Erasmus MC, Department of Cell Biology, PO Box 1738, 3000 DR Rotterdam, The Netherlands
* Author for correspondence (e-mail: j.philipsen{at}erasmusmc.nl)
Accepted 22 March 2004
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SUMMARY |
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Key words: Erythropoiesis, Gata1, Homotypic signalling, REDS
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Introduction |
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Gata1 belongs to the GATA family of zinc-finger transcription factors
(Evans and Felsenfeld, 1989;
Patient and McGhee, 2002
;
Tsai et al., 1989
;
Yamamoto et al., 1990
). It is
mainly expressed in haematopoietic cells (erythroid cells, megakaryocytes,
eosinophils and mast cells) (Hannon et al.,
1991
; Martin and Orkin,
1990
; Patient and McGhee,
2002
; Romeo et al.,
1990
; Weiss and Orkin,
1995a
) but also in Sertoli cells of the testis
(Ito et al., 1993
;
Yomogida et al., 1994
). Gata1
recognises a consensus binding motif that is present in the regulatory
elements of all erythroid-specific genes examined, including the
Gata1 gene itself (Ohneda and
Yamamoto, 2002
). Correct regulation of Gata1 levels appears
crucial for normal primitive and definitive erythropoiesis. Erythroid cells
null for Gata1 undergo apoptosis at the relatively immature proerythroblast
stage (Pevny et al., 1995
;
Pevny et al., 1991
;
Weiss et al., 1994
;
Weiss and Orkin, 1995b
) and
Gata1 knockout mice die of anaemia at around 11.5 dpc
(Fujiwara et al., 1996
). The
Gata1 gene is X-linked (Zon et
al., 1990
) and, owing to X-inactivation, female mice heterozygous
for a functional Gata1 gene have two populations of erythroid cells
with respect to Gata1 expression, one that is wild type and one that is
Gata1 null. These mice are transiently anaemic during gestation, but
recover during the neonatal period, probably owing to the in vivo selection of
progenitors able to express Gata1. Mutations resulting in reduced levels of
Gata1 also inhibit erythroid differentiation
(McDevitt et al., 1997
;
Takahashi et al., 1997
).
Interestingly, overexpression of Gata1 in erythroid cells inhibits
erythroid differentiation both in vitro and in vivo
(Whyatt et al., 2000;
Whyatt et al., 1997
). In order
to study overexpression in vivo, mice were generated that express Gata1 from
an X-linked transgene under the control of the erythroid-specific
ß-globin gene promoter and locus control region. Transgenic males display
pancellular Gata1 overexpression in the erythroid lineage and die of anaemia
at around 13.5 dpc, because of a block in definitive erythroid differentiation
(Whyatt et al., 2000
).
Furthermore, Gata1-overexpressing erythroid colonies grown from single
precursors (colony forming units-erythroid, CFU-Es) fail to differentiate
normally in vitro. By contrast, heterocellular overexpression of Gata1, as
occurs in chimeric mice or in the heterozygous transgenic females because of
X-inactivation, results in live transgenic mice that are phenotypically
normal. Remarkably, all erythroid cells, both wild type and overexpressing
Gata1, contribute normally to the differentiated erythrocyte pool in these
animals. This shows that the defect generated by overexpression of Gata1 is
cell-nonautonomous (Whyatt and Grosveld,
2002
). The explanation of this phenomenon is a signal, that we
tentatively termed red cell differentiation signal (REDS), that is supplied by
wild-type cells and directs Gata1-overexpressing cells to differentiate
normally (Whyatt et al.,
2000
). However, Gata1-overexpressing CFU-Es isolated from the
heterocellularly overexpressing mice fail to differentiate in vitro, even
though their differentiation is normal in vivo. Thus, the defect generated by
overexpression of Gata1 is intrinsic to the erythroid cells. This in vitro
assay suggests that REDS cannot be mediated by a soluble factor and that
cell-cell contact is required for REDS signalling. Thus, the erythroblastic
island structure is an absolutely necessary context for REDS to act.
The fact that overexpressing males do not show erythroid differentiation suggests that the source of REDS must be a cell type that is reduced in numbers or absent in these males. In light of the highly organised structure of the erythroblastic island, the most likely source of REDS would be the wild-type erythroid cells in a late stage of differentiation. However, we could not exclude that a cell type other than erythroid is supplying REDS.
We therefore set out to distinguish whether REDS is a signalling mechanism
involving cells of the same type (erythroid), defined as homotypic signalling,
or a mechanism involving a different cell type other than erythroid, i.e. a
heterotypic mechanism (Fig. 1)
(Whyatt and Grosveld, 2002).
In order to substantiate our model, we decided to ablate the wild-type
erythroid cells in heterocellularly Gata1-overexpressing mice. As mentioned
above, the survival of the earliest committed erythroid precursors is
dependent on Gata1 function (Weiss and
Orkin, 1995b
) and, hence, deletion of the Gata1 gene
would result in loss of this population. As the Gata1 gene itself is
on the X-chromosome, we have exploited an erythroid specific Cre/loxP
recombination system and the process of X-inactivation to generate such mice.
Compound transgenic mice expressing the Cre recombinase under the control of
the erythroid-specific ß-globin gene promoter and locus control region
(pEV-Cre), carrying a Gata1 gene flanked by two loxP
recombination sites on one X-chromosome and carrying the Gata1
overexpression transgene on the other X-chromosome were generated. These
transgenic females display two erythroid populations because of
X-inactivation, one population overexpressing Gata1 and one population that is
Gata1 null. If the cells supplying REDS are erythroid, which would be
consistent with a homotypic mechanism for REDS, Gata1-overexpressing cells
should no longer differentiate in such compound animals, which leads us to
predict that these animals would die in utero because of anaemia caused by
impaired differentiation. However, if the cells supplying REDS are not
erythroid, which would be consistent with an heterotypic model for REDS,
Gata1-overexpressing cells would still be able to differentiate in such
compound animals. In this case, these animals would develop with half of the
erythroid progenitors, probably showing the same phenotype as heterozygous
Gata1 knockout females, which are anaemic during gestation but survive
normally to term with half of the erythroid progenitors
(Fig. 1). In summary, if the
compound females show a phenotype worse than heterozygous Gata1 knockout
females, the mechanism would be homotypic. However, if the compound females
show a phenotype equal to heterozygous Gata1 knockout females, the
mechanism would be heterotypic. In this way, we wished to identify the cell
type supplying REDS.
|
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Materials and methods |
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Foetal blood analysis
Blood samples were collected by bleeding dissected foetuses in 5 ml of
phosphate-buffered saline (PBS). Cell numbers were determined by counting in a
hemocytometer. Blood samples were also analysed in an electronic cell counter
(CASY-1, Schärfe Systems) to determine the proportion of primitive
(nucleated) and definitive (enucleated) erythrocytes in blood.
Hanging drop culture
Half of the liver from each foetus was collected and placed in 0.5 ml of
Dulbeccos modified Eagles medium (DMEM) with 20% foetal calf
serum (FCS). Foetal livers were disaggregated into single-cell suspension and
cells counted. For hanging drop cultures, 5x104 cells were
resuspended in 20 µl hanging drop medium (DMEM supplemented with 20% FCS,
0.1% ß-mercaptoethanol, 2x104 M hemin, 5 µg/ml
penicillin/streptomycin, 2 U/ml erythropoietin, 5 µg/ml insulin) and
cultured for 2 days (F.L., unpublished). Anti-mouse Fas antibody (Jo2) was
purchased from BD Pharmingen (Catalogue number 554254) and used in hanging
drop cultures at 20 µg/ml and 40 µg/ml concentrations.
Histological staining
Foetal blood and foetal liver single-cell suspension samples from each
foetus were cytocentrifuged, and the preparations were stained with neutral
benzidine and histological dyes as described
(Beug et al., 1982). Cells
cultured in hanging drops in the presence or absence of Jo2 were also
collected after 2 days of culture, cytocentrifuged and stained. Images were
acquired in an Olympus BX40 microscope. The lenses used were Plan 40X/0.65 and
Olympus Plan 100X/1.25. The acquisition software used was Viewfinder Lite
Version 1.0.125 and Studio Lite Version 1.0.124, Pixera Corporation. Image
processing was done in Adobe Photoshop 5.5.
FACS analysis
FACS analysis was performed in every foetal liver with
5x104 events taken per sample at day of collection and after
two days of hanging drop culture. Single-cell suspensions were incubated with
R-PE-conjugated TER119 antibody and 7-aminoactinomycin-D (7AAD). Cell
populations were divided as follows: non-viable (7AAD+), erythroid
(TER119+), small erythroid (TER119+/FSClow).
Ex vivo differentiation results were compared between the different
genotype/phenotypes.
Western blot analysis
The same number of cells from 13.5 dpc foetal livers at day 0, 1 and 2 of
hanging drop culture were lysed with 2xLaemmli buffer and these whole
cell extracts were analysed by western blot. The N6 Gata1 rat monoclonal
(sc-265) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz,
CA). Mouse monoclonal antibody against B23 nucleophosmin was a kind gift from
Pui K. Chan (Baylor College of Medicine, Houston, TX). Secondary antibodies
conjugated to horseradish peroxidase were purchased from Dako (DakoCytomation,
Denmark). Enhanced chemoluminescence (ECL) was performed to develop the blots
as described by the manufacturer (Amersham Pharmacia).
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Results |
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|
Analysis of the recombination driven by pEV-Cre
Southern blot analysis of foetal liver DNA from XKOX females
demonstrates that the vast majority of floxed alleles have undergone
recombination (Fig. 2B, lane
3). Although the foetal liver at this stage of development consists mainly of
erythroid cells, other haematopoietic and non-haematopoietic cells are
present. As we have found that pEV-Cre is also expressed in other tissues
(L.G., unpublished) it is important to assess recombination activity in other
Gata1-dependent cells present in the foetal liver, as well as in macrophages
(the central cells of the erythroblastic island), to avoid misinterpretation
of the data. First we determined the percentage of erythroid and non-erythroid
haematopoietic cells present in the foetal liver by sorting cell populations
by fluorescence-activated cell sorting (FACS) on the basis of antigen
expression specific for the different haematopoietic lineages. The results are
presented in Table 1A and
demonstrate that eosinophils, mast cells, megakaryocytes and macrophages
represent only a small fraction of the haematopoietic cells present in the
liver, while the vast majority of cells were classified as erythroid. In order
to assess pEV-Cre transgene encoded recombination activity in these cells we
crossed the pEV-Cre transgene and also the CAG-Cre
(Sakai and Miyazaki, 1997) (as
ubiquitous control) with ROSA26-lacZ reporter mice
(Soriano, 1999
). lacZ
expression (from the recombined ROSA26 allele) in the different lineages was
determined by staining with fluorescein di-ß-D-galactopyranoside (FDG),
which is hydrolysed into a fluorescent product by ß-galactosidase, and
analysed by FACS (Table 1B).
From this analysis, we conclude that the pEV-Cre recombination activity, after
subtraction of the background staining, is present only in 5-20% of the
non-erythroid haematopoietic cells in the foetal liver. The floxed
non-recombined allele present in non-erythroid cells does not show up in the
Southern because the contribution of these cell types in the foetal liver to
the total cell population is very low. As the recombined Gata1 gene
is X-linked, only half of the cells would have the recombined allele active.
Thus, if 20% of the mast cells express pEV-Cre through a position effect, only
10% would be Gata1-null. Assuming that all of the cKit+
SSChigh cells are mast cells (the real number is lower), 2% of the
foetal liver cells would be mast cells. This brings us to the estimation that
only 0.2% of the foetal liver cells would be Gata1-null mast cells.
Even lower numbers are obtained for the other cell types. In the erythroid
lineage, and as would be expected from the Southern blot analysis
(Fig. 2), recombination driven
by pEV-Cre was complete as was observed with CAG-Cre (data not shown). We
therefore consider that the phenotypes observed are caused by the deletion of
Gata1 in erythroid cells. Thus, pEV-Cre-mediated deletion is appropriate to
assess the nature of REDS by analysing the phenotype of compound females.
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In order to quantify how the switch from primitive to definitive erythropoiesis was affected, blood samples were monitored for cell size in an electronic cell counter (CASY-1, Schärfe Systems). The large nucleated cells are mostly primitive erythroid cells (also non-erythroid cells and possible erythroid precursors), while the small enucleated cells are definitive erythrocytes. The ratio of definitive versus primitive cell counts was plotted to measure a shift in the balance amongst the two cell types (Fig. 4A). The ratio is just above one at 13.5 dpc and increases to above two at 14.0 dpc in wild-type animals, which illustrates the cessation of primitive erythropoiesis in the yolk sac and the beginning of definitive erythropoiesis in the foetal liver starting around 11 dpc. XFLXOX females are delayed compared with wild-type animals with a lower ratio at both 13.5 and 14.0 dpc. The replacement of primitive erythrocytes in the circulation is significantly repressed in XOXY males and XKOX females. XKOXOX females are the most severely affected, displaying in blood at 14.0 dpc at least two times more primitive cells compared with definitive erythrocytes, suggesting a major block in definitive erythropoiesis.
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Analysis of the foetal liver, the site of definitive erythropoiesis, of Gata1 mutant mice
To examine the process of differentiation at the source of definitive
erythroid cells at this stage and confirm the anaemias, cytospins of
disaggregated foetal livers were also stained and analysed. Foetal livers from
all genotypes analysed have similar size, suggesting that colonisation of the
liver by erythroid precursors is not affected in any genotype. An example
cytospin for each genotype at 14.0 dpc is shown in
Fig. 5A. Wild-type and
XFLXOX transgenic female foetal livers contain erythroid
precursors at all stages of differentiation. As previously reported
(Whyatt et al., 2000),
XOXY male foetal livers contain fewer cells late in the
differentiation process, i.e. fewer orthochromatic erythroblasts and
enucleated definitive cells. XKOX female foetal liver samples
differ from wild-type samples in that they contain fewer cells beyond the
polychromatic erythroblast stage. Similarly, XKOXOX
female foetal liver samples contain fewer benzidine-positive cells. In these
three genotypes, there is a clear impairment in definitive erythropoiesis. The
reason for this in the XOXY male is that Gata1-overexpressing cells
are intrinsically defective, as described previously. In the case of the
XKOX female, the data suggest that owing to the loss of half of the
precursors (Gata1-null cells), the wild-type population has an
altered balance favouring proliferation versus differentiation in order to
increase the numbers of progenitors to normal levels. In the case of the
XKOXOX females, the data show that the
Gata1-overexpressing cells are not rescued by another cell type. Thus REDS
appears to be blocked, favouring the notion of REDS acting through a homotypic
signalling mechanism.
|
In order to determine the ex vivo differentiation capability of erythroid
progenitors, hanging drop assays were performed. In this assay, 14.0 dpc
foetal livers were disaggregated to single cells and then cultured for 2 days
in hanging drops. After culture, cells were analysed by FACS to measure
differentiation (Whyatt et al.,
2000). Differentiation is scored as the percentage of alive (7AAD
negative), erythroid (TER119 positive) and low forward scattered cells
(FSClow), i.e. small cells corresponding to enucleated
erythrocytes. Because the erythroblastic island is disrupted in this assay
REDS signalling is lost (Whyatt et al.,
2000
), which means that the REDS-dependent cells will not
differentiate. Differentiation rates in wild-type animals are between 15 and
23% of the total (Fig. 6,
light-grey bars). As expected, male XOXY foetal liver cells fail to
differentiate. XFLXOX female foetal liver cells display
an intermediate phenotype relative to wild-type animals and XOXY
males, reflecting that on average half of the erythroid cells are wild type
(the rest is Gata1 overexpressing) (Whyatt
et al., 2000
). Similarly, differentiation rates of XKOX
female foetal liver cells are comparable with those of
XFLXOX mice, as half of these cells are wild type (the
rest are Gata1 null). XKOXOX female-derived cells
differentiated as poorly as XOXY males, in agreement with the fact
that there are no wild-type cells, i.e. 50% of these cells are Gata1-null and
the rest are overexpressing Gata1. The percentage of differentiation relative
to the alive cell counts is depicted in
Fig. 6 in dark grey bars.
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Discussion |
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The crossing strategy generated a set of transgenic mice that are either
wild type, or overexpress Gata1 in all erythroid cells (XOXY
males), or overexpress Gata1 in 50% of cells (XFLXOX
females), or are null for Gata1 in 50% of cells (XKOX females), or
overexpress Gata1 in 50% of cells and are null for Gata1 in the other 50%
(XKOXOX females)
(Fig. 1A,
Fig. 3A). We showed that
recombination was efficient, and Gata1-null male foetuses generated using this
Cre/loxP system are extremely anaemic by 12.5 dpc and die in utero
(D.W. and F.L., unpublished). Interestingly, XKOX females display
no reduction in survival, whereas classical Gata1-null heterozygous females
are clearly affected (Fujiwara et al.,
1996). This is expected as the XKOX females lose the
Gata1 gene during differentiation (as the Cre transgene is
activated), which presumably results in residual levels of Gata1 and a less
severe phenotype. Such a situation is similar to the mice where Gata1 levels
have been reduced by a promoter knockout, males die and heterozygous females
survive normally (Takahashi et al.,
1997
).
The different genotypes obtained from the breeding were essential controls for comparison. The compound female XKOXOX has lost 50% of the erythroid precursors (Gata1-null cells that apoptose), while the remaining population is overexpressing Gata1. These females are therefore pancellularly overexpressing Gata1 in the remaining erythroid compartment. If a non-erythroid cell type supplied REDS, the remaining Gata1 overexpressing population (50%) would still be rescued by REDS in the compound female, as happens in the XFlXOX female. The phenotype of the compound female would be comparable with that of XKOX females, i.e. transient anaemia, born normal. Conversely, if erythroid cells supply REDS, the source of the signal is ablated in the compound females and hence, the Gata1-overexpressing erythroid cells will not be rescued. The resulting phenotype would be expected to be more severe than that found in XKOX females and this is what is observed.
We have found that XKOX females undergo a transient anaemia, but survive to birth. By contrast, XKOXOX females are anaemic and die by 14.0 dpc, none survive to birth. In addition, the number of erythrocytes in blood in the XKOXOX females was consistently lower than that found in XKOX females. The shift from primitive to definitive erythropoiesis was impaired in both XKOXOX and XKOX females, though XKOXOX females appeared to be consistently affected while XKOX females displayed some variability. This variability may be due to variation in the X-inactivation balance in the XKOX females. Consistent with the inhibition of definitive erythroid differentiation, XKOXOX females had significantly fewer viable erythroid cells in the foetal liver than did XKOX females.
These results demonstrate that the cell type supplying REDS is the normally differentiating erythroid cell. In the compound female, the early cells are present (Gata1-overexpressing cells) and the only erythroid population missing is the more mature cells. Thus, the signal must be provided by erythroid cells in a late stage of differentiation.
These experiments do not address the identity of REDS itself. At present,
the best candidates are the death receptor family of signalling molecules.
Differentiating erythroid cells express death receptors and mature erythroid
cells express their ligands (Barcena et
al., 1999; Dai et al.,
1998
; De Maria et al.,
1999a
; De Maria et al.,
1999b
; Josefsen et al.,
1999
; Maciejewski et al.,
1995
; Oda et al.,
2001
; Silvestris et al.,
2002
; Zamai et al.,
2000
). Death receptors activate caspases and caspase activation is
thought to be required for terminal erythroid differentiation
(Kolbus et al., 2002
;
Zermati et al., 2001
). It has
been demonstrated that death receptor activation can induce the
caspase-dependent degradation of Gata1 (De
Maria et al., 1999b
). Furthermore, in zebrafish, expression of a
dominant-negative form of a haematopoietic death receptor dysregulates
erythroid cell production (Long et al.,
2000
). Thus, death receptor-mediated activation of Gata1
degradation may be a component of REDS. As we have demonstrated that REDS is a
homotypic signalling mechanism that takes place between erythroid cells,
identification of the signalling molecules involved is focussed on molecules
expressed by differentiating erythroid cells.
We induced one of the known death receptor pathways and showed that these can act at the last stages of differentiation of erythroid cells. At present, we do not know which of the pathways is used by REDS. Although erythroid cells can differentiate ex vivo under the appropriate conditions, they are arranged differently in hanging drops when compared with erythroblastic islands. The ex vivo differentiation of wild-type cells is improved by the Jo2-mediated induction of FasR, as shown by an increase in the number of enucleated cells. Gata1 levels decrease during differentiation and these levels decrease even more in FasR-activated cells. We therefore conclude that terminal differentiation of erythroid cells is enhanced by Jo2 mimicking the action of REDS, that occurs in the erythroblastic island in vivo. However, when the levels of Gata1 are very high, the Jo2 treatment ex vivo cannot provide a sufficient decrease in the levels of Gata1 to rescue overexpressing cells, while the REDS pathway can rescue the same cells in the erythroblastic island (in XOXX females). Hence, we conclude that the required decrease of Gata1 levels in vivo is achieved by a pathway similar to that of FasR.
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ACKNOWLEDGMENTS |
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