1 Laboratory of Molecular Cell Biology, Ecole Normale Supérieure de Lyon,
UMR CNRS 5161, INRA 1237, IFR128 Biosciences Lyon-Gerland, 46 Allée
d'Italie, 69364 Lyon Cedex 07, France
2 Department of Gynecologic Endocrinology and Reproductive Medicine, Medical
University of Vienna, Vienna, Austria
3 Research Institute of Molecular Pathology (IMP), Dr Bohr Gasse 7, 1030 Vienna,
Austria
4 Université Claude Bernard Lyon I, 43 bd du 11 Novembre 1918, 69622
Villeurbanne cedex, France
* Author for correspondence (e-mail: jacques.samarut{at}ens-lyon.fr)
Accepted 16 December 2004
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SUMMARY |
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Key words: Thyroid hormone, Nuclear receptor, Erythropoiesis, Mouse
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Introduction |
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Here, we have studied the role of TH and TRs on mouse erythropoiesis in vivo under the hypothesis that they play a transient role during early postnatal development.
Soon after birth the TH blood level, extremely low in the fetus, increases
sharply by about 2000-fold, peaks at 2 weeks after birth and then decreases to
the low levels observed under normal conditions in adults
(Morreale de Escobar et al.,
1994, Campos-Barros et al.,
2000
, Hadj-Sahraoui et al.,
2000
). During the transition from fetal to newborn life, which
resembles TH-mediated amphibian metamorphosis, TR
s switch from
apo-receptors, due to the hypothyroid fetal environment, into holo-receptors
after the sudden burst of TH production at birth, and turn on target gene
expression and physiologic functions, as recently demonstrated in the heart
(Mai et al., 2004
).
Using mice lacking TRs, or the congenital hypothyroid
Pax8-/- mice lacking TH
(Mansouri et al., 1998), we
now provide evidence that TH and TR
, but not TRß, are specifically
required for normal spleen erythropoiesis during early postnatal development.
Moreover, we show that the effect of TH is exerted on spleen erythrocytic
precursor cells in a cell-autonomous fashion. The precursors lacking TR
do not recognize the spleen environment to complete their differentiation.
These data demonstrate that T3, signaling through its receptor TR
, is
necessary for the development of erythropoiesis within the postnatal spleen
environment.
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Materials and methods |
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Flow cytometry
Single cell suspensions obtained from spleens and bone marrow were analyzed
with the following antibodies: FITC-B220, PE-TER119, FITC-CD71 and AnnexinV
(Pharmingen, San Diego). Cells were analyzed on a FACScan machine (Becton
Dickinson) using Cell Quest software. The four different erythrocytic
populations were defined according to Socolovsky et al.
(Socolovsky et al., 2001): I:
Ter119medCD71high; II:
Ter119highCD71high; III:
Ter119highCD71med; IV:
Ter119highCD71low. For the calibration and
standardization of the procedures, 60 000 cells were analyzed. For the
comparative analyses between animals the number of analyzed cells was reduced
to 10,000 and was the same for all genotypes and experimental conditions. No
significant differences were observed in the frequencies of the different
populations between the two numbers of tested cells. The reduction in the
number of tested cells was decided to preserve most of the cells for other
purposes, mainly molecular biology studies.
KI-67 nuclear staining to identify proliferating cells was performed as described at http://icg.cpmc.columbia.edu/cattoretti/Protocol/flowcytometry/IntracellularStaining.html.
Primary cell isolation, culture and differentiation
Primary splenocytes and bone marrow cells were isolated from 10-day-old
(P10) SV129 mice, passed through a 70 µm cell strainer and washed. To
analyze their proliferation potential, the cells were seeded and expanded in
the proliferation medium containing serum-free erythroid medium (StemPro34TM;
Life Technologies) supplemented with 2 units/ml human recombinant
erythropoietin (EPO; Janssen-Cilag), 100 ng/ml murine recombinant stem cell
factor (R&D Systems), 10-6 mol dexamethasone (Sigma) and 40
ng/ml insulin-like growth factor 1 (Promega), as described for fetal liver and
bone marrow cells (Dolznig et al.,
2001; von Lindern et al.,
2001
; Kolbus et al.,
2002
). Cultures were kept between 3 and 5x106
cells/ml, counted daily and cultured for 6 days to enrich for erythrocytic
progenitors.
To induce differentiation, cells were centrifuged (7 minutes at 700 g) to remove dead and differentiated cells and to obtain a 99% homogenous population of proliferating erythroid progenitors. Cells were washed with PBS and reseeded at 3x106 cells/ml in differentiation medium containing serum-free erythroid medium (StemPro34TM; Life Technologies) supplemented with 10 units/ml Epo, insulin (4x10-4 IE=10 ng/ml, Actrapid® HM; Novo Nordisk), and 1 mg/ml iron-saturated human transferrin (Sigma). When indicated, 10-7 mol of T3 (Sigma-Aldrich) was added to the cultures. The percentage of viable cells was determined as a ratio of the number of Trypan-Blue-excluding cells over total cell number.
CFU-Es and BFU-Es colony assay
For evaluation of burst-forming unit erythrocytic (BFU-Es) and
colony-forming unit erythrocytic (CFU-Es), spleen cells were seeded at various
concentrations (2x105 to 2x106 cells/well)
into semisolid medium MethoCultM334 (Stem Cell Technologies) in 12 well
dishes. For evaluation of CFU-Es, small compact colonies were counted 48 hours
later and for BFU-Es burst were counted 6 days later.
Cell morphology and histological staining
Erythroblasts at various stages of differentiation were cytocentrifuged
onto glass slides and stained with neutral benzidine (Sigma), to detect
hemoglobin-expressing cells, and with May-Gruenwald Giemsa (MGG) staining.
Differentiated and undifferentiated cells were distinguished in the following
manner: differentiated hemoglobin-positive cells are either nucleated cells
with brown cytoplasm, or enucleated brown stained erythrocytes;
undifferentiated hemoglobin-negative cells are larger cells with blue
cytoplasm. Following these criteria, differentiated and undifferentiated cells
were counted after visual inspection under the microscope, evaluating >300
cells per sample on multiple, randomly selected fields.
Reconstitution of lethally irradiated mice
SV129 wild-type or TR0/0 mice (8-12 weeks old) were
lethally irradiated with 10 Gy and received a retro-orbital injection of
spleen suspension in PBS from 10-day-old mice. Eleven days after grafting,
mice were sacrificed and reconstituted spleen analyzed by histology
(hematoxilin and eosin staining of paraffin sections), counting total cell
number, determining CFU-E numbers and cell morphology analysis on cytospin
smears after MGG staining.
Statistical analyses
Values reported are means ± standard deviation (s.d.).
P-values were calculated by two-way ANOVA when mice of different
genotypes and treatments were compared and by Student's t-test when
comparisons were made within the same genotype.
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Results |
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Late basophilic erythroblasts are the spleen cells mainly affected by TH/TR deficiency
The expression of the surface markers CD71 and TER119, which define
specific steps of differentiation of erythroblast precursors toward terminally
differentiated enucleated red blood cells, was used to identify the population
affected by TH/TR deficiency. CD71highTER119med
(fraction I) defines pro-erythroblasts;
CD71highTER119high (fraction II) defines basophilic
erythroblasts; CD71medTER119high (fraction III) defines
late basophilic erythroblasts; and CD71lowTER119high
(fraction IV) defines the ortochromatophilic erythroblasts
(Socolovsky et al., 2001
). A
strong defect in the distribution of TER119+ cells was observed in
splenocytes of 2-week-old hypothyroid Pax8-/- and
TR
0/0 mice (Fig.
2A,B): fraction III (CD71medTER119high)
representing late basophilic erythroblasts was reduced by at least 5-fold in
these mutant mice. TH injection into Pax8-/- mice, but not
into TR
0/0, resulted in an increase of this fraction after
only 48 hours, suggesting that this is the erythrocytic population mostly
affected by TH. Cytological analysis confirmed the decreased number of late
basophilic erythroblasts in Pax8-/- mice and their
reappearance upon TH treatment (Fig.
2C, arrows). These results suggest that TH through TR
influences the late and not the early phases of erythrocytic differentiation.
To have an overview of the whole sequence of differentiation, we determined
the relative frequencies of each differentiation step, including CFU-Es and
BFU-Es (these two last progenitors being determined by in-vitro colony
assays). The respective frequencies of all erythrocytic progenitors were
normalized to that of the BFU-Es. As shown in
Fig. 2D, the
Pax8-/- mice showed a specific defect in the relative
number of late basophilic erythroblasts (fraction III) compared with control
mice. By contrast, Pax8-/- mice treated with TH for 48
hours showed a strong enhancement in the representation of these same cells.
This observation confirms that the defect in erythropoiesis in the mutant
Pax8-/- mice resides at the level of production of late
basophilic erythroblasts. To determine if TH controls proliferation or
survival of late basophilic erythroblasts, TER119+ splenocytes
obtained from control, Pax8-/-, and
Pax8-/- mice treated with TH, were stained with antibodies
against the Ki-67 antigen or Annexin V, markers of proliferation and apoptosis
respectively. Although not statistically different, the number of apoptotic
cells was slightly decreased in Pax8-/- mutant mice
compared with controls (Fig.
2F). Clearly the major effect of the mutation was a decrease in
the number of TER119+ Ki67+ proliferating cells
(Fig. 2E).
TER119+Ki67+ double positive cells strongly increased
upon TH treatment of Pax8-/- mice. Thus, TH affects mostly
the proliferation of late basophilic erythroblasts.
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TR affects the ability of erythrocytic progenitors to develop in spleens of lethally irradiated mice
The results described above suggest a direct effect of TH on spleen
erythropoiesis in vivo and ex vivo. However, the situation may be more complex
in vivo, due to the pleiotropic effects of TH and the possibility that TH
influences erythropoiesis indirectly by acting on the spleen environment. To
address this question, lethally irradiated adult wild-type and
TR0/0 mice were reconstituted with splenocyte suspensions
isolated from 10-day-old TR
0/0 or wild-type mice. Eleven
days after reconstitution, grafted mice were sacrificed and their spleens
analyzed. At this time, the hematopoietic reconstitution consisted mainly of
cells of the erythrocytic lineage, as observed by benzidine and MGG staining
(Fig. 6D-F) and by FACS
analysis (not shown). Macroscopically, spleens obtained from wild-type mice
reconstituted with TR
0/0 splenocytes were smaller than
spleens reconstituted with wild-type cells, while no major differences were
observed in TR
0/0 mice reconstituted with wild-type cells
(not shown). In wild-type recipient mice, grafted with TR
0/0
splenocytes, spleen cellularity was decreased by about 40% compared with
wild-type mice grafted with wild-type cells. By contrast, no major defects
were observed in spleen cellularity of recipient TR
0/0 mice
grafted with wild-type splenocytes (Table
1). In agreement with this observation, histological examination
on recipient spleen sections after hematoxilin and eosin coloration, showed
significantly smaller hematopoietic colonies when wild-type mice had been
reconstituted with TR
0/0 splenocytes
(Fig. 6B); whereas no major
difference was observed in TR
0/0 or wild-type recipient
spleen grafted with wild-type splenocytes
(Fig. 6A,C). These results
indicate that TR
exerts a direct effect on erythrocytic cells and not
on the spleen environment.
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Discussion |
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In mouse, erythropoiesis is regulated timely throughout life in three
different organs (reviewed by Godin and
Cumano, 2002). It starts in the yolk sac at embryonic day (E) 7.5
to give rise to primitive erythropoiesis, then in fetal liver to produce
definitive erythrocytic cells. By E12, the fetal liver, colonized by
hematopoietic stem cells (HSCs), becomes the main erythropoietic organ until
birth (Delassus and Cumano,
1996
; Robb, 1997
;
Dzierzak et al., 1998
;
Keller et al., 1999
;
Wolber et al., 2002
). The bone
marrow initiates production of red blood cells at birth and then constantly
raises its production until adult age. The spleen is colonized at E12 by HSCs
originating in the aorta-gonad-mesonephros area
(Godin et al., 1999
), but its
erythropoietic potential is confined to the first two postnatal weeks. During
this period, the spleen contains a number of early erythrocytic progenitors
comparable to bone marrow, but its contribution to blood cell production might
not be as much as bone marrow, and it is considered as an additional
erythropoietic organ for stress conditions
(Wolber et al., 2002
). Even if
BFU-Es are still found in constant numbers in the spleen between the pup and
adult stages, the bone marrow remains by far the major erythropoietic organ
during adult life (Wolber et al.,
2002
). The physiological mediators of the switch of erythropoietic
activity from one organ to another are not yet known.
The results presented here demonstrate that TH via TR is required
specifically for normal spleen, and not bone marrow, erythropoiesis during
early postnatal development. Both mice deficient in TH production
(Pax8-/- congenital hypothyroid), and mice devoid of
TR
receptors (TR
0/0), show a defect in spleen
erythropoiesis soon after birth, which could be quickly rescued in
Pax8-/- mice upon injection of TH. Interestingly, the
defect in red cell production takes place at a late stage of erythrocytic
differentiation. Indeed, Pax8-/- and
TR
0/0 mice showed only minor changes in the relative
frequencies of BFU-Es, CFU-Es and early erythroblasts, but a strong decrease
in late basophilic erythroblasts. This population was also the one that
increased sharply upon TH injection, as a consequence of enhanced
proliferation. Yet, there was no major effect on the population at the next
developmental stage, the ortochromatophilic erythroblasts. Several hypotheses
may be put forward. First, this population is close to the terminal
differentiation step and might have a longer turn over time. Second, many of
these cells might be circulating cells coming from the bone marrow, thus
masking a decreased production from the spleen. In agreement with this
hypothesis, the defect in spleen erythropoiesis did not result in major anemia
in mutant animals because of bone marrow erythropoiesis compensation.
One major question about the role of TH signaling on spleen erythropoiesis
was to know whether it results from a direct effect on erythrocytic
progenitors or from an indirect effect on the splenic environment. Two
approaches showed that TH and TR work directly on erythrocytic cells.
First, when cultured ex vivo, wild-type spleen erythroblasts showed enhanced
proliferation and differentiation in the presence of T3, while T3 had no
effect on splenic erythroblasts derived from TR
0/0 mice.
Second, when TR
0/0 spleen cell progenitors were grafted into
a wild-type spleen environment, the production of mature red cells was
impaired, whereas the reverse grafting of wild-type progenitors into a
TR
0/0 spleen environment gave rise to normal erythropoietic
production. These grafting experiments demonstrate that the defect is
intrinsic to TR
-expressing erythrocytic progenitors. This conclusion is
consistent with the observation that spleen erythroblasts do express TR
mRNAs and presumably the receptor (not shown).
We ignore at present the reasons for the specificity of TH on spleen
erythrocytic cells, as TR mRNA expression was also detected in bone
marrow erythroblasts (not shown). It is possible that TR
is active only
in splenic erythroblasts because of the presence of specific transcription
co-activators or co-repressors, themselves under the control of specific
splenic signals. Alternatively, different progenitors may exist in the spleen
and in the bone marrow. Proof of the presence of a specific erythroblast
precursor in the spleen has been obtained so far only under conditions of
stress-induced erythropoiesis in adult anemic mice
(Bauer et al., 1999
). However,
it is not clear if this precursor is already present in the spleen during
early postnatal development and if it is under TH control. Finally, it is
possible that erythrocytic differentiation normally depends on interactions
with the spleen environment and that this interaction is defective in
TH/TR
mutant mice. Our data support this last possibility. Indeed,
whereas TR
0/0 erythrocytic progenitors cannot completely
achieve differentiation within the wild-type spleen environment, they can
undergo full red cell differentiation when explanted in the in-vitro CFU-E
assay (see Table 1), suggesting
that the mutant CFU-Es do not recognize the wild-type spleen environment
necessary for differentiation.
Whereas in neonatal spleen endogenous TR0/0 progenitors
show a limited differentiation, they show a higher extent of erythrocytic
differentiation when grafted into irradiated wild-type recipient spleens. One
explanation could be that the splenic environment of irradiated adult
recipient animals might allow some differentiation of TR
0/0
erythrocytic progenitors in contrast to the normal newborn spleen
environment.
Together, these data lead us to propose the following model for the role of
TH/TR signaling in erythropoiesis of the mouse. In the bone marrow,
TH/TR
signaling is not necessary for the pluripotent hematopoietic stem
cells to achieve erythrocytic differentiation. On the contrary, in the spleen
the stem cells give rise to erythrocytic progenitors (BFU-Es and CFU-Es) whose
final differentiation depends on functional interactions with the spleen
microenvironment. T3 via TR
would induce in red cell progenitors the
synthesis of TR
-target gene products that would mediate this
environmental interaction. Integrins are probable candidates, as homing of
hematopoietic progenitors in different organs is regulated by integrin
expression, as for example expression of
4/ß1 integrins and
hematopoietic progenitors homing in the bone marrow
(Scott et al., 2003
). Thus,
TR
may act by modulating the expression of integrins in erythrocytic
progenitors that home and differentiate specifically in the spleen.
Interestingly, in chicken erythrocytic progenitors blocked by the ErbA
oncoprotein, we have shown that the oncoprotein abrogates the expression of
the gene encoding the
2/ß1 integrin expression
(Mey et al., 2002
). However,
we did not observe any inhibition in the expression of this integrin in the
spleen erythrocytic cells of TR
0/0 mice (data not shown),
suggesting that in mouse a different set of integrins or proteins interacting
with the extracellular matrix might be involved. Alternatively, one could also
imagine that the wild-type spleen environment expresses compounds that inhibit
final differentiation of erythrocytic progenitors and that wild-type
progenitors but not TR
0/0 progenitors have an intact
signaling mechanism to overcome or abrogate this inhibition.
The fact that spleen-derived erythroblasts explanted in liquid cultures
could be stimulated by T3 suggests that besides controlling recognition of the
homing environment, T3/TR signaling activates other physiological
functions in spleen erythrocytic cells. It should be noticed that this direct
effect of T3 seen in vitro is not a determinant for the final differentiation
under these experimental conditions.
The observation that alteration of spleen erythropoiesis is more severe in
the Pax8-/- hypothyroid mutant than in the
TR0/0 mice is consistent with the hypothesis that in the
hypothyroid mutant erythroblasts, these target genes are strongly repressed by
the TR
apo-receptors. This assumption is supported by the fact that the
spleen defect phenotype in Pax8-/- mice is partially
overcome by inactivation of the TR
gene in these same mutants
(Flamant et al., 2002
).
This model in which T3/TR signaling controls spleen erythropoiesis
through spleen environment recognition would explain why spleen erythropoiesis
develops at birth in the mouse. Indeed, at this time the level of T3 in the
body of the pups sharply increases, peaking at the second week
(Hadj-Sahraoui et al., 2000
).
This level then decreases to reach the adult basal level. The role of T3 would
then be to support the development of a transient erythropoietic activity
between the declining fetal liver erythropoiesis and the starting bone marrow
erythropoiesis. Thus, while it is clear that T3 is not absolutely required for
general erythropoiesis, our results show that it plays an important role in
accelerating red blood production at an important changing state during
development. Our data and the model we propose add one more example after
intestine (Plateroti et al.,
2001
), heart (Mai et al.,
2004
) and brain (Morte et al.,
2002
), showing that TR
plays a major role in mouse ontogeny
by managing the transition between fetal and postnatal life. Therefore, the
early postnatal period represents a physiologic hyperthyroid situation that
aims presumably at switching on, via the transcription factor TR
, gene
expression programs involved in the maturation of many developmental processes
and physiological functions in an overall mechanism that recalls
TH/TR
-dependent amphibian metamorphosis
(Shi, 1999
;
Sachs et al., 2002
;
Buchholz et al., 2003
).
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ACKNOWLEDGMENTS |
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