1 Monash Immunology and Stem Cell Laboratories, Monash University, Clayton, VIC,
3800, Australia
2 The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, 3050,
Australia
Author for correspondence (e-mail:
andrew.elefanty{at}med.monash.edu.au)
Accepted 10 December 2004
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SUMMARY |
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Key words: Mixl1, BMP4, Haematopoiesis, Kdr
![]() |
Introduction |
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Key targets of TGFß signalling in gastrulating Xenopus laevis
embryos include the Mix/Bix homeobox genes that regulate mesoderm and
endoderm formation in response to nodal/activin and BMP4
(Rosa, 1989;
Vize, 1996
). Mix.1 is
induced by BMP4 and can ventralise mesoderm
(Mead et al., 1996
), while a
number of other Mix/Bix genes induce endoderm
(Henry and Melton, 1998
;
Latinkic and Smith, 1999
;
Lemaire et al., 1998
;
Tada et al., 1998
). The two
zebrafish Mix-related homeobox genes, bon and mezzo
(og9x - Zebrafish Information Network) are immediate-early targets of
nodal signalling that are transiently expressed in precursors of
mesoderm and endoderm (Kikuchi et al.,
2000
; Poulain and Lepage,
2002
; Trinh et al.,
2003
). The avian Mix gene is expressed in the epiblast
and posterior marginal zone endoderm just prior to gastrulation and in the
primitive streak, excluding Hensen's node
(Peale et al., 1998
;
Stein et al., 1998
).
Similarly, expression of the single mouse Mix gene homologue, Mixl1,
is restricted to the visceral endoderm of the pre-gastrulation embryo and the
primitive streak (Pearce and Evans,
1999
; Robb et al.,
2000
). Indeed, gene targeting has confirmed the important role
that Mixl1 plays during gastrulation. Mixl1-null mutants
display an enlarged primitive streak and subsequently exhibit abnormalities in
axial morphogenesis and formation of definitive endoderm that result in death
at embryonic day (E) 8.5 (Hart et al.,
2002
).
In mice, the first mesoderm to emerge from the primitive streak migrates
extra-embryonically and forms the blood islands of the yolk sac at E7-7.5
(Kinder et al., 1999). The
first wave of haematopoiesis occurs concurrently with the formation of
extra-embryonic vasculature, consistent with the development of these two
lineages from a common progenitor (Keller
et al., 1999
; Lacaud et al.,
2001
). The in vitro differentiation of embryonic stem (ES) cells
recapitulates many aspects of early haematopoietic development and represents
a valuable model system to study a process occurring at a relatively
inaccessible period of embryogenesis (Dang
et al., 2002
; Desbaillets et
al., 2000
; Keller,
1995
; Maye et al.,
2000
; Takahashi et al.,
2003
). Indeed, the Flk1 (Kdr - Mouse Genome
Informatics)-positive blast colony forming cell (BL-CFC) capable of giving
rise to both haematopoietic and endothelial lineages was first isolated from
embryoid bodies (EBs), thus providing tangible evidence for the existence of
an haemangioblast (Choi et al.,
1998
; Kennedy et al.,
1997
; Nishikawa et al.,
1998
).
We have used mouse ES cell lines in which Mixl1-coding sequences
on one (Mixl1GFP/w) or both
(Mixl1GFP/GFP) alleles were replaced by the gene encoding
green fluorescent protein (GFP) (Hart et
al., 2002) to investigate the role of Mixl1 in ventral
mesoderm patterning and haematopoiesis. We have shown that a large proportion
of differentiating Mixl1GFP/w ES cells transiently
expressed both GFP and Flk1 and that this doubly-positive population
was enriched for BL-CFCs. However, in differentiating Mixl1-null ES
cells, Flk1 expression was delayed and reduced and the frequency of
haematopoietic CFCs was decreased. Differentiation of ES cells under
serum-free (SF) conditions demonstrated that induction of Mixl1- and
Flk1-expressing haematopoietic mesoderm required medium supplemented
with BMP4 or activin A. Therefore, this study has revealed an important role
for Mixl1 in haematopoietic development and demonstrated the utility
of the Mixl1GFP/w ES cells for evaluating growth factors
influencing mesendodermal differentiation.
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Materials and methods |
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ES cell culture and differentiation
ES cells were cultured as described
(Barnett and Köntgen,
2001) and differentiated using the method of Kennedy et al.
(Kennedy et al., 1997
). For
differentiation of ES cells under SF conditions, ES cells were resuspended at
5000 cells/ml in modified Chemically Defined Medium (CDM)
(Johansson and Wiles, 1995
)
comprising IMDM/Ham's F12 with Glutamax (Gibco) supplemented with 5 mg/ml
bovine serum albumin (Sigma), 1 U/ml LIF (Chemicon), 4.5x10-4
M
-MTG (Sigma), a 1% Chemically-Defined Lipid Concentrate (Gibco), 1%
Insulin-Transferrin-Selenium-X Supplement (Gibco) and antibiotics. Activin A
(0.1-100 ng/ml) or BMP4 (0.5-20 ng/ml) (R&D Systems) were added at the
time of cell plating. Cultures were maintained at 37°C in a humidified
environment of 8% CO2 in air. Phase-contrast and fluorescent
microscopy images of EB cultures were acquired using a Zeiss Axiocam mounted
on an Axiovert 200 microscope and processed with Axiovision software. Single
optical sections of EBs were taken using a Leica confocal scanning
microscope.
Flow cytometry
Embryoid bodies were dissociated to single cells using trypsin/EDTA (Gibco)
containing 1% chicken serum (Hunter). Cells were resuspended in a block
solution (phosphate-buffered saline supplemented with 2% FCS, 1% goat serum
and 1% rabbit serum) and incubated with primary antibodies directed against
E-cadherin (ECCD-2, Zymed), FLK1 (VEGF-R2, Ly-73) conjugated to phycoerythrin
(PE) (Avas 121, BD Biosciences), Ter-119 (Ly-76) conjugated to PE (BD
Biosciences) and CD34 (RAM34) conjugated to biotin (BD Biosciences). Anti
E-cadherin antibodies were detected with either PE or allophycocyanin
(APC)-conjugated anti-rat IgG (BD Biosciences) while biotinylated anti-CD34
antibodies were detected with streptavidin-conjugated PE or APC. Cells were
analysed using a FACSCalibur (Becton Dickinson) running CellQuest software
(Becton Dickinson). For cell-sorting and reculture experiments,
differentiating Mixl1GFP/w EBs were dissociated, stained
with antibodies against FLK1 and sorted according to GFP and FLK1 expression
using a FACStar Plus (Becton Dickinson).
Haematopoietic colony forming assays
Haematopoietic colonies were generated by plating
2.5x104-105 dissociated EB cells into 1.5 ml of 1%
methylcellulose in IMDM supplemented with 10% FCS, 25% D4T endothelial cell
conditioned medium, 25 µg/ml ascorbic acid, 2 mM L-glutamine
(Kennedy et al., 1997). To
assay BL-CFCs, methylcellulose cultures were supplemented with 5 ng/ml
Vascular Endothelial Growth Factor (VEGF) (R&D systems) and 50 ng/ml Stem
Cell Factor (SCF) (R&D systems). For detection of BL-CFCs and primitive
erythroid colonies (EryP), the growth factor combination used was 5 ng/ml
VEGF, 50 ng/ml SCF, 5 U/ml EPO (Janssen Cilag) and IL3 (1% of a supernatant
from a cell line producing mIL3)
(Karasuyama and Melchers,
1988
). Colonies were scored after 5-7 days. Blast colonies were
expanded in liquid culture supplemented with the same combination of growth
factors and analysed after 7 days. Morphology was assessed by
May-Grunwald-Giemsa staining of cytocentrifuge preparations and endothelial
cells were identified by staining wells fixed with 4% paraformaldehyde (Sigma)
with anti-PECAM1 antibodies (MEC13.3, BD Biosciences).
Gene expression analysis
Total RNA was prepared using an RNeasy kit (Qiagen) according to the
manufacturer's instructions. DNase I treated samples were reverse transcribed
using Superscript II (Invitrogen) and the resultant cDNA preparations
standardized as described (Elefanty et
al., 1997). Primer sequences and annealing temperatures are shown
in Table 1. References for
Brachyury, FLK1, ßH1 globin and HPRT primer sequences can be found in
Elefanty et al. (Elefanty et al.,
1997
). PCRs were performed for 30 cycles with reaction conditions
as described (Elefanty et al.,
1997
). PCR products were analysed by electrophoresis on a 2%
agarose gel.
|
![]() |
Results |
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Examination of differentiating Mixl1GFP/w ES cells revealed that most day 3 and 4 embryoid bodies (EBs) expressed GFP, coinciding with endogenous Mixl1 expression (Figs 1, 2 and data not shown). Confocal images demonstrated that GFP was present in the outer, flattened endoderm-like cells and in the inner core of the EBs (Fig. 2C,D).
|
|
GFP is co-expressed with the stem cell marker E-cadherin in Mixl1GFP/w and Mixl1GFP/GFP cells
Examination of the gene expression profile of differentiating ES cells
demonstrated that day 3 EBs simultaneously expressed stem cell, primitive
streak and mesodermal genes (Fig.
1). We used flow cytometry to correlate the expression of GFP with
that of the stem cell marker E-cadherin (E-cad) and the ventral mesoderm
marker FLK1 in differentiating Mixl1GFP/w and
Mixl1GFP/GFP ES cells. In differentiating EBs from all
Mixl1 genotypes, E-cad was expressed in over 90% of cells up until
day 2 of differentiation (Fig.
4A and data not shown), consistent with its expression in the
inner cell mass, epiblast and primitive streak in vivo
(Ciruna and Rossant, 2001;
Huber et al., 1996
). Between
day 2 and day 4, the proportion of E-cad+ cells fell to under 20%,
reflecting loss expression in emerging mesoderm
(Fig. 4B). The first cells
expressing GFP were invariably E-cad+, consistent with the expected
phenotype of primitive streak cells (Ciruna
and Rossant, 2001
; Huber et
al., 1996
) (Fig.
4B). As differentiation progressed, an increasing proportion of
GFP+ cells lost E-cad expression, suggesting that they had `passed
through' the streak. There was no significant difference in the E-cad
expression profiles between Mixl1GFP/w and
Mixl1GFP/GFP EBs (Fig.
4A and data not shown).
|
|
|
Blast colony forming cells are enriched in the GFP+FLK1+ population from day 4 Mixl1GFP/w EBs
In order to further explore the relationship between Mixl1
expression and haematopoietic differentiation, the blast colony-forming
ability of sorted fractions of anti-FLK-1 labelled
Mixl1GFP/w day 3 and day 4 EBs were compared. FACS sorted
GFP-FLK1-, GFP+FLK1-,
GFP+FLK1+ and GFP-FLK1+
populations were cultured in methylcellulose in the presence or absence of
vascular endothelial growth factor (VEGF) and stem cell factor (SCF) in order
to detect haematopoietic blast colonies
(Kennedy et al., 1997).
Interestingly, BL-CFCs (Fig.
7A-D) were cultured from all sorted fractions from day 3 EBs,
although the highest frequency was observed in the GFP+ fractions
(219 colonies/5x104 GFP+FLK1- cells and
225/5x104 GFP+FLK1+ cells)
(Fig. 7E). Irrespective of
their population of origin, all the blast cell colonies displayed similar
morphology, dependence on VEGF and SCF and ability to give rise to
haematopoietic and adherent vascular cells. The endothelial nature of the
adherent cells was verified by staining with antibodies to PECAM1 or FLK1 (see
Fig. 7A-D; data not shown).
Consistent with the transient nature of BL-CFCs
(Kennedy et al., 1997
), their
frequency was considerably decreased in day 4 EBs
(Fig. 7F). At this time, the
majority of BL-CFCs were found in the GFP+FLK1+
fraction, although some were present in the GFP-FLK1+
population (Fig. 7F).
|
|
BMP4 induces GFP and FLK1 expression and augments survival in Mixl1GFP/w ES cells differentiated in SF medium
Studies in Xenopus laevis demonstrated that Mix-family genes were
directly induced by both activin and BMP4
(Mead et al., 1996;
Rosa, 1989
;
Vize, 1996
). Therefore, we
examined the ability of these factors to induce GFP expression in
Mixl1-heterozygous and Mixl1-null ES cells. ES cells
differentiated in SF medium (Johansson and
Wiles, 1995
) in the absence of growth factor supplements failed to
express GFP or FLK1, but lost expression of the stem cell marker E-cad
(Fig. 9C,E and data not shown),
consistent with a default to neurectodermal differentiation
(Wiles and Johansson,
1999
).
|
Comparison of GFP induction in Mixl1GFP/w cells in serum containing and SF media revealed that the appearance of GFP-expressing cells was delayed in SF media supplemented with BMP4 (SF/BMP4) and that the peak percentage of GFP+ cells was reduced (Fig. 9C). Interestingly, supplementation of SF medium with BMP4 significantly increased the viability of cells at day 4 and 5 of differentiation (Fig. 9D). Moreover, the augmentation of cell survival in SF medium by BMP4 occurred prior to the induction of GFP expression (data not shown).
Induction of GFP expression was examined in Mixl1GFP/w
and Mixl1GFP/GFP EBs cultured in SF medium in the absence
or presence of BMP4 (Fig. 9E).
GFP expression was seen in 46% of Mixl1GFP/w and 55% of
Mixl1GFP/GFP cells in day 4 EBs cultured in SF/BMP4. FLK1
expression was reduced and delayed in the Mixl1GFP/GFP
cells, as observed in serum containing medium. In contrast to the results for
serum-containing cultures in which the highest percentage of FLK1+
cells coincided with maximal GFP expression
(Fig. 5A), the frequency of
FLK1+ cells in SF/BMP4 medium continued to increase for at least 3
days after the peak in GFP expression (Fig.
9E). In day 4 EBs, clonogenic haematopoietic progenitor cells were
30-fold less frequent in SF/BMP4 than in serum differentiated EBs,
further indicating that the addition of 5 ng/ml BMP4 was insufficient to
completely replicate the effects of serum (compare
Fig. 6B with
Fig. 9F). One tenth the number
of CFCs were observed in the Mixl-null cell lines, consistent with
the results obtained from experiments using serum containing medium
(Fig. 6B). Despite the
observation that, by day 7, Mixl1GFP/w and
Mixl1GFP/GFP EBs contained a similar percentage of
FLK1+ cells, the frequency of CFCs was still lower in EBs derived
from Mixl1-null cells (Fig.
9F). These data establish a link between activin A and BMP4
signalling and Mixl1 expression in mammalian cells. Furthermore,
analysis of Mixl1-null cells in SF medium demonstrates that
Mixl1 is required for normal BMP4-dependent expression of
Flk1 and development of haematopoietic CFCs.
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Discussion |
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The appearance of GFP+ cells in Mixl1-null EBs mirrored
that seen in EBs from the Mixl1-heterozygous line, consistent with
the assumption that `pre gastrulation' events would be unchanged in the
absence of Mixl1. The higher peak percentage of GFP+ cells
and the prolongation of GFP expression seen in Mixl1-null cells could
be explained by the presence of two GFP alleles. However, an alternative
hypothesis is that these features represented an in vitro correlate of the
expanded primitive streak and delayed egress of GFP+ nascent
mesendodermal cells observed in Mixl1-null embryos
(Hart et al., 2002).
Definitive exclusion of this scenario will require analysis of
Mixl1-null ES cells that carry only one GFP allele.
In order to place Mixl1 expression during ES cell differentiation
into a developmental context, flow cytometry was used to correlate the
expression of GFP in Mixl1GFP/w EBs with the expression of
E-cadherin, a cell-adhesion molecule expressed in the epiblast of the mouse
embryo (Burdsal et al., 1993).
Experiments by Ciruna and Rosssant showed that expression of E-cadherin was
downregulated as cells passed through the primitive streak and was lost by the
time they underwent the epithelial mesenchymal transition required for
migration from the streak (Ciruna and
Rossant, 2001
). Therefore, cells coexpressing E-cadherin and
Mixl1 during EB differentiation could be regarded as a primitive
streak like population. Indeed, flow cytometric analysis of E-cadherin and GFP
during differentiation of Mixl1GFP/w ES cells allowed the
identification of cells reminiscent of the embryonic stages of epiblast
(GFP-E-cad+), primitive streak
(GFP+E-cad+) and nascent mesoderm
(GFP+E-cad-). By day 4 of ES differentiation, most
GFP+ cells expressed the haematopoietic marker Flk1 and
only
20% of cells were still E-cad+. In fact, as reported by
Nishikawa et al. (Nishikawa et al.,
1998
), we found that FLK1+ and E-cad+
populations were mutually exclusive (data not shown).
Mixl1 is required for efficient haematopoiesis
In heterozygous Mixl1GFP/w ES cells, Flk1 was
expressed immediately following GFP and both genes were maximally expressed in
day 4 EBs. In Mixl1-null cells, the onset of Flk1 expression
was delayed and, even though the frequency of FLK1+ cells increased
by day 4, normal haematopoiesis was not established, irrespective of whether
the cells were differentiated in serum-based or SF medium (compare
Fig. 5 with
Fig. 9). This was most
obviously evidenced by the failure of visible haemoglobinisation of day 7
Mixl1-null EBs. Also, the large reduction in the frequency of blast
and erythroid colonies generated from day 4 Mixl1GFP/GFP
EBs suggested that the haematopoietic defect could not simply be explained by
the lower percentage of FLK1+ cells at that time. This conclusion
is consistent with analysis of Flk1-null ES cells showing that
Flk1 was not necessary for the development of haematopoietic cells in
vitro (Ema et al., 2003;
Schuh et al., 1999
). These
data indicated that Mixl1 expression was required for the efficient
generation of haematopoietic cells and placed Mixl1 functionally
upstream of a FLK1+ haematopoietic precursor. Interestingly, recent
studies showed that the haematopoietic defect associated with deficiency of
the transcription factor Scl could only be rescued by expressing
Scl by day 3 of ES cell differentiation, contemporaneous with the
expression of Mixl1 but antedating expression of Flk1
(Endoh et al., 2002
).
Collectively, these data predict that the specification of haematopoietic
precursors occurs early in gastrulation and requires the input of a number of
transcription factors, including Mixl1 and Scl.
The consequences of Mixl1-deficiency were reminiscent of the
phenotype observed in the absence of Fgfr1 or Fgf8, the
predominant FGF family member expressed in the gastrulating embryo.
Specifically, embryos lacking Fgfr1 exhibited an enlarged primitive
streak due to a failure of progenitor cell migration
(Ciruna and Rossant, 2001;
Ciruna et al., 1997
;
Deng et al., 1994
;
Yamaguchi et al., 1994
) and
Fgfr1-deficient ES cells displayed a marked impairment in
haematopoietic colony formation in vitro
(Faloon et al., 2000
).
Similarly, Fgf8-null embryos also displayed a cell migration defect
in the primitive streak and showed evidence of perturbed haematopoiesis, with
reduced expression of the erythroid and endothelial markers Fog and
PECAM in the yolk sac (Sun et al.,
1999
). Collectively, these data speak to the requirement for both
Mixl1 expression and FGF signalling in normal streak morphogenesis
and haematopoietic specification. As the expression patterns of Fgf8
and Mixl1 overlapped both in the primitive streak
(Crossley and Martin, 1995
;
Pearce and Evans, 1999
;
Robb et al., 2000
) and in
differentiating EBs (C. Hirst, A. Mossman and A.G.E., unpublished), we are
investigating the possibility of a direct link between these two
molecules.
Haemangioblasts arise from a Mixl1-expressing mesodermal progenitor
Primitive erythroid and endothelial cells arise from a common mesodermal
precursor, the haemangioblast, most convincingly identified as a transient
FLK1+ population in day 2.75-4.00 EBs
(Choi et al., 1998;
Chung et al., 2002
;
Fehling et al., 2003
;
Kennedy et al., 1997
;
Nishikawa et al., 1998
).
Furthermore, Fehling et al. (Fehling et
al., 2003
) showed that in day 3.5 EBs, haemangioblasts arose
exclusively from brachyury-positive precursors and that
brachyury+FLK1+ cells from day 2.5 EBs were enriched for
BL-CFCs. Given the overlapping expression of brachyury and Mixl1 in
differentiating ES cells (Kubo et al.,
2004
) (this study) and during embryogenesis, we anticipated that
haemangioblasts would arise from a Mixl1-positive precursor and be
enriched in GFP+FLK1+ cells from
Mixl1GFP/w EBs. Indeed, when cells were sorted from day 4
Mixl1GFP/w EBs, most BL-CFCs were found in the
GFP+FLK1+ fraction. Interestingly, some BL-CFCs were
detected in the GFP-FLK1+ population from day 4 EBs.
This might have represented an artefact of the sorting process, with some
GFP-dull cells inadvertently included in the GFP-FLK1+
fraction. Alternatively, BL-CFCs did not express Mixl1 and might have
expressed alternate `gastrulation' genes such as brachyury. Indeed, in ES
cells in which GFP was targeted to the brachyury locus, essentially all the
FLK1+ cells were GFP+
(Fehling et al., 2003
).
The frequency of BL-CFCs was much higher in cells sorted from day 3 EBs but
there was not a statistically significant enrichment in the
GFP+FLK1+ population. Because overnight culture of
sorted cells demonstrated a narrow window between day 2.8 and day 4, during
which GFP-FLK1- and GFP+FLK1-
cells could continue to differentiate, cells that fell into the either of
these populations at the time of sorting at day 3 could have upregulated
Mixl1 and/or Flk1 expression after seeding in
methylcellulose and produced VEGF-dependent blast cell colonies. However, in
other studies that compared the BL-CFC content of FLK1+ and
FLK1- populations sorted from day 3 EBs, a clearer enrichment of
BL-CFC in the FLK1+ cells was observed
(Chung et al., 2002;
Faloon et al., 2000
;
Fehling et al., 2003
). It is
possible that differences in the kinetics of FLK1 expression between the
various ES cell lines used or differences in the ability of the
methylcellulose cultures to support further differentiation may explain this
discrepancy.
BMP4 and activin A induce Mixl1 in vitro
We used a SF culture system in order to determine which growth factors
regulate `molecular gastrulation' and subsequent haematopoietic commitment in
differentiating ES cells. In this context, the Mixl1GFP/w
ES cells provided a simple means to identify live cells at the primitive
streak stage by fluorescence microscopy and by flow cytometry. Previous
studies in Xenopus have shown that Mix.1, the closest
homologue to mammalian Mixl1, was induced by either activin A or BMP4
(Mead et al., 1996;
Rosa, 1989
). In fact, more
recently, Kubo et al. (Kubo et al.,
2004
) used a two-step SF culture system to show that
Mixl1 RNA could be induced by activin A. In our culture system,
although both activin and BMP4 induced GFP with similar kinetics in
Mixl1GFP/w EBs, the percentage of GFP+ cells
was much greater in BMP4-treated cultures. The modest expression of
Mixl1 in response to activin A contrasted with the robust induction
of brachyury GFP by this factor reported by Keller and colleagues
(Fehling et al., 2003
).
Indeed, in their original description of the SF medium used in our studies,
Johansson and Wiles demonstrated that brachyury was more readily induced by
activin than by BMP4 (Johansson and Wiles,
1995
). These data suggest that aspects of TGFß signalling may
be conserved between mouse ES cells and Xenopus embryos, in which
activin induced both brachyury and Mix.1, and BMP4 treatment of
animal pole cells induced Mix.1
(Cunliffe and Smith, 1992
;
Mead et al., 1996
;
Smith et al., 1991
).
The addition of BMP4 to ES cells differentiated in SF cultures led to an
increase in total cell numbers and an improvement in cell viability that
antedated Mixl1 expression (Fig.
9D) (E.S.N. and A.G.E., unpublished), corresponding to the
growth-promoting effect of BMP4 on epiblast cells prior to gastrulation
(Beppu et al., 2000;
Mishina et al., 1995
;
Winnier et al., 1995
).
Park et al. (Park et al.,
2004) recently described Flk1 induction by BMP4 in a
different SF medium. However, because brachyury was expressed in their EBs
prior to the addition BMP4, it was unclear whether BMP4 induced mesoderm or
simply acted to ventrally pattern existing mesoderm. We also observed the
emergence of Flk1+ ventrally patterned mesoderm in
Mixl1GFP/w EBs cultured in SF/BMP4 medium. Our data showed
unambiguously that induction of the primitive streak marker Mixl1 was
completely dependent on the inclusion of BMP4 in the culture medium,
strengthening the link between BMP4 and mesoderm induction in mammalian cells
suggested by Johansson and Wiles
(Johansson and Wiles, 1995
;
Wiles and Johansson, 1997
).
Nevertheless, as others have shown (Park
et al., 2004
), additional growth factors, such as VEGF, are
required to efficiently generate haematopoietic CFCs from BMP4-induced
FLK1+ ventral mesoderm.
In conclusion, we have shown that differentiating ES cells express Mixl1 as they transit through a stage equivalent to the primitive streak of the gastrulating mouse embryo. The most primitive haematopoietic BL-CFCs arise from Mixl1-expressing cells and absence of Mixl1 disrupts normal haematopoiesis. We have demonstrated that BMP4 augments survival, induces Mixl1 expression and ventrally patterns mesoderm in differentiating EBs. Finally, the Mixl1GFP/w ES cell lines will be valuable tools in further elucidating the factors that regulate mesoderm and endoderm formation.
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ACKNOWLEDGMENTS |
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![]() |
Footnotes |
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REFERENCES |
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---|
Barnett, L. D. and Köntgen, F. (2001). Gene targeting in a centralized facility. Methods Mol. Biol. 158,65 -82.[Medline]
Beppu, H., Kawabata, M., Hamamoto, T., Chytil, A., Minowa, O., Noda, T. and Miyazono, K. (2000). BMP type II receptor is required for gastrulation and early development of mouse embryos. Dev. Biol. 221,249 -258.[CrossRef][Medline]
Burdsal, C. A., Damsky, C. H. and Pedersen, R. A.
(1993). The role of E-cadherin and integrins in mesoderm
differentiation and migration at the mammalian primitive streak.
Development 118,829
-844.
Choi, K., Kennedy, M., Kazarov, A., Papadimitriou, J. C. and
Keller, G. (1998). A common precursor for hematopoietic and
endothelial cells. Development
125,725
-732.
Chung, Y. S., Zhang, W. J., Arentson, E., Kingsley, P. D.,
Palis, J. and Choi, K. (2002). Lineage analysis of the
hemangioblast as defined by FLK1 and SCL expression.
Development 129,5511
-5520.
Ciruna, B. and Rossant, J. (2001). FGF signaling regulates mesoderm cell fate specification and morphogenetic movement at the primitive streak. Dev. Cell 1, 37-49.[Medline]
Ciruna, B. G., Schwartz, L., Harpal, K., Yamaguchi, T. P. and
Rossant, J. (1997). Chimeric analysis of fibroblast growth
factor receptor-1 (Fgfr1) function: a role for FGFR1 in morphogenetic movement
through the primitive streak. Development
124,2829
-2841.
Conlon, F. L., Lyons, K. M., Takaesu, N., Barth, K. S., Kispert,
A., Herrmann, B. and Robertson, E. J. (1994). A primary
requirement for nodal in the formation and maintenance of the primitive streak
in the mouse. Development
120,1919
-1928.
Corish, P. and Tyler-Smith, C. (1999). Attenuation of green fluorescent protein half-life in mammalian cells. Protein Eng. 12,1035 -1040.[CrossRef][Medline]
Crossley, P. H. and Martin, G. R. (1995). The
mouse Fgf8 gene encodes a family of polypeptides and is expressed in regions
that direct outgrowth and patterning in the developing embryo.
Development 121,439
-451.
Cunliffe, V. and Smith, J. C. (1992). Ectopic mesoderm formation in Xenopus embryos caused by widespread expression of a Brachyury homologue. Nature 358,427 -430.[CrossRef][Medline]
Dang, S. M., Kyba, M., Perlingeiro, R., Daley, G. Q. and Zandstra, P. W. (2002). Efficiency of embryoid body formation and hematopoietic development from embryonic stem cells in different culture systems. Biotechnol. Bioeng. 78,442 -453.[CrossRef][Medline]
Deng, C. X., Wynshaw-Boris, A., Shen, M. M., Daugherty, C., Ornitz, D. M. and Leder, P. (1994). Murine FGFR-1 is required for early postimplantation growth and axial organization. Genes Dev. 8,3045 -3057.[Abstract]
Desbaillets, I., Ziegler, U., Groscurth, P. and Gassmann, M. (2000). Embryoid bodies: an in vitro model of mouse embryogenesis. Exp. Physiol. 85,645 -651.[Abstract]
Elefanty, A. G., Robb, L., Birner, R. and Begley, C. G.
(1997). Hematopoietic-specific genes are not induced during in
vitro differentiation of scl-null embryonic stem cells.
Blood 90,1435
-1447.
Ema, M., Faloon, P., Zhang, W. J., Hirashima, M., Reid, T.,
Stanford, W. L., Orkin, S., Choi, K. and Rossant, J. (2003).
Combinatorial effects of Flk1 and Tal1 on vascular and hematopoietic
development in the mouse. Genes Dev.
17,380
-393.
Endoh, M., Ogawa, M., Orkin, S. and Nishikawa, S.
(2002). SCL/tal-1-dependent process determines a competence to
select the definitive hematopoietic lineage prior to endothelial
differentiation. EMBO J.
21,6700
-6708.
Faloon, P., Arentson, E., Kazarov, A., Deng, C. X., Porcher, C.,
Orkin, S. and Choi, K. (2000). Basic fibroblast growth factor
positively regulates hematopoietic development.
Development 127,1931
-1941.
Fehling, H. J., Lacaud, G., Kubo, A., Kennedy, M., Robertson,
S., Keller, G. and Kouskoff, V. (2003). Tracking mesoderm
induction and its specification to the hemangioblast during embryonic stem
cell differentiation. Development
130,4217
-4227.
Gu, H., Marth, J. D., Orban, P. C., Mossman, H. and Rajewsky, K. (1994). Deletion of a DNA polymerase beta gene segment in T cells using cell type-specific gene targeting. Science 265,103 -106.[Medline]
Hart, A. H., Hartley, L., Sourris, K., Stadler, E. S., Li, R.
L., Stanley, E. G., Tam, P. P. L., Elefanty, A. G. and Robb, L.
(2002). Mixl1 is required for axial mesendoderm morphogenesis and
patterning in the murine embryo. Development
129,3597
-3608.
Henry, G. L. and Melton, D. A. (1998). Mixer, a
homeobox gene required for endoderm development.
Science 281,91
-96.
Huber, O., Bierkamp, C. and Kemler, R. (1996). Cadherins and catenins in development. Curr. Opin. Cell Biol. 8,685 -91.[CrossRef][Medline]
Johansson, B. M. and Wiles, M. V. (1995). Evidence for involvement of activin A and bone morphogenetic protein 4 in mammalian mesoderm and hematopoietic development. Mol. Cell Biol. 15,141 -151.[Abstract]
Kabrun, N., Buhring, H. J., Choi, K., Ullrich, A., Risau, W. and
Keller, G. (1997). Flk-1 expression defines a population of
early embryonic hematopoietic precursors. Development
124,2039
-2048.
Karasuyama, H. and Melchers, F. (1988). Establishment of mouse cell lines which constitutively secrete large quantities of interleukin 2, 3, 4 or 5, using modified cDNA expression vectors. Eur. J. Immunol. 18, 97-104.[Medline]
Keller, G. M. (1995). In vitro differentiation of embryonic stem cells. Curr. Opin. Cell Biol. 7, 862-869.[CrossRef][Medline]
Keller, G., Kennedy, M., Papayannopoulou, T. and Wiles, M. V. (1993). Hematopoietic commitment during embryonic stem cell differentiation in culture. Mol. Cell Biol. 13,473 -486.[Abstract]
Keller, G., Lacaud, G. and Robertson, S. (1999). Development of the hematopoietic system in the mouse. Exp. Hematol. 27,777 -787.[CrossRef][Medline]
Kennedy, M., Firpo, M., Choi, K., Wall, C., Robertson, S., Kabrun, N. and Keller, G. (1997). A common precursor for primitive erythropoiesis and definitive haematopoiesis. Nature 386,488 -493.[CrossRef][Medline]
Kikuchi, Y., Trinh, L. A., Reiter, J. F., Alexander, J., Yelon,
D. and Stainier, D. Y. (2000). The zebrafish bonnie and clyde
gene encodes a Mix family homeodomain protein that regulates the generation of
endodermal precursors. Genes Dev.
14,1279
-1289.
Kinder, S. J., Tsang, T. E., Quinlan, G. A., Hadjantonakis, A.
K., Nagy, A. and Tam, P. P. (1999). The orderly allocation of
mesodermal cells to the extraembryonic structures and the anteroposterior axis
during gastrulation of the mouse embryo. Development
126,4691
-4701.
Kubo, A., Shinozaki, K., Shannon, J. M., Kouskoff, V., Kennedy,
M., Woo, S., Fehling, H. J. and Keller, G. (2004).
Development of definitive endoderm from embryonic stem cells in culture.
Development 131,1651
-1662.
Lacaud, G., Robertson, S., Palis, J., Kennedy, M. and Keller,
G. (2001). Regulation of hemangioblast development.
Ann. New York Acad. Sci.
938,96
-107.
Lacaud, G., Gore, L., Kennedy, M., Kouskoff, V., Kingsley, P.,
Hogan, C., Carlsson, L., Speck, N., Palis, J. and Keller, G.
(2002). Runx1 is essential for hematopoietic commitment at the
hemangioblast stage of development in vitro. Blood
100,458
-466.
Latinkic, B. V. and Smith, J. C. (1999).
Goosecoid and mix.1 repress Brachyury expression and are required for head
formation in Xenopus. Development
126,1769
-1779.
Lemaire, P., Darras, S., Caillol, D. and Kodjabachian, L.
(1998). A role for the vegetally expressed Xenopus gene Mix.1 in
endoderm formation and in the restriction of mesoderm to the marginal zone.
Development 125,2371
-2380.
Maye, P., Becker, S., Kasameyer, E., Byrd, N. and Grabel, L. (2000). Indian hedgehog signaling in extraembryonic endoderm and ectoderm differentiation in ES embryoid bodies. Mech. Dev. 94,117 -132.[CrossRef][Medline]
Mead, P. E., Brivanlou, I. H., Kelley, C. M. and Zon, L. I. (1996). BMP-4-responsive regulation of dorsal-ventral patterning by the homeobox protein Mix.1. Nature 382,357 -360.[CrossRef][Medline]
Mishina, Y., Suzuki, A., Ueno, N. and Behringer, R. R. (1995). Bmpr encodes a type I bone morphogenetic protein receptor that is essential for gastrulation during mouse embryogenesis. Genes Dev. 9,3027 -3037.[Abstract]
Mohn, D., Chen, S. W., Dias, D. C., Weinstein, D. C., Dyer, M. A., Sahr, K., Ducker, C. E., Zahradka, E., Keller, G., Zaret, K. S. et al. (2003). Mouse Mix gene is activated early during differentiation of ES and F9 stem cells and induces endoderm in frog embryos. Dev. Dyn. 226,446 -459.[CrossRef][Medline]
Nishikawa, S. I., Nishikawa, S., Hirashima, M., Matsuyoshi, N.
and Kodama, H. (1998). Progressive lineage analysis by cell
sorting and culture identifies FLK1+VE-cadherin+ cells at a diverging point of
endothelial and hemopoietic lineages. Development
125,1747
-1757.
Park, C., Afrikanova, I., Chung, Y. S., Zhang, W. J., Arentson,
E., Fong Gh, G., Rosendahl, A. and Choi, K. (2004). A
hierarchical order of factors in the generation of FLK1- and SCL-expressing
hematopoietic and endothelial progenitors from embryonic stem cells.
Development 131,2749
-2762.
Peale, F. V., Jr, Sugden, L. and Bothwell, M. (1998). Characterization of CMIX, a chicken homeobox gene related to the Xenopus gene mix.1. Mech. Dev. 75,167 -170.[CrossRef][Medline]
Pearce, J. J. and Evans, M. J. (1999). Mml, a mouse Mix-like gene expressed in the primitive streak. Mech. Dev. 87,189 -192.[CrossRef][Medline]
Poulain, M. and Lepage, T. (2002). Mezzo, a
paired-like homeobox protein is an immediate target of Nodal signalling and
regulates endoderm specification in zebrafish.
Development 129,4901
-4914.
Robb, L. and Tam, P. P. (2004). Gastrula organiser and embryonic patterning in the mouse. Semin. Cell Dev. Biol. 15,543 -554.[CrossRef][Medline]
Robb, L., Hartley, L., Begley, C. G., Brodnicki, T. C., Copeland, N. G., Gilbert, D. J., Jenkins, N. A. and Elefanty, A. G. (2000). Cloning, expression analysis, and chromosomal localization of murine and human homologues of a Xenopus Mix gene. Dev. Dyn. 219,497 -504.[CrossRef][Medline]
Robertson, S. M., Kennedy, M., Shannon, J. M. and Keller, G.
(2000). A transitional stage in the commitment of mesoderm to
hematopoiesis requiring the transcription factor SCL/tal-1.
Development 127,2447
-2459.
Rosa, F. M. (1989). Mix.1, a homeobox mRNA inducible by mesoderm inducers, is expressed mostly in the presumptive endodermal cells of Xenopus embryos. Cell 57,965 -974.[Medline]
Schuh, A. C., Faloon, P., Hu, Q. L., Bhimani, M. and Choi,
K. (1999). In vitro hematopoietic and endothelial potential
of flk-1(-/-) embryonic stem cells and embryos. Proc. Natl. Acad.
Sci. USA 96,2159
-2164.
Smith, J. C., Price, B. M., Green, J. B., Weigel, D. and Herrmann, B. G. (1991). Expression of a Xenopus homolog of Brachyury (T) is an immediate-early response to mesoderm induction. Cell 67,79 -87.[Medline]
Stein, S., Roeser, T. and Kessel, M. (1998). CMIX, a paired-type homeobox gene expressed before and during formation of the avian primitive streak. Mech. Dev. 75,163 -165.[CrossRef][Medline]
Sun, X., Meyers, E. N., Lewandoski, M. and Martin, G. R.
(1999). Targeted disruption of Fgf8 causes failure of cell
migration in the gastrulating mouse embryo. Genes Dev.
13,1834
-1846.
Tada, M., Casey, E. S., Fairclough, L. and Smith, J. C.
(1998). Bix1, a direct target of Xenopus T-box genes, causes
formation of ventral mesoderm and endoderm.
Development 125,3997
-4006.
Takahashi, T., Lord, B., Schulze, P. C., Fryer, R. M., Sarang,
S. S., Gullans, S. R. and Lee, R. T. (2003). Ascorbic acid
enhances differentiation of embryonic stem cells into cardiac myocytes.
Circulation 107,1912
-1916.
Tam, P. P. and Behringer, R. R. (1997). Mouse gastrulation: the formation of a mammalian body plan. Mech. Dev. 68,3 -25.[CrossRef][Medline]
Tam, P. P., Gad, J. M., Kinder, S. J., Tsang, T. E. and Behringer, R. R. (2001). Morphogenetic tissue movement and the establishment of body plan during development from blastocyst to gastrula in the mouse. BioEssays 23,508 -517.[CrossRef][Medline]
Tam, P. P., Kanai-Azuma, M. and Kanai, Y. (2003). Early endoderm development in vertebrates: lineage differentiation and morphogenetic function. Curr. Opin. Genet. Dev. 13,393 -400.[CrossRef][Medline]
Trinh, L. A., Meyer, D. and Stainier, D. Y.
(2003). The Mix family homeodomain gene bonnie and clyde
functions with other components of the Nodal signaling pathway to regulate
neural patterning in zebrafish. Development
130,4989
-4998.
Vize, P. D. (1996). DNA sequences mediating the transcriptional response of the Mix.2 homeobox gene to mesoderm induction. Dev. Biol. 177,226 -231.[CrossRef][Medline]
Wiles, M. V. and Johansson, B. M. (1997). Analysis of factors controlling primary germ layer formation and early hematopoiesis using embryonic stem cell in vitro differentiation. Leukemia Suppl. 11,454 -456.[CrossRef]
Wiles, M. V. and Johansson, B. M. (1999). Embryonic stem cell development in a chemically defined medium. Exp. Cell Res. 247,241 -248.[CrossRef][Medline]
Winnier, G., Blessing, M., Labosky, P. A. and Hogan, B. L. (1995). Bone morphogenetic protein-4 is required for mesoderm formation and patterning in the mouse. Genes Dev. 9,2105 -2116.[Abstract]
Yamaguchi, T. P., Harpal, K., Henkemeyer, M. and Rossant, J. (1994). fgfr-1 is required for embryonic growth and mesodermal patterning during mouse gastrulation. Genes Dev. 8,3032 -3044.[Abstract]