was subcloned
into a PCS2 vector that contained sequences encoding six
consecutive myc epitopes (generous gift from David Turner and
Hal Weintraub, Fred Hutchinson Cancer Research Center),
so that six myc-epitopes were expressed from the
carboxyl terminus of Jagged-1
(Fig 1). An EcoRI fragment
containing Jagged-1 plus the myc tags was subcloned
into LXSN, a retroviral vector.30 Viruses were made
and NIH-3T3 cells were infected. Five G418-resistant clones were then
assessed for Jagged-1 expression with Western blots using 9E10,
a monoclonal antibody (MoAb) that recognizes the myc tag
epitopes.32
To generate the extracellular Jagged-1ext,
Jagged-1 sequences encoding from amino acid 1047 to the
carboxy terminus including the transmembrane and cytoplasmic domain
were deleted. The remaining sequence encoding the extracellular domain
was then subcloned into the PCS2 vector with the myc
tag. A sequence encoding six consecutive histidines and a stop codon
was then added in frame to the 3 end (Fig 1). The cDNA encoding
the extracellular domain of Jagged-1 plus the myc tags
and the poly-histidine tail was then subcloned into the expression
vector pcDNA 3.1 (Invitrogen, San Diego, CA). COS cells were
transfected with Jagged-1ext using a modified
method developed by Hirano et al.33 COS cells to be
transfected were plated onto 150-mm dishes in 20 mL of serum-free medium (Dulbecco's modified Eagle's medium [DMEM]
supplemented with Nutridoma HU; Boehringer Mannheim, Indianapolis, IN;
at the recommended concentration) containing 10 mmol/L chloroquine
diphosphate (Sigma, St Louis, MO). A solution of diethyl
aminoethyl dextran (1 mg/mL final concentration;
Pharmacia Biotech, Piscataway, NJ) and DNA (1.5 µg/mL
final concentration) in phosphate-buffered saline (PBS) was added.
After a 2- to 3-hour incubation at 37°C, transfected cells were
washed with serum-free medium and subsequently maintained in serum-free
medium. Control cells were treated in an identical manner, but DNA was
not added. Conditioned medium was collected from both cells expressing
Jaggedext and control cells after 3 and 5 days.
Conditioned medium from each cell type was then concentrated and
dialyzed against PBS (10 mmol/L Na2HPO4, 100 mmol/L NaCl, pH 7.2). Jagged-1ext-containing medium and
control medium were then bound to a Nickle column (Ni-NTA Agarose;
Qiagen, Chatsworth, CA) using the His-Bind buffer Kit (Novagen,
Madison, WI) according to manufacturers' instructions. After elution
with imidizol (5 mmol/L to 1 mol/L), fractions that contained
Jagged-1ext were identified with Western blots using the
9E10 antibody. Positive fractions were then pooled, concentrated, and
dialyzed with PBS and analyzed by polyacrylamide gel electrophoresis
(PAGE) in reducing conditions. The same fractions from the control
elutions were also pooled, concentrated, and dialyzed.
To further purify Jagged-1ext, concentrated and dialyzed
pooled fractions were added to 9E10 crosslinked Sepharose
beads (Sigma) overnight at 4°C in a tube roller.
About 10 µg of ligand was added to about 20 µL of beads in 0.5 mL
of a 10 mg/mL solution of bovine serum albumin (BSA) in PBS. To make
9E10 crosslinked beads, 1 mg 9E10 antibody was crosslinked to 0.9 g
cyanogen bromide activated Sepharose 4B (Sigma) according to
manufacturer's instructions. Briefly, beads were swollen in 1 mmol/L
HCl for 15 minutes and washed two times with coupling buffer (0.1 mol/L
NaHCO3 pH 8.3, 0.5 mol/L NaCl). Antibody was then
resuspended in sterile coupling buffer to a final concentration of 2 mg/mL then mixed with 0.5 mL of a 50% bead solution and incubated for
2 hours at room temperature. To block remaining active groups, 0.2 mol/L glycine, pH 8.0, was added for 2 hours at 4°C. Beads were
then washed according to manufacturer's instructions. Antibody beads
were blocked with 10 mg/mL BSA (Sigma) in PBS before use.
Antibodies, Western blots, immunoprecipitations.
MoAbs GR-1 (clone RB6-8C5) and Mac-1 (clone M1/70), CD2 (clone RM2.2),
CD3 (clone KT3-1.1), CD5 (clone 53-7.3), CD8 (clone 53-6.7), B220
(clone RA36B2), and TER-119 were a generous gift from G. Spangrude
(University of Utah, Salt Lake City). The methods for
developing the rat monoclonals BHN6 and 18G are described elsewhere.34 To generate rabbit polyclonal antibody SER10,
a fragment (bp 3241-3550) of the portion of C-Serrate-1 cDNA
that encodes the cytoplasmic domain of C-Serrate-1 was subcloned into pet23b vector (Novagen), expressed in Escherichia
coli, and purified on a Nickel column under nondenaturing
conditions according to the manufacturer's instructions (Novagen).
For Western blot analysis, total cell lysates were prepared from 1 to
5 × 106 cells (per lane) using lysis buffer
(50 mmol/L Tris, pH 8.0, 0.15 mol/L NaCl, 20 mmol/L EDTA, 1.0%
Triton-X-100). Triton soluble proteins from lysates were separated
using either an 8% or a 6% sodium dodecyl sulfate (SDS)-PAGE using a
mini-gel apparatus (Owl Scientific, Woburn, MA). Before loading gels,
lysates were resuspended in reducing sample buffer (.06 mol/L Tris, pH
6.8, 1% SDS, 12.5% glycerol, 1.25%  -mercaptoethanol, 0.025%
bromophenol blue). Separated proteins were transferred to
nitrocellulose (Schleicher and Schull, Keene, NH) and
immunoblotted with the respective MoAbs. Immunoreactivity was detected
using a horseradish peroxidase (HRP)-conjugated sheep anti-mouse IgG
antibody (Amersham, Arlington Heights, IL) and enhanced
chemiluminescence Western blot reagents (Amersham).
To biotinylate surface proteins before immunoprecipitation, 1 × 106 cells were incubated with sulfo-NHS-biotin (Pierce,
Rockford, IL) in 0.1 mol/L HEPES in PBS, pH 8.0 according to the
manufacturer's instructions. Cells were removed and washed three times
with TBS (25 mmol/L Tris, pH 8.0, 140 mmol/L NaCl, 2 mmol/L
KCl) and lysed for 15 minutes on ice with lysis buffer plus protease
inhibitors (2 µg/mL aprotinin, 1 µmol/L pepstatin A, 100 µmol/L
leupeptin [all from Boehringher Mannheim, Indianapolis, IN], and 1 mmol/L phenylmethylsulfonyl fluoride [PMSF; Calbiochem, San Diego,
CA]).
For immunoprecipitation of Jagged-1 with 9E10, biotinylated cell
lysates were first incubated with beads crosslinked with a control
antibody. Lysates were then incubated with 9E10 crosslinked beads (see
below) for 2 hours at 4°C. Beads were washed once with Lysis buffer
and then with RIPA buffer (50 mmol/L Tris, pH 8.3, 0.45 mol/L NaCl, 0.5 % NP-40). Beads were then resuspended in reducing sample buffer and
proteins were separated using an 8% SDS-PAGE. Separated proteins were
transferred and biotinylated proteins were detected with HRP
streptavidin (Amersham) and enhanced chemiluminescence reagents
(Amersham).
Hematopoietic cells and flow immunocytometry.
C57BL/6J (Ly5.2) female mice were obtained from The Jackson Laboratory
(Bar Harbor, ME). All animals were housed in specific pathogen-free
conditions and maintained on acidified drinking water and autoclaved
chow ad libitum. Mice were used at 8 to 12 weeks of age. For enrichment
of hematopoietic precursor cells, suspensions of BM cells were obtained
by flushing femurs and passaging the marrow through a 23-gauge needle
into PBS containing 2% heat-inactivated fetal calf serum (PBS/FCS).
Low-density cells were enriched by equilibrium centrifugation over a
cushion of Ficoll-Hypaque (Pharmacia and
Nycomed-Amersham, Princeton, NJ) at a density of 1.077 g/mL. Retrieved
cells were washed, then resuspended at a density of 5 × 107 cells/mL in PBS/FCS containing a predetermined
saturating solution of MoAbs specific for murine T lymphocytes (CD2,
CD3, CD5, CD8), B lymphocytes (B220), macrophages (Mac-1), granulocytes
(Gr-1), and erythrocytes (TER-119).
After 30 minutes of incubation on ice, the cells were washed,
resuspended in PBS/FCS at a concentration of 108 cells/mL,
and transferred to a 50-mL conical tube. Twice-washed immunomagnetic
particles (Dynabeads M-450, sheep anti-rat IgG specificity; Dynal Inc,
Great Neck, NY) were slowly added to the cells to a final ratio of 4 beads/cell. The cell/bead suspension was allowed to stand for 5 minutes
at room temperature, then centrifuged at 400 rpm for 3 minutes. The cells and bead mixture was resuspended in 1 mL of PBS/FCS.
The magnetic-particle-free fraction was retrieved by exposure to a
magnetic field, and the magnetic depletion procedure was repeated on
this fraction.
The bead-free cells were then preincubated with Fc RII block (clone
2.4G2; Pharmingen, San Diego, CA) for 10 minutes on ice and then
stained with biotinylated anti-Ly6A/E (Sca-1, rat IgG2a, clone
E13-161.7) and fluorescein isothiocyanate (FITC)-conjugated anti-CD117
(c-kit, rat IgG2b, clone 2B8) MoAbs (Pharmingen) for 30 minutes
on ice. Aliquots of the bead-free cells were also stained with
isotype-matched controls. The cells were then washed with 10 mL
PBS/FCS, stained with streptavidin-phycoerythrin (Pharmingen) for 30 minutes on ice, washed, and resuspended in PBS/FCS containing 1 mg/mL
propidium iodide. The sample was filtered through a 70-µm pore-size
nylon screen.
Fluorescence-activated cell sorting was performed on a Vantage (Becton
Dickinson, Mountain View, CA), using the CloneCyt direct cloning application (Becton Dickinson). Cells sorted were propidium iodide (PI)-negative, c-kit+,
Sca-1+, with intermediate forward- and right-angle
scatters.
For 7-day primary culture, 50 purified precursors were added to a
single well of a round-bottom 96-well plate (Corning, Corning, NY)
containing beads (about 1,000 beads per well) and culture medium
(Iscove's medium, 20% FCS) plus cytokines (stem cell factor [SCF], Flt3-ligand, interleukin-6
[IL-6]) (each at 100 ng/mL), and IL-11 (10 ng/mL). At 7 days of
culture, cells were removed and 1/12 (approximately 10,000 cells) of
the culture was replated into a 35-mm culture dish (Nunc, Naperville,
IL) containing semisolid medium (40%  -MEM-based methylcellulose
[Stem Cell Technologies, Vancouver, BC, Canada], 10% WEHI
conditioned medium,35 30% FCS, and 1 × 104 mol/L 2-mercaptoethanol [2-ME], 100 ng/mL IL-6
and SCF, 50 ng/mL megakaryocyte growth and development factor
[MGDF], and 3 U/mL erythropoietin).
After 10 days of culture in semisolid medium, colonies were viewed with
an inverted microscope and colony types determined and counted.
Colonies were designated HPP-mix if they consisted of granulocytes,
macrophages, and erythroid clusters and were larger than 1.5 mm.
Colonies were evaluated by preparing cytocentrifuge slides. In each
colony evaluated the presence of each of these cell types as well as
megakaryocytes was confirmed. A mean number of all colony types was
determined for all 10 wells and then a final mean for five experiments.
Statistical differences were assessed using analysis of
variance.36
For time-course experiments, 200 purified precursors were added to a
single well of a round-bottom 96-well plate containing beads and
culture medium as described above. In experiment 1 there were 8 replicate wells and in experiment 2 there were 10. After primary
culture for 5 and 8 days, cells were harvested, counted, and 25,000 cells were replated with fresh beads for further culturing. At the same
time 1/12 of the culture (approximately 10,000 cells) was replated into
35-mm plates containing semisolid medium so that there were about 50 colonies in each plate. Each well was plated in this manner in
quadruplicate. After 10 days in secondary culture, types and size of
colonies were assessed.
For replating experiments all HPP-mix colonies from day 0, 5, and
8 primary cultures were picked and replated into fresh semisolid medium. After 5 to 7 days in the tertiary cultures, types and size of
colonies were assessed.
To analyze Notch-1 or Notch-2 expression, suspensions
of BM cells were enriched for low-density cells as described above. Cells were washed twice, then fixed and permeabilized by incubation with PermeaFix (Ortho, Raritan, NJ), washed twice, and then incubated at room temperature with the rat anti-human Notch-1 antibody 18G or
anti-human Notch-2 antibody BHN6. Cells were washed twice and incubated
at 4°C with mouse-absorbed FITC-conjugated monoclonal mouse
anti-rat IgG1 antibody (Pharmingen). In addition, whole marrow was
enriched for hematopoietic precursor cells by magnetic depletion using
lineage-specific antibodies as described above. Enriched cells were
then stained with anti-Ly6A/E (Sca-1) directly conjugated to
phycoerythrin (Pharmingen) and anti-CD117 (c-kit) directly
conjugated to biotin (Pharmingen). Cells were washed twice and
incubated with RED613 conjugated to streptavidin (GIBCO-BRL, Grand
Island, NY) and again washed twice. Cells were then fixed and
permeabilized and stained with either Notch-1 or Notch-2 antibodies as
previously described. Control staining was measured using isotype matched directly labeled or unlabeled control antibody. Cells were
analyzed using a Vantage flow cytometer (Becton Dickinson). Cells with
a surface antigen fluorescence intensity greater than 99% of control
staining were considered positive while cells with a surface antigen
fluorescence intensity less than the top 5% of control staining were
considered negative.
Stromal cells.
Methods are previously described for culturing fetal liver
stroma.28 Briefly, fetal livers at day 14 of gestation from
C57Bl/6J were dissociated mechanically with repeated pipetting and
passed through a 70-µm nylon filter. The retentate was cultured in
5% CO2 in modified Dexter media (DMEM, 10% FCS, 10%
horse serum, 50 µmol/L 2-ME, 0.1 µmol/L hydrocortisone). Murine
stromal cell lines derived from fetal liver were generated and
maintained as previously described.28,29
To culture BM stroma, suspensions of BM cells were
obtained by flushing femurs from two C57Bl/6J mice and two Balb-C mice. Cells were cultured at an initial density of 2 to 3 × 107 in a 25-cm culture flask in 5% CO2 in
long-term culture medium (Iscove's medium, 12.5% FCS, 12.5% horse
serum, 2% MEM-amino acids [GIBCO], 1% MEM-nonessential amino acids
[GIBCO], 1% MEM-vitamins [GIBCO], 106 mol/L
hydrocortisone, 101 mol/L 2-ME, 24 mmol/L
NaHCO3, 100 mmol/L Na-Pyruvate). Cultures were harvested at 3 weeks and lysates prepared for Western
blotting.
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RESULTS |
Expression of Notch by murine hematopoietic cells.
To assess Notch expression in hematopoietic cells, whole-mouse
marrow and precursors
(LinSca-1+c-kit+)
were analyzed with flow cytometry, using MoAbs that specifically recognize Notch-1 (18G) and Notch-2 (BHN6)
(Fig 2). Although little or no Notch-1 was
seen in whole marrow, Notch-1 was detected in precursors
(LinSca-1+c-kit+).
Notch-2 was detected in whole marrow and, as with Notch-1, more was
seen in precursors.
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| Fig 2.
Expression of Notch-1 and Notch-2 by
mouse BM cells. (A) Fluorescence histograms of
Ficoll-Hypaque-separated marrow cells stained with MoAbs that
recognize Notch-1 (18G) or Notch-2 (BHN6). (B) Fluorescence histograms
of precursor cells
(LinSca-1+c-kit+)
stained with the same MoAbs as in (A). The x-axis represents log
fluorescence intensity and the y-axis represents cell number. The solid
line represents staining with 18G or BHN6 antibodies and the dashed
line represents staining with an isotype-matched nonspecific
antibody.
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Generation of a 3T3 cell line expressing Jagged-1.
Fibroblast cell lines expressing Jagged-1 were generated by
infecting a parental NIH-3T3 cell line with a retrovirus vector containing full-length human Jagged-1. Sequences encoding an
myc epitope recognized by an MoAb (9E10) were added to the
3 end of Jagged-1 (Fig 1). Two of five G418-resistant
3T3 clones expressed ligand detectable by Western analysis using 9E10
as a probe (data not shown). To confirm cell-surface expression of
Jagged-1, cells were biotinylated and lysates were prepared and
immunoprecipitated with 9E10. A band of molecular weight
(Mr) 150 kD, the expected Mr for Jagged-1, was seen in
the expressing line but not in the parental 3T3 cell line
(Fig 3A).
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| Fig 3.
Immunoprecipitates of biotinylated 3T3 cells to detect
surface expression of Jagged (A) and Westerns of hematopoietic
cell lysates to detect expression of Jagged-1 (B, C, and D).
(A) Lysates were prepared from 3T3 cells infected with a retroviral
vector containing sequences encoding full-length human Jagged-1 (lane 1) or parental 3T3 cells (lane 2) that had been previously biotinylated to label surface proteins. Lysates were immunoprecipitated with 9E10
antibody and resulting proteins were separated on an 8% SDS-PAGE, transferred to nitrocellulose, and probed with HRP-streptavidin and
developed using ECL. (B) Protein lysates were prepared from a 3T3 cell
line expressing Jagged-1 (lane 1), a parental 3T3 cell line
(lane 2 ), and murine hematopoietic stromal cell lines AFT024, CFC034,
2058, 2012, and 2018 (lanes 3 through 7, respectively). (C) Protein
lysates were prepared from a 3T3 cell line expressing Jagged
(lane 1), primary cultured murine fetal liver stoma (lane 2), murine BM
cells (lane 3), primary cultured BM stroma from C57BL/6J (lane 4), and
from Balb-C mice (lane 5). (D) Protein lysates were prepared from a 3T3
cell line expressing Jagged (lane 1), Ficoll-Hypaque-separated
murine BM cells (lane 2), and sorted precursors
(LinSca-1+c-kit+)
(lane 3). For gels in (B), (C), and (D), proteins were separated with
either an 8% SDS-PAGE (B and D) or a 6% SDS-PAGE (C), transferred to
nitrocellulose, and probed with SER10, a polyclonal antibody raised
against C-Serrate 1.
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Expression of Jagged-1.
To assess expression of Jagged-1 in hematopoietic cells,
proteins from marrow cells and stroma, fetal liver stroma, and
hematopoietic stromal cell lines derived from fetal liver were analyzed
by Western blot using a rabbit polyclonal antibody (SER10) that was
raised against the cytoplasmic domain of C-Serrate-1. In 3T3 cells
expressing Jagged-1, a band of Mr 150 kD, the correct molecular
weight for Jagged-1, was detected (Fig 3B), whereas no bands were
detected in the parental 3T3 cell line. In addition, a clear band of Mr 150 kD was seen in mouse hematopoietic stromal cell lines AFT024, CFC034, 2058, 2012, and 2018 derived from fetal liver (Fig 3B). Also, a
band of Mr 150 kD was seen in lysates prepared from cultured primary
fetal liver stroma and cultured primary BM stroma from two different
strains of mice (Fig 3C). However, a 150-kD band was not seen in whole
mouse marrow (Fig 3C, lane 3), Ficoll-Hypaque separated marrow, or
purified precursors
(LinSca-1+c-kit+,
Fig 3D).
The effect of cell-bound Jagged-1 on mouse marrow progenitor cells.
To assess the effect of Jagged-1 on marrow precursor cells, 100 enriched mouse marrow
LinSca-1+c-kit+
cells were sorted into wells containing either mitomycin-treated 3T3
cell layers expressing Jagged-1 or mitomycin-treated parental cell layers. Cultures were supplemented with IL-11, IL-6, SCF, and
Flt3-ligand. After 7 days of incubation in primary culture, hematopoietic cells were harvested from individual wells and replated in secondary culture with semisolid medium containing multiple growth
factors. Similar numbers of cells were found in cultures containing
cell layers expressing Jagged-1 compared to cultures with
parental 3T3 cell layers (5.4 × 105 cells/well ± 0.16 × 105 compared to 3.3 × 105
cells/well ± 0.6 × 105, respectively, mean for
three experiments ± SEM). There was no difference in the types or
numbers of mature myeloid cells found after 7 days in primary culture,
as shown by flow analysis using antibodies that recognize granulocyte
(Gr-1) and macrophage (Mac-1) associated antigens (data not shown).
However, cell-bound Jagged-1 affected the generation or maintenance of
a very early precursor cell, HPP-mix. HPP-mix colonies consisted of
cells derived from multiple lineages, including granulocytes,
macrophages, and erythroid clusters and were larger than 1.5 mm in
diameter. There were about four times more HPP-mix colonies formed from
cultures previously incubated with layers expressing Jagged-1
compared to those without Jagged-1
(Fig 4A). However, there were very few
HPP-mix remaining in either group, suggesting that under both
conditions most HPP-mix had developed into CFU-C. Indeed,
cells from the 3T3-Jagged-1 cultures produced four times more
colonies of all types compared to cells cultured with parental layers
(Fig 4B, P < .05).
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| Fig 4.
Effect of Jagged exogenously expressed from 3T3
fibroblasts on purified hematopoietic precursors. Enriched mouse marrow
cells (LinSca-1+c-kit+)
were added to wells containing cytokines (IL-11, IL-6, SCF, and
Flt3-ligand) and either mitomycin-treated Jagged expressing 3T3 cell
layers ( ) or mitomycin-treated parental cell layers ( ). After 7 days of incubation, hematopoietic cells were obtained from individual
wells and replated in semisolid medium containing growth factors. After
10 days of incubation, the colony types were counted and the mean
number of colonies per well was derived for each experiment. Each bar
represents the mean number of HPP-mix (A) or CFU-C (B) colonies per
well from three separate experiments ± SEM. HPP-mix refers to
colonies larger than 1.5 mm in diameter and consisting of multiple
lineages including at least granulocyte/macrophage and erythroid
clusters.
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The effect of Jagged-1ext on mouse marrow progenitor
cells.
Because the outcome of coculture experiments could be influenced by
possible differences between the 3T3-Jagged-1 cell lines and
the parental 3T3 cell line in the types or amounts of cytokines made,
adhesion molecules expressed, or other factors unrelated to
Jagged-1 expression, we examined the effect of a
Jagged-1ext, consisting of the extracellular domain of
Jagged-1, on hematopoietic precursor cells. An myc tag epitope
and six histidines were added to the carboxy terminus of the molecule
to aid in purification. Jagged-1ext was purified from the
supernatant of transiently transfected COS cells using a nickle column.
Because several lower molecular weight contaminants were observed,
further affinity purification was necessary. Hence, a fraction from the
nickle column containing Jagged-1ext was added to Sepharose
beads crosslinked with 9E10. To avoid possible denaturation of
Jagged-1ext after dissociation from the antibody, cells
were incubated directly with the Jagged-1ext-9E10-Sepharose
beads. Supernatant from mock-transfected COS cells was treated with the
same purification protocol and added to the 9E10 crosslinked Sepharose
beads as a control.
To quantitate the effect of Jagged-1ext on hematopoietic
precursor cells, 50 enriched mouse
LinSca-1+c-kit+
marrow cells were deposited into wells containing either
Jagged-1ext-9E10-beads or control-9E10-beads in the
presence of IL-11, IL-6, SCF, and Flt3-ligand. After 7 days of
incubation in primary culture, cells from each well were obtained and
plated in semisolid medium to assess clonogenicity. In primary
cultures, no difference in cell number was seen between the two culture
conditions (data not shown). By 7 days there were between 1 and 5 × 105 cells/well in both culture conditions. Because
there were too few cells for flow analysis, the types of mature cells
found in the primary cultures were assessed by preparing
cyto-centrifuge slides followed by Wright-Giemsa staining. Most mature
cells were macrophages and granulocytes and no obvious differences in
the distribution of these types of cells were detected between cells removed from the two conditions (data not shown). However, there were
twice as many HPP-mix colonies formed from cultures incubated with
Jagged-1ext than with control proteins
(Fig 5A, P < .025). In addition,
in four of five experiments, wells with Jagged-1ext beads
contained more CFU-C than control wells (Fig 5B). The mean increase in
CFU-C was 21%.
View larger version (16K):
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[in a new window]
| Fig 5.
Effect of Jagged coated beads on purified hematopoietic
precursors. Enriched mouse marrow cells
(Linsca-1+c-kit+)
were added to wells containing cytokines (IL-11, IL-6, SCF, and
Flt3-ligand) and beads coated with Jagged-1ext () or
control beads (). After 7 days of incubation cells were harvested
and replated in semisolid medium containing growth factors. After 10 days of incubation the colony types were quantitated and a mean number
of colonies per well was derived from each experiment. Each bar
represents the mean number of HPP-mix (A) or CFU-C (B) colonies per
well from five separate experiments ± SEM. HPP-mix refers to colonies
larger than 1.5 mm in diameter and consisting of multiple lineages
including at least granulocyte/macrophage and erythroid clusters.
|
|
Time course of precursor cell generation.
To assess whether Jagged-1ext altered the day of maximal
expression of CFU-C or HPP-mix, 200 precursor cells
(LinSca-1+c-kit+)
were plated in suspension culture in the presence of IL-11, IL-6, SCF,
and Flt3-ligand either with Jagged-1ext-9E10-beads or with
control-9E10-beads. To maintain density in the primary culture at below
1 × 106 cells/mL during the course of the experiment,
cell numbers were reduced on days 5 and 8 by replating 25,000 cells to
a new well with fresh medium and fresh Jagged-1ext or
control beads. To measure clonogenicity, an aliquot of cells was
replated in semisolid medium on 0, 5, 8, and 11 days of culture. Jagged-1ext had no effect on the number of cells generated
(Fig 6A). However, cultures with
Jagged-1ext produced more HPP-mix than did cultures without
Jagged-1ext throughout the course of the experiment (Fig
6B). The largest differences were observed at days 8 and 11. In
addition, Jagged-1ext affected CFU-C. In both conditions,
the number of CFU-C increased until about day 8, when their numbers
stopped increasing and started to decrease (Fig 6C). At day 5 there
were similar numbers of CFU-C in both conditions, whereas by day 8 there were about 25% more CFU-C in wells containing
Jagged-1ext and at day 11 there was 1.5- to 3-fold more
CFU-C in the cultures containing Jagged-1ext.
View larger version (35K):
[in this window]
[in a new window]
| Fig 6.
Effect of Jagged-1ext coated beads on the
time course of formation of clonogenic cells. Two hundred enriched
mouse marrow cells (Linsca-1+c-kit+)
were added to wells containing cytokines (IL-11, IL-6, SCF, and
Flt3-ligand) and beads coated with Jagged-1ext () or
control beads ( ). Cells were removed at time intervals and replated
in semi-solid medium. After 10 days the total number of cells per 200 starting cells (A), HPP-mix (B), and CFU-C (C) were determined for each
well. Each point represents the mean from 8 wells in experiment 1 and
10 wells in experiment 2.
|
|
Secondary colony formation.
We replated HPP-mix colonies derived from the primary cultures
incubated with Jagged-1ext or control beads. Because all
colonies from the two conditions were replated, there were more HPP-mix
replated from wells where Jagged-1ext was in the primary
culture and hence there were more HPP-mix that were replated at the two
time points that were evaluated. However, there was very little
difference in the characteristics of the HPP-mix colonies derived from
the two types of cultures. The percentage of colonies which replated
into granulocyte/macrophage or erythroid colonies was the same from the
two conditions and the number of granulocyte/macrophage or erythrocyte
colonies formed in the tertiary culture was also roughly the same
(Table 1).
| |
DISCUSSION |
Previously it was shown that Notch-1 mRNA was detected in human
precursors (CD34+ Lin), indicating a
potential role for Notch interactions in hematopoiesis.11 In the present study we further assess protein expression of both Notch-1 and Notch-2 in murine whole marrow and in
precursors. Although little or no Notch-1 was seen in whole marrow, it
was detected in precursors. Notch-2 was detected in whole marrow and, again, more was seen in precursors. Interestingly, more Notch-2 was
detected in murine marrow than Notch-1. Until other antibodies are used
to compare Notch-1 and Notch-2 expression, it is unclear if this result
is caused by differences between the two antibodies used rather than
differences in Notch-1 and Notch-2 protein levels. However, these data
indicate clearly that Notch protein is expressed by murine
hematopoietic precursors, supporting previous suggestions of a
potential role for Notch in hematopoiesis.
In this report we also examine expression of the Notch ligand
Jagged-1 in mouse hematopoietic cells. Interestingly, no
detectible Jagged-1 was seen in whole marrow,
Ficoll-Hypaque-separated marrow, or in sorted precursor cells
(LinSca-1+c-kit+).
However, Jagged-1 was detected in murine stroma and in stromal cell
lines, again supporting suggestions for a potential role for
Notch-Notch-ligand interactions in hematopoiesis. Additionally, a
recent paper reported Jagged-1 expression in human stromal cell lines that supported hematopoiesis.37
To address whether Notch has a role in hematopoiesis, we determined the
effect of Notch-ligand interactions on primitive hematopoietic precursors using transfected fibroblasts expressing the full-length human Jagged-1. Previously it was shown that L-cell fibroblasts expressing full-length rat Jagged-1 activated full-length
Notch-1 expressed in cells of a C2 myoblast cell line and
inhibited differentiation of those cells.17 In this report
we have transfected a similar fibroblast cell line with a cDNA encoding
full-length human Jagged-1. Murine Jagged-1 has not yet
been cloned. However, rat Jagged-1 is very similar to human
Jagged-1 (95% of the amino acids are identical). In fact, the DSL
domain, a region deemed critical for Notch function, is 98% identical
between rat and human, differing by only one amino acid. Therefore, it
is likely that human Jagged-1 would produce comparable effects to
murine Jagged-1.
When we cocultured sorted murine precursor cells
(LinSca-1+c-kit+)
that endogenously express Notch-1 and Notch-2 with
fibroblasts expressing human Jagged-1, we found increased
numbers of CFU-C and of a primitive precursor, HPP-mix. The primitive
nature of these cells was shown by their formation of colonies of
macroscopic size indicative of high proliferative potential, their
multi-lineage potential, and their high replating efficiency. These
results suggest that Notch ligand promotes the maintenance or expansion of precursor cells in culture presumably by activating the receptor in
these cells.
Because the outcome of coculture experiments could be influenced by
possible differences between the 3T3-Jagged-1 cell lines and
the parental 3T3 cell line in the types or amounts of cytokines made,
adhesion molecules expressed, or other factors unrelated to
Jagged-1 expression, we further examined the effect of the extracellular domain of the Jagged-1 molecule on hematopoietic precursors. In this report we have used Jagged-1ext bound
to a substrate, consisting of beads covalently linked to an antibody
that recognizes the ligand. We found the same effect on hematopoietic
precursors with ligand bound beads as 3T3 cells expressing Jagged-1. In
both cases, we saw an increase in the numbers of precursors including
total CFU-C and HPP-mix. These results would suggest that in this case,
Jagged-1ext bound to beads-activated Notch.
Previous analyses involving truncated forms of Notch ligand would not
necessarily predict such an outcome. In C elegans it was shown
that soluble ligand (Apx 1) exogenously expressed in vivo rescued a
ligand null mutant, indicating that the soluble ligand is able to
activate the C elegans Notch homologs Lin-12 and Glp-1.38 It was also possible to activate the
Notch homologs in some but not all developmental processes in
C elegans using a construct that expressed only the DSL domain.
In other studies it was shown that a soluble form of human Jagged-1
inhibited the differentiation of 32D cells that were exogenously
expressing murine Notch-1.37 However, the opposite
result was found in Drosophila, where it was shown that the
soluble ligand (Serrate or Delta) or membrane-bound truncated ligand
exogenously expressed in vivo inhibits Notch
activity.25,26 In addition, exogenous expression of the
extracellular domain plus the transmembrane domain of Xenopus
or chicken Delta also inhibits Notch activity during
Xenopus eye development or chicken retinal development, respectively.27,28 In our situation, it is conceivable that the binding of Jagged-1ext to bivalent antibody on the
beads may affect the structure in such a way that it can mimic the
wild-type ligand and thereby activate Notch.
We saw no effect on the types of mature cells formed in our cultures,
although we have detected Notch expression in developing myeloid cells and mature monocytes (Flowers et al,
submitted). It is possible that Jagged-1 is unable to
activate Notch on these cell types whereas Jagged-2, Delta-1,
or Delta-3 may be able to activate Notch. It has been shown
that in wing development in Drosophila, Notch
activation by Serrate is inhibited by the presence of Fringe, a
secreted protein, whereas activation by Delta is not
inhibited.39 Consistent with this is a recent report
showing the expression of Fringe in hematopoietic
cells.40
Overall, our studies show that interactions of Notch expressed on the
surface of isolated hematopoietic precursors with its ligand results in
increased numbers of a primitive precursor, HPP-mix. These results may
indicate that Notch interactions may promote self-renewal of HPP-mix or
it may promote self-renewal of an even earlier precursor cell, the
transplantable stem cell. Experiments are now in progress to test these
possibilities.
| |
FOOTNOTES |
Submitted October 23, 1997;
accepted January 17, 1998.
Supported in part by National Institutes of Health (NIH) Grants No.
HL54881, NS26084, and the Howard Hughes Medical Institute. I.L.R. is
supported by the Human Capital Mobility Program No. CHBICT930657.
K.A.M. is supported by NIH Grant No. HL58739-01 and National Cancer
Institute Grant No. DHP-144/01. I.D.B. is also supported
as a Clinical Research Professor by the American Cancer Society.
Address reprint requests to Irwin D. Bernstein, MD,
Pediatric Oncology, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, C1-169, Seattle, WA 98109-1024.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank John E.J. Rasko and Robert G. Andrews for reading the
manuscript and for helpful suggestions. We thank David Ish-Horowitz for
helpful discussions and help in preparing SER10.
| |
REFERENCES |
1.
Fairbairn LJ,
Cowling GJ,
Reipert BM,
Dexter TM:
Suppression of apoptosis allows differentiation and development of a multipotent hemopoietic cell line in the absence of added growth factors.
Cell
74:823,
1993[Medline]
[Order article via Infotrieve]
2.
Simpson P:
The Notch connection.
Nature
375:736,
1995[Medline]
[Order article via Infotrieve]
3.
Nye JS,
Kopan R:
Vertebrate ligands for Notch.
Curr Biol
5:966,
1995[Medline]
[Order article via Infotrieve]
4.
Artavanis-Tsakonas S,
Matsuno K,
Fortini ME:
Notch signaling.
Science
268:225,
1995[Abstract/Free Full Text]
5.
Heitzler P,
Simpson P:
The choice of cell fate in the epidermis of Drosophila.
Cell
64:1083,
1991[Medline]
[Order article via Infotrieve]
6.
Doherty D,
Feger G,
Younger-Shepherd S,
Jan LY,
Jan YN:
Delta is ventral to dorsal signal complimentary to Serrate, another Notch ligand in Drosophila wing formation.
Genes Dev
10:421,
1996[Abstract/Free Full Text]
7.
Cagan RL,
Ready DF:
Notch is required for successive cell decisions in the developing Drosophila retina.
Development
121:2407,
1989[Abstract]
8.
Ruohola H,
Bremer KA,
Baker D,
Swedlow JR,
Jan LY,
Jan YN:
Role of neurogenic genes in establishment of follicle cell fate and oocyte polarity during oogenesis in Drosophila.
Cell
66:433,
1991[Medline]
[Order article via Infotrieve]
9.
Greenwald I,
Rubin GM:
Making a difference: The role of cell-cell interactions in establishing separate identities for equivalent cells.
Cell
68:271,
1992[Medline]
[Order article via Infotrieve]
10.
Dorsky RI,
Chang WS,
Rapaport DH,
Harris WA:
Regulation of neuronal diversity in the Xenopus retina by Delta signalling.
Nature
385:67,
1997[Medline]
[Order article via Infotrieve]
11.
Milner LA,
Kopan R,
Martin DI,
Bernstein ID:
A human homolog of the Drosophila developmental gene, Notch, is expressed in CD34+ hematopoietic precursors.
Blood
83:2057,
1994[Abstract/Free Full Text]
12.
Ellison LW,
Bird J,
West DC,
Soreng AL,
Reynolds TC,
Smith SD,
Sklar J:
TAN-1, the human homolog of Drosophila Notch gene, is broken by chromosomal translocations in T lymphoblastic neoplasms.
Cell
66:649,
1991[Medline]
[Order article via Infotrieve]
13.
Weinmaster G,
Roberts VJ,
Lemke G:
A homolog of Drosophila Notch is expressed during mammalian development.
Development
113:199,
1991[Abstract]
14.
Weinmaster G,
Roberts FJ,
Lemke G:
Notch2: A second mammalian Notch gene.
Development
116:931,
1992[Abstract]
15.
Lardeli M,
Dahlstrand J,
Lendahl U:
The novel Notch homolog mouse Notch3 lacks specific epidermal growth factor-repeats and is expressed in proliferating neuroepithelium.
Mech Dev
46:123,
1994[Medline]
[Order article via Infotrieve]
16.
Uytetendaele H,
Marazzi G,
Wu G,
Yan Q,
Sassoon D,
Kitajewski J:
Notch4/int-3, a mammary proto-oncogene, is an endothelial cell-specific mammalian Notch gene.
Development
122:2251,
1996[Abstract]
17.
Lindsell CE,
Whawber CJ,
Boulter J,
Weinmaster G:
Jagged: A mammalian ligand that activates Notch1.
Cell
80:909,
1995[Medline]
[Order article via Infotrieve]
18.
Myat A,
Henrique D,
Ish-Horowicz,
Lewis J:
A chick homologue of Serrate and its relationship with Notch and Delta homologues during central neurogenesis.
Dev Biol
174:23,
1996
19.
Shawber C,
Boulter J,
Lindsell CE,
Weinmaster G:
Jagged2: A Serrate-like gene expressed during rat embryogenesis.
Dev Biol
180:370,
1996[Medline]
[Order article via Infotrieve]
20.
Luo B,
Aster JC,
Hasserjian RP,
Kuo F,
Sklar J:
Isolation and functional analysis of a cDNA for human Jagged2, a gene encoding a ligand for the Notch1 receptor.
Mol Cell Biol
17:6057,
1997[Abstract]
21.
Henrique D,
Adam J,
Myat A,
Chitnis J,
Lewis J,
Ish-Horowicz:
Expression of a Delta homologue in prospective neurons in the chick.
Nature
375:787,
1995[Medline]
[Order article via Infotrieve]
22.
Bettenhausen B,
de Angelis MH,
Simon D,
Guenet JL,
Gossler A:
Transient and restricted expression during mouse embryogenesis of DLL1, a murine gene closely related to Drosophila Delta.
Development
121:2407,
1995
23.
Dunwoodie SL,
Henrique D,
Harrison SM,
Beddington RSP:
Mouse Dll3: A novel divergent Delta gene which may complement the function of other Delta homologues during early pattern formation in the mouse embryo.
Development
124:3065,
1997[Abstract]
24.
Tax FT,
Yeargers JJ,
Thomas JH:
Sequence of C. elegans Lag-2 reveals a cell signaling domain shared with Delta and Serrate of Drosophila.
Nature
368:150,
1994[Medline]
[Order article via Infotrieve]
25.
Sun X,
Artavanis-Tsakonas S:
Secreted forms of Delta and Serrate define antagonists of Notch signaling in Drosophila.
Development
124:3439,
1997[Abstract]
26.
Hukriede NA,
Gu Y,
Fleming RJ:
A dominant-negative form of Serrate acts as a general antagonist of Notch activation.
Development
124:3427,
1997[Abstract]
|
Abstract
Introduction
Abstract