1 Department of Immunology, Medical Faculty/University Clinics Ulm,
Germany
2 Carl C. Icahn Center for Gene Therapy and Molecular Medicine, Mount Sinai
School of Medicine, New York, NY 10029, USA
3 Department of Molecular Biology, UT Southwestern Medical Center, Dallas, TX
75390, USA
* Author for correspondence (e-mail: gordon.keller{at}mssm.edu)
Accepted 12 May 2003
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SUMMARY |
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Key words: ES cell, Brachyury, Mesoderm, Hemangioblast
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INTRODUCTION |
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Direct demonstration for the existence of a progenitor with hemangioblast
properties has been provided by experiments using a model system based on the
in vitro differentiation potential of embryonic stem (ES) cells
(Choi et al., 1998;
Nishikawa et al., 1998
).
Following the initiation of differentiation in culture, ES cells will form
colonies known as embryoid bodies (EBs), that generate hematopoietic and
endothelial progeny in a temporal pattern recapitulating the development of
these populations in the yolk sac (Keller
et al., 1993
; Palis et al.,
1999
; Vittet et al.,
1996
). Analysis of early EBs, prior to the hematopoietic and
endothelial commitment stages, revealed the presence of a progenitor with
hemangioblast potential. In response to VEGF, these progenitors generate blast
colonies that display both hematopoietic and endothelial potential
(Choi et al., 1998
). Kinetic
studies demonstrated that this progenitor or blast colony-forming cell
(BL-CFC) represents a transient population that is present within the EBs for
approximately 36 hours, between day 2.5 and 4 of differentiation, preceding
the onset of primitive erythropoiesis. The characteristics of the BLCFC,
namely its early development and its potential to generate primitive and/or
definitive hematopoietic as well as endothelial progeny, suggests that it
represents the in vitro equivalent of the yolk sac hemangioblast. More recent
studies have shown that most BL-CFC express Flk1 (VEGF receptor 2; Kdr
Mouse Genome Informatics)) and that a subpopulation of Flk1+ cells
also expresses the transcription factor Scl
(Chung et al., 2002
;
Faloon et al., 2000
). The early
development of the BL-CFC, prior to hematopoietic commitment, suggests that it
could be a direct descendent of a mesodermal progenitor, or possibly a
subpopulation of mesoderm. In the ES/EB model system, mesoderm, as defined by
expression of the T-box gene brachyury, is induced within 48 hours of the
onset of differentiation and persists until day 4
(Robertson et al., 2000
). This
mesodermal window overlaps with the onset of Flk1 expression, initiated as
early as day 2.5 of differentiation and with the BL-CFC stage of development,
typically found between day 2.5 and 4.0 of differentiation.
In the mouse embryo, mesoderm is generated from the epiblast or embryonic
ectoderm through the process of gastrulation that is initiated at
approximately day 6.5 of gestation (reviewed by
Tam and Behringer, 1997). At
the onset of gastrulation, the epiblast cells in the region that defines the
posterior part of the embryo undergo an epithelial to mesenchymal transition
and form a transient structure known as the primitive streak from which the
mesoderm emerges. The newly formed mesoderm migrates away from the primitive
streak, moves laterally and anteriorly and is patterned into various
populations with distinct developmental fates. Brachyury is expressed in all
nascent mesoderm and downregulated as these cells undergo patterning and
specification into the derivative tissues including skeletal muscle, cardiac
muscle and connective tissues in addition to blood and endothelium
(Herrmann, 1991
;
Kispert and Herrmann,
1994
).
The first mesodermal cells to develop within the embryo contribute
predominantly to the extra-embryonic tissues, giving rise to the hematopoietic
and vascular cells of the yolk sac (Kinder
et al., 1999). Hematopoietic progenitors are first found in the
developing yolk sac as early as day 7.0 of gestation,
12 hours after the
beginning of gastrulation (Palis et al.,
1999
). Flk1 is expressed in the yolk sac at this stage and is
essential for the establishment of the blood cell and vascular lineages
(Schuh et al., 1999
;
Shalaby et al., 1995
).
Although the yolk sac hemangioblast has not yet been identified, the rapid
commitment to the hematopoietic and endothelial lineages following the
induction of mesoderm suggests that this putative in vivo progenitor should
also be closely related to mesoderm.
Our understanding of hematopoietic and endothelial development has been
greatly enhanced by the identification and characterization of cell
populations representing the earliest stages of commitment towards these
lineages (Nishikawa et al.,
1998; Chung et al.,
2002
; Faloon et al.,
2000
; Lacaud et al.,
2002
; Robertson et al.,
2000
). By contrast, however, developmental stages earlier than the
hemangioblast remain difficult to study as there are few known cell surface
markers that enable one to isolate these populations or subsets of progenitors
within these populations. To access prehemangioblast cell populations and
define their relationship with respect to the BL-CFC, we targeted the GFP cDNA
to the brachyury locus. In this report, we show that GFP is an effective
marker for the mesodermal populations that develop within the EBs. Analysis of
GFP and Flk1 expression led to the identification of three subpopulations
GFPFlk1,
GFP+Flk1 and GFP+Flk1+ that
represent a developmental progression from pre-mesodermal cells to the
hemangioblast.
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MATERIALS AND METHODS |
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Generation of brachyury/EGFP knock-in ES cells
Mouse embryonic stem cells (E14.1, 129/Ola) were electroporated with the
NotI-linearized targeting vector. Clones that had undergone a
homologous recombination event were identified by PCR with one primer
(5'-CAGGTAGAACCCACAACTCCGAC-3') annealing to genomic sequences in
the 5' region of the brachyury gene, upstream of the `short arm of
homology', the other primer (5'-CCGGACACGCTGAACTTGTGGC-3') to the
5' region of EGFP. Correctly targeted clones were confirmed by Southern
blot analysis. Out of 384 singly selected and 80 doubly selected colonies,
four and three correctly targeted clones were identified, respectively. Two
positive clones (#164 and #201) were transiently transfected with a modified
Cre recombinase expression vector (H.J.F., unpublished) to excise the
neo gene. Neo-deficient clones were identified due to loss of G418
resistance. The intactness of the targeted locus before and after Cre-mediated
excision of Neo was confirmed by Southern blot analysis. The absence of the
neo cassette in Cre-treated G418-sensitive clones was verified by
Southern blotting using the Neo cassette as probe (not shown).
ES cell growth and differentiation
ES cells were maintained on irradiated embryonic feeder cells in Dulbecco's
Modified Eagle Medium (DMEM) supplemented with 15% fetal calf serum (FCS),
penicillin, streptomycin, LIF (1% conditioned medium) and
1.5x10-4 M monothioglycerol (MTG; Sigma). Two days prior to
the onset of differentiation, cells were transferred on gelatinized plates in
the same media. For the generation of EBs, ES cells were trypsinized and
plated at various densities in differentiation cultures. Differentiation of
EBs was carried out in 60 mm petri grade dishes in IMDM supplemented with 15%
FCS, 2 mM L-glutamine (Gibco/BRL), transferrin (200 µg/ml), 0.5 mM ascorbic
acid (Sigma), and 4.5x10-4 M MTG. For reaggregation, sorted
cells were cultured in the media used to differentiate EBs. Cells were
cultured for 20 hours at a density of 4x105/ml in ultra low
attachment 24-well plates (Costar). Cultures were maintained in a humidified
chamber in a 5% CO2/air mixture at 37°C.
Colony assays
For the generation of blast cell colonies (BL-CFC assay), EB cells were
plated in 1% methylcellulose supplemented with 10% FCS (Summit), vascular
endothelial growth factor (VEGF; 5 ng/ml), Kit ligand (KL; 1% conditioned
medium), IL6 (5 ng/ml) and 25% D4T endothelial cell conditioned medium
(Kennedy et al., 1997). For
the growth of hematopoietic precursors, cells were plated in 1%
methylcellulose containing 10% plasma-derived serum (PDS; Antech), 5%
protein-free hybridoma medium (PFHM-II; Gibco-BRL) and the following
cytokines: KL (1% conditioned medium), TPO (5 ng/ml), erythropoietin (2 U/ml),
IL11 (25 ng/ml), IL3 (1% conditioned medium), GM-CSF (3 ng/ml), G-CSF (30
ng/ml), M-CSF (5 ng/ml) and IL6 (5 ng/ml). Cultures were maintained at
37°C, 5% CO2. LIF and Kit ligand were derived from media
conditioned by CHO cells transfected with LIF and KL expression vectors,
respectively (kindly provided by Genetics Institute). IL3 was obtained from
medium conditioned by X63 AG8-653 myeloma cells transfected with a vector
expressing IL3 (Karasuyama and Melchers,
1988
). VEGF, GM-CSF, M-CSF, G-CSF, TPO, IL6 and IL11 were
purchased from R&D Systems.
Neuronal differentiation
Both GFP positive and negative populations were isolated from day 2.5 EBs
by cell sorting. Pre-sort and sorted cells were reaggregated at 105
cells/ml in ultra low attachment 24-well plates (Costar) in IMDM supplemented
with 15% serum replacement media (Gibco BRL). Twenty-four hours later, the
reaggregated EB-like structures were moved to 60 mm petri dishes in the same
medium and cultured for an additional 3.5 days. At this stage, the EBs were
harvested and transferred to gelatin-coated dishes for the evaluation of
neurite formation or to gelatin-coated cover slip for specific staining. Four
days later, the proportion of EBs that generated neurites was scored. For
immunohistochemical staining, EBs were fixed in 2% paraformaldehyde for 20
minutes, washed twice in PBS, permeabilized in 0.2% Triton X-100/PBS, washed
in 10% FCS, 0.2% Tween 20/PBS, and then blocked with 10% FCS/PBS for 10
minutes. EBs were incubated for 1 hour with an antibody against the neuronal
class III ß-tubulin (TuJ1; Babco). Bound antibodies were visualized using
a secondary Cy3-conjugated goat anti-mouse IgG antibody (Jackson
Immunoresearch Laboratories).
Gene expression analysis
The preparation and analysis of 3' UTR cDNA was performed as
previously described (Robertson et al.,
2000). For gene-specific PCR, total RNA was extracted from each
sample with an RNeasy mini kit and treated with Rnase-free DNase (Qiagen). Two
micrograms of total RNA was reverse-transcribed into cDNA with random hexamer
using an Omniscript RT kit (Qiagen). The PCR reactions were performed with 2.5
U of Taq polymerase (Promega), PCR buffer, 2.5 mM MgCl2, 0.2 µM
of each primers and 0.2 mM dNTP. Cycling conditions were as follows: 94°C
for 5 minutes followed by 35 cycles of amplification (94°C denaturation
for 1 minute, 60°C annealing for 1 minute, 72°C elongation for 1
minute) with a final incubation at 72°C for 7 minutes. PCR was carried out
using the following gene specific oligonucleotides: ß-actin, 5'ATG
AAG ATC CTG ACC GAG CG3' (sense) and 5'TAC TTG CGC TCA GGA GGA
GC3' (antisense); brachyury, 5'CAT GTA CTC TTT CTT GCT GG3'
(sense) 5'GGT CTC GGG AAA GCA GTG GC3' (antisense); Runx1,
5'CCA GCA AGC TGA GGA GCG GG3' (sense) 5'CGG ATT TGT AAA GAC
GGT GA3' (antisense); Flk1, 5'CAC CTG GCA CTC TCC ACC TTC3'
(sense) 5'GAT TTC ATC CCA CTA CCG AAA G3' (antisense); Nodal,
5'CCG TCC CCT CTG GCG TAC ATG3' (sense) 5'GAC CTG AGA AGG
AAT GAC GG3' (antisense); Pax6, 5'GCT TCA TCC GAG TCT TCT CCG TTA
G3' (sense) 5'CCA TCT TTG CTT GGG AAA TCC G3' (antisense);
Rex1, 5'CGT GTA ACA TAC ACC ATC CG3' (sense) 5'GAA ATC CTC
TTC CAG AAT GG3' (antisense); Fgf5, 5'AAA GTC AAT GGC TCC CAC
GAA3' (sense) 5'CTT CAG TCT GTA CTT CAC TGG3' (antisense);
Bmp2, 5'GAA TCA GAA CAC AAG TCA GT3' (sense) 5'GTT TGT GTT
TGG CTT GAC GC3' (antisense); Bmp4, 5'TGT GAG GAG TTT CCA TCA
CG3' (sense) 5'CAG CGA AGG ACT GCA GGG CT3' (antisense);
Wnt3a, 5'GGA ATG GTC TCT CGG GAG TTT G3' (sense) 5'AGG TTC
GCA GAA GTT GGG TGA G3' (antisense); Wnt8a, 5'CTG CCT GGT
CAG TGA ACA ACT TC3' (sense) 5'GAG TCT GGA GAT TTT TTC CCC
G3' (antisense).
Flow cytometry and cell sorting
EBs were harvested, trypsinized and the single cell suspension analyzed on
a Facscalibur flow cytometer (Becton Dickinson) or sorted on a Moflo cell
sorter (Cytomation Systems). Staining with mAb Flk1 bio, Kit-PE or CD31-bio
(PharMingen) was performed as previously described
(Kouskoff et al., 2000).
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RESULTS |
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Development and characterization of an ES cell line carrying GFP
targeted to the brachyury locus
Two different vectors were designed for targeting GFP to the brachyury
locus. The vector that gave the highest level of expression contained a mini
gene locus, which consisted of the GFP cDNA, followed by a splice donor site,
an artificial intron and an exon encoding the SV40 polyadenylation signal
sequence to prevent transcription of regions downstream of the brachyury gene
(Fig. 2A). A translational stop
codon was positioned downstream of the artificial intron, as it has been
reported that primary transcripts with stop codons preceding intronic
sequences can be recognized as aberrant messages, thereby rendering them
subject to rapid degradation (Maquat,
2002). The vector was designed to replace approximately two-thirds
of the first exon of the brachyury gene with the GFP expression cassette,
resulting in the disruption of the targeted brachyury allele. The targeting
construct was electroporated into E14.1 ES cells and several positive clones
were identified. The Neo selection marker was subsequently removed from the
targeted ES cells by Cre/loxP-mediated recombination
(Gu et al., 1993
) to minimize
the impact of foreign DNA sequences and ensure that expression of the inserted
GFP cassette was under the control of native brachyury regulatory elements.
Two GFP targeted, Neo-deleted ES clones, referred as GFP-Bry cells, were
differentiated in vitro and analyzed for GFP expression. EBs generated from
these two clones expressed readily detectable levels of GFP when observed
under a fluorescence microscope (Fig.
2C) and were used in the subsequent analyses. The second vector,
which did not result in significant GFP expression after targeting, consisted
of an insertion of a simple GFP cDNA instead of the minigene into the same
segment of the brachyury locus (not shown). The comparison of these vectors
highlight the importance of vector design as relatively small differences in
targeting constructs resulted in significant differences in levels of GFP
expression.
|
Correlation between expression of the targeted GFP and transcription
of the endogenous brachyury gene
As a marker of mesoderm formation, GFP expression must reflect the
expression pattern of the endogenous gene. To determine if this is the case,
EBs derived from GFP-Bry ES cells were harvested at daily intervals over a
6-day differentiation period and analyzed for brachyury transcription by
RT-PCR and for GFP expression by flow cytometry
(Fig. 2D,E). As shown in
Fig. 2D, brachyury expression
was detected between day 2 and 4 of differentiation, with the highest levels
present at day 3, consistent with the previously described pattern for this
gene (Robertson et al., 2000).
FACS analysis revealed the presence of low numbers of GFP+ cells as
early as day 2 of differentiation. The number of GFP+ cells
increased dramatically over the next 48 hours, representing 65% and 85% of the
total day 3 and 4 EB populations, respectively. Following this peak, the
number of GFP+ cells dropped sharply to undetectable levels in day
6 EBs. The high levels of GFP detected by FACS analysis demonstrate that a
large proportion of the day 3-4 EB cells express brachyury indicative of
extensive mesoderm development in our differentiation conditions. The findings
from this comparative analysis strongly suggest that GFP expression faithfully
recapitulates brachyury expression in differentiating EBs and as such,
provides a unique marker for the identification and isolation of cells
expressing this gene.
Segregation of mesoderm and neuroectoderm lineages by GFP
expression
In early development, brachyury expression is restricted to the primitive
streak and nascent mesoderm in the region that will define the posterior part
of the embryo. The epiblast cells in the anterior region of the embryo acquire
a neuroectoderm fate and do not express brachyury
(Herrmann, 1991). To determine
if brachyury expression could also distinguish these primary germ cell
populations within the ES differentiation cultures, EBs were fractionated into
GFP+ and GFP populations by cell sorting and
assayed for mesoderm and neuroectoderm potential
(Fig. 3). GFP+ and
GFP populations were isolated from day 3.5 EBs and subjected
to gene expression and BL-CFC analysis
(Fig. 3A). As shown in
Fig. 3B, cells expressing
brachyury segregated to the GFP+ fraction indicating that it is
possible to isolate mesodermal cells based on GFP expression. Genes associated
with the earliest stages of hematopoietic and endothelial commitment,
including Flk1 and Runx1
(Okuda et al., 1996
;
Wang et al., 1996
),
co-segregated with brachyury to the GFP+ fraction. By contrast,
markers of primitive ectoderm (Rex1; Zfp42 Mouse
Genome Informatics) (Rogers et al.,
1991
) and neuroectoderm (Pax6)
(Walther and Gruss, 1991
) were
detected only in the GFP population. BL-CFC analysis
indicated that most progenitors were found in the GFP+ fraction.
Segregation of the BL-CFC potential to GFP+ fraction demonstrates
that it contains mesodermal derivatives and that the BL-CFC itself may retain
some level of brachyury expression (Fig.
3C).
|
Temporal expression of brachyury relative to markers indicative of ES
cell differentiation and BLCFC development
As shown in Fig. 2, GFP is
expressed in a dynamic temporal pattern that reflects expression of the
endogenous brachyury gene. To further define the stages of ES differentiation
to mesoderm and subsequent specification to the hemangioblast lineages, we
compared the kinetic of GFP expression with that of CD31, Kit and Flk1.
Although Kit and CD31 are best known for their expression on hematopoietic
(Ogawa et al., 1991) and
endothelial (Vecchi et al.,
1994
) populations from fetal and adult tissues, they are also
expressed on undifferentiated ES cells
(Robson et al., 2001
;
Vittet et al., 1996
). As both
are downregulated after the onset of ES cell differentiation, their expression
patterns can be used to track the early commitment steps in the formation of
EBs. Undifferentiated GFP-Bry ES cells did not express significant levels of
GFP or Flk1, but did have high levels of CD31
(Fig. 4A) and intermediate
level of Kit (Fig. 4B). Day 2
EBs contained a small population of GFP+ cells, a fraction of which
also expressed Flk1. The overall levels of CD31 on day 2 EBs were somewhat
reduced compared with that found on the undifferentiated ES population. The
expression patterns of all markers changed dramatically over the next 24
hours. At day 3 of differentiation, more than 40% of the EB population
expressed GFP with no Flk1, while 18% of the cells expressed both GFP and
Flk1. CD31 levels were significantly reduced on the entire EB population and
none was expressed on the GFP+ cells. Three colors analysis
revealed that the double positive GFP+Flk1+ cells had
low level of Kit expression (Fig.
4B). Both the GFP+Flk1 and the
GFPFlk1 populations had intermediate
levels of Kit. By day 4 of differentiation, the EB cells appear to have down
regulated the levels of GFP and Flk1 expression, although a significant
portion of the populations still expressed both markers. Relatively few cells
expressed CD31 (Fig. 4A) or Kit
(not shown) at this stage of development. These analyses clearly demonstrate
that using the GFP-Bry ES line together with cell-surface markers, it is
possible to track the differentiation of ES cells to brachyury-positive
mesoderm and subsequently to cell populations that express Flk1 together with
brachyury.
|
|
Tracking the induction of mesoderm and its specification to the
BL-CFC
If the three fractions defined by GFP and Flk1 represent distinct steps
within a developmental program, then it should be possible to demonstrate that
those representing the early stages are able to give rise to the more mature
populations. To address this issue, each fraction was isolated from day 3 EBs
and allowed to reaggregate at high cell density in culture for 20 hours
(Fig. 6A). Although cell
numbers did not change significantly during this time, the developmental
potential of the populations did. In the reaggregated presort control, the
GFP+Flk1+ fraction increased in size from 16% to 40% of
the total population, the GFP+Flk1 fraction
remained relatively constant in size whereas the
GFPFlk1 fraction decreased in size during
this time. After the 20-hour culture period, the
GFPFlk1 sorted population gave rise to a
significant number of GFP+Flk1 cells (27% of the
total culture) and also to a small emerging population (3.5%) that expressed
Flk1. In this same period, the GFP+Flk1 fraction
generated a large GFP+Flk1+ population that represented
66% of the total cells recovered from the reaggregation culture. A
subpopulation of cells did not acquire Flk1 and retained relatively high
levels of GFP. Expression of both Flk1 and GFP was downregulated after culture
of the GFP+Flk1+ fraction. Analysis of the BL-CFC and
hematopoietic progenitor potential of the fractions prior to (pre-culture) and
following (post-culture) the culture revealed changes that were consistent
with the changes observed in Flk1 and GFP expression. Prior to culture, most
of the blast colony-forming potential was found in the
GFP+Flk1+ fraction
(Fig. 6B). After culture, the
BL-CFC potential of the two GFP+ fractions changed dramatically.
The GFP+Flk1 population acquired the potential to
generate blast colonies, a finding consistent with the fact that these cells
upregulated Flk1 expression level during this time period. The
GFP+Flk1+ population, conversely, lost BL-CFC activity
but developed significant hematopoietic potential during this culture step
(Fig. 6C). These changes in
progenitor cell potential together with the downregulation of both Flk1 and
GFP within this population are an indication of maturation beyond the
hemangioblast stage to the early hematopoietic stage of development. The
majority of hematopoietic progenitors that developed in the reaggregation
culture of the GFP+Flk1+ fraction were of the primitive
erythroid lineage, the earliest population to develop in EBs. The
GFPFlk1 fraction contained no BL-CFC prior
to or following culture, consistent with the lack of significant numbers of
Flk1-expressing cells in either population.
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DISCUSSION |
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One of the original goals of this targeting approach was to further
characterize the developmental relationship between the BL-CFC and mesoderm by
determining whether or not this hemangioblast-like progenitor expressed
brachyury. The outcome of this study clearly demonstrates that the entire
Flk1+ population at the BL-CFC stage of development expresses GFP.
Although all BL-CFC are GFP+, the RT-PCR analyses in
Fig. 5 indicate that the levels
of brachyury are reduced compared with the
GFP+Flk1 population suggesting that these
progenitors are downregulating brachyury as they differentiate to the
hematopoietic and endothelial lineages. Previous findings demonstrating that
progeny of the BLCFC no longer express brachyury
(Kennedy et al., 1997;
Robertson et al., 2000
) are
consistent with this interpretation. Collectively, these observations would
position the BL-CFC at a stage of development that represents mesodermal cells
committed to the hematopoietic and vascular lineages.
In the early mouse embryo, the first mesodermal cells generated within the
primitive streak migrate to the extra-embryonic region where they
differentiate and form the hematopoietic and endothelial lineage of the blood
islands (Moore and Metcalf,
1970; Kinder et al.,
1999
). Although most cells within the primitive streak are
brachyury positive, expression is rapidly downregulated as they exit the
streak and begin migrating (Wilkinson et
al., 1990
). Flk1 is expressed in these migrating cells and
subsequently in the blood islands and the developing vasculature of the yolk
sac (Shalaby et al., 1995
).
Given that brachyury is widely expressed in the primitive streak, it is
assumed that the Flk1+ cells and ultimately the hematopoietic and
endothelial lineages derive from brachyury expressing mesoderm. The findings
in this report formally demonstrate that brachyury+ mesodermal
progenitors do indeed give rise to the BL-CFC and to cells of the
hematopoietic lineage within the ES/EB model system.
In addition to providing further characterization of the BLCFC, the GFP-Bry ES cell line has enabled us to segregate the Flk1 fraction of day 3.0-3.5 EBs into GFP and GFP+ populations, representing cells with pre-mesoderm and prehemangioblast mesoderm potential, respectively. The strongest evidence in support of this interpretation is provided by the experiment using reaggregation cultures in which each isolated population was found to differentiate rapidly to the subsequent stage of development. One of the most striking developmental changes was observed with the GFP+Flk1 population that contained little, if any, BL-CFC potential prior to culture. After culture, a substantial number of the cells upregulated Flk1 and with this change in expression the population acquired the capacity to generate blast cell colonies. This is an important observation as it enables one, for the first time, to access the immediate progenitors of the BL-CFC.
If the GFP+Flk1+ population is indicative of cells
emerging from the primitive streak, then the
GFP+Flk1 population should represent cells within
the primitive streak, whereas the GFPFlk1
population would be equivalent to the pregastrulation, pre-streak stage of
development. The gene expression profiles of the three populations are
consistent with this interpretation. Two of the most restricted expression
patterns were observed with the Wnt genes that are upregulated with the onset
of brachyury expression and downregulated with the acquisition of Flk1. In the
early mouse embryo, expression of Wnt3a and Wnt8a are
overlapping with that of brachyury, both are expressed in the primitive streak
and then downregulated with migration and patterning associated with the
formation of extra-embryonic mesoderm of the yolk sac
(Bouillet et al., 1996;
Takada et al., 1994
;
Yamaguchi et al., 1999
).
Studies in the chick embryo (Marvin et
al., 2001
) and in Xenopus
(Schneider and Mercola, 2001
)
have implicated Wnt3a and Wnt8 as important molecules in the
specification of mesoderm to a hematopoietic fate. When expressed in cells of
the cardiac crescent, Wnt3a displayed the potential to respecify
these cells to a hematopoietic fate. Conversely, when the function of both was
blocked in cells fated to the hematopoietic lineage, these cells acquired
cardiac potential. Expression of both Wnt3a and Wnt8a in the
GFP+Flk1 population that contains cells
undergoing commitment to the BL-CFC suggests that these factors could have a
similar role in the specification of hematopoietic mesoderm in the mouse. The
expression pattern of Fgf5 and Nodal is also consistent with
the assigned developmental potential of the three populations. In the mouse
embryo, Fgf5 is initially expressed in cells of the epiblast and
following gastrulation is found in cells that form the primitive streak and
subsequently in the paraxial subpopulation of mesoderm
(Haub and Goldfarb, 1991
;
Hebert et al., 1991
).
Nodal is expressed in the epiblast and primitive endoderm, the
primitive streak and ultimately in a subset of cells found in the node
(Varlet et al., 1997
).
Expression of both genes in the GFPFlk1
and GFP+Flk1 populations but not in the
GFP+Flk1+ cells would be consistent with a progression
from epiblast-like cells to cells representing the primitive streak and
finally specification to the hemangioblast.
The isolation of cell populations based on brachyury expression described here together with our previous studies and findings from others provide the basis for a model of mesoderm induction and specification as outlined in Fig. 7. Based on surface marker analysis, the undifferentiated ES cells can be defined as CD31+ Kit+ brachury Flk1. Over a 2.5-3.0-day period of differentiation, the ES cells differentiate and give rise to three different populations, the most immature of which is considered as pre-mesoderm and represented as GFPFlk1. The majority of these cells has downregulated CD31 (Fig. 4), but retain some expression of Kit. This population also expressed Fgf5, Nodal and low levels of Wnt8a. The next stage of development, the pre-hemangioblast mesoderm has upregulated brachyury (GFP+) and continues to express Kit. Fgf5, Nodal, Wnt3a, Wnt8a and Bmp2 are expressed at readily detectable levels at this stage. The most mature population, the hemangioblast, continues to express some brachyury, has upregulated Flk1 and downregulated Kit. These cells no longer express Fgf5 or the Wnt genes, but do express Bmp2 and Bmp4 as well as Runx1 and Scl.
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ACKNOWLEDGMENTS |
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