From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
RED CELLS, IRON, AND ERYTHROPOIESIS
Maintenance of the BMP4-dependent stress erythropoiesis pathway in the murine
spleen requires hedgehog signaling
John M. Perry,1 Omid F. Harandi,2 Prashanth Porayette,3 Shailaja Hegde,4,5 Arun K. Kannan,4 and Robert F. Paulson1-5
1Cell
and Developmental Biology Option, 2Graduate Program in Genetics, 3Molecular Medicine Option, 4Department of Veterinary and Biomedical Sciences,
Huck Institutes of the Life Sciences, and the 5Center for Molecular Immunology and Infectious Disease, Pennsylvania State University, University Park
The production of mature cells necessitates that lineage-committed progenitor
cells be constantly generated from multipotential progenitors. In addition, the ability to respond rapidly to physiologic
stresses requires that the signals that
regulate the maintenance of progenitor
populations be coordinated with the signals that promote differentiation of progenitors. Here we examine the signals
that are necessary for the maintenance of
the BMP4-dependent stress erythropoi-
esis pathway. Our previous work demonstrated that BMP4, stem cell factor, and
hypoxia act in concert to promote the
expansion of a specialized population of
stress erythroid progenitors in the spleen
during the recovery from acute anemia.
Our analysis shows that acute anemia
leads to an almost complete mobilization
of BMP4-responsive stress erythroid
burst-forming units; therefore, new stress
progenitors must be recruited to the
spleen to replenish this system. We show
that bone marrow cells can home to the
spleen and, in response to a signal in the
spleen microenvironment, Hedgehog,
they develop into BMP4-responsive stress
progenitors. Hedgehog induces the expression of BMP4, and together these 2
signals are required for the development
of BMP4-responsive stress progenitors.
These data demonstrate that the interplay
between these 2 signals is crucial for
maintenance of this stress response pathway. (Blood. 2009;113:911-918)
Introduction
Acute anemia induces a systemic response designed to increase the
transport of oxygen to hypoxic tissues. One aspect of this response
is the increased production of erythrocytes.1 Our previous analysis
has demonstrated that acute anemia leads to the rapid expansion
and differentiation of a specialized population of stress erythroid
progenitors in the spleen.2 These progenitors are resident in the
spleen. However, their differentiation is tightly regulated and only
occurs at times of acute erythropoietic need. Part of this regulation
stems from the fact that this response requires 3 signals (BMP4,
stem cell factor [SCF], and hypoxia), and the expression BMP4 is
in part regulated by hypoxia.2,3 In response to these 3 signals, stress
erythroid burst-forming units (BFU-E) are rapidly expanded in the
spleen, which in vivo translates into a 45-fold increase in stress
BFU-E. These progenitors are ideally suited to respond to acute
anemia in that they exhibit a greater potential to generate new
erythrocytes than bone marrow steady-state progenitors.2,3
Stress response pathways by definition must be able to transiently mount a response to alleviate a physiologic stress, but once
equilibrium is restored, the pathway is inactivated.1 In addition,
stress response pathways must have a mechanism by which they
are able to maintain readiness for subsequent challenges. In this
work, we extend our analysis of the BMP4-dependent stress
erythropoiesis pathway by investigating the mechanisms that
regulate the maintenance of stress erythroid progenitors in the
murine spleen. Here we find that acute anemia mobilizes essentially all progenitors that can respond to BMP4, which results in a
loss of responsiveness to immediate rechallenge with acute anemia.
We observe that, after recovery from anemia, the BMP4-responsive
stress progenitors are replenished. Although our previous work
showed that bone marrow cells do not respond to BMP4 like spleen
stress erythroid progenitors,2 here we show that bone marrow cells
can home to the spleen and differentiate into splenic BMP4responsive stress erythroid progenitors. These data suggest that the
spleen microenvironment contains a signal that promotes the
development of stress erythroid progenitors. We have identified
that signal as Hedgehog. Treatment of bone marrow cells with
Hedgehog induces BMP4 expression, and these 2 signals act in
concert to promote the development of BMP4-responsive stress
erythroid progenitors. Furthermore, mutations that block Hedgehog signaling compromise the development of BMP4-responsive
stress erythroid progenitors in the spleen after recovery from acute
anemia and render the mice incapable of responding to subsequent
anemic challenges. Taken together, our data show that BMP4 and
Hedgehog are specific signals in the spleen that are required to
maintain extramedullary stress erythroid progenitors.
Submitted March 27, 2008; accepted September 24, 2008. Prepublished
online as Blood First Edition paper, October 16, 2008; DOI 10.1182/blood2008-03-147892.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The online version of this article contains a data supplement.
© 2009 by The American Society of Hematology
BLOOD, 22 JANUARY 2009 䡠 VOLUME 113, NUMBER 4
Methods
Mice
C57BL/6 and B6.SJLPtprcaPep3b/BoyJ (CD45.1) mice were bred in our
colony. The WBB6F1-KitW/Wv mice and the Smoothened (Smo) conditional
allele Smotm2amc/J4 were purchased from JAX Mice and Services (Bar
Harbor, ME). The conditional Patched allele (Ptcfx) was provided by
Brandon Wainwright (Institute for Molecular Bioscience, University of
Queensland, Brisbane, Queensland, Australia).5 Smo and Ptc mutant alleles
were crossed onto the C57BL/6 background at least 5 generations.
C57BL/6-Smotm2amc/J mice were crossed with the interferon-inducible Cre
recombinase transgenic mouse line, B6.Cg-Tg(Mx1-cre)1Cgn/J mice6 to
911
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
912
BLOOD, 22 JANUARY 2009 䡠 VOLUME 113, NUMBER 4
PERRY et al
A
B
P < .05
Stress BFU-E
Fold Increase
in BFU-E
10
5
0
0
12
24
36
48
4
Hours
After PHZ Injection
6
P < .05
150
100
8
50
0
36Hr
7
14
21
Days after initial
PHZ Injection
Days
Figure 1. Analysis of BMP4R stress erythroid progenitors during and after the recovery from acute anemia. (A) C57BL/6 mice were injected with PHZ to induce acute
anemia. Spleen cells were isolated on the indicated days. BMP4R cells were measured by determining the fold increase in stress BFU-E when the number of stress BFU-E
generated when cells were plated in Epo (3 U/mL) plus BMP4 (15 ng/mL) was compared with the number of stress BFU-E generated when cells were plated in Epo alone.
(B) C57BL/6 mice were treated with PHZ to induce anemia and allowed to recover for 7 days. The mice were then challenged with a second dose of PHZ as indicated. At
36 hours after the second dose, stress BFU-E were measured by plating spleen cells in methylcellulose media containing Epo (3 U/mL) alone. The response of an untreated
mouse 36 hours after the initial dose of PHZ is shown in black. For all assays, at least 3 mice were used at each time point.
generate C57BL/6-Smotm2amc;Mx1cre. C57BL/6-Ptcfx mice were crossed
with the tamoxifen-inducible Cre recombinase transgenic line, C57BL/6CAGGCre-ER7 to generate C57BL/6-Ptcfloxed; CAGGCre-ER mice. Deletion of Smo using poly(I)poly(C) injection to induce MX1-cre expression
and deletion of Ptc using 4-hydroxytamoxifan to activate CreER were done
as previously described.8,9 The efficiency of deletion was measured by
polymerase chain reaction (PCR) analysis as described.4,5 Induction of
acute anemia by phenylhydrazine injection was done as previously
described.2,3 All procedures were approved by the Institutional Animal Care
and Use Committee of the Pennsylvania State University.
The analysis of Shh, Desert Hedgehog (Dhh), and Indian Hedgehog (Ihh)
was analyzed by reverse-transcribed PCR (RT-PCR) using the following
primers.11 Shh (570 bp): forward, 5⬘-TCC GAA TTT AAG GAA CTC
ACC-3⬘; reverse, 5⬘-GGC TCC AGC GTC TCG ATC ACG TAG-3⬘. Dhh
(791 bp): forward, 5⬘-GAC CTC GTA CCC AAC TAC AAC CCC G-3⬘;
reverse, 5⬘-ACG TCG TTG ACC AGC AGC GTC C-3⬘. Ihh (668 bp):
forward, 5⬘-CAA GCT CGT GCC TCT TGC CTA CAA G-3⬘; reverse,
5⬘-GCA CAT CAC TGA AGG TGG GGG TCC-3⬘. Expression of
Smoothened in deleted floxed strains was done by RT-PCR using the
following primers: forward, 5⬘-AAC TAT CGG TAC CGT GCT GG-3⬘;
reverse, 5⬘-GCT GAA GGT GAT GAG CAC AA-3⬘.
Analysis of stress BFU-E
Stress BFU-E and steady-state BFU-E were assayed as previously described.2,3,10 In some experiments, other factors were added to the
methylcellulose BFU-E cultures. For experiments investigating the ability
of Sonic Hedgehog (Shh) to induce stress BFU-E, bone marrow cells were
preincubated in Iscove modified Dulbecco medium plus 5% fetal calf
serum supplemented 200 ng/mL Shh (R&D Systems, Minneapolis, MN) for
24 hours before plating cells in methylcellulose media. For experiments
using the BMP4 antagonist Noggin, 200 ␮g/mL Noggin (R&D Systems)
was added.
Transplantation of bone marrow or spleen cells into WBB6F1
KitW/Wv mice
A total of 2 ⫻ 106 bone marrow or spleen cells were isolated from C57BL/6
and injected into the tail vein of WBB6F1 KitW/Wv mice. Bone marrow and
spleen cells were isolated on the indicated days and the presence of stress
BFU-E, BMP4-responsive stress progenitors, BMP4R cells, and total
BFU-E were assayed. Additional transplantations were done as described
using bone marrow cells from CD45.1, poly(I)poly(C)-treated C57BL/6Smotm2amc/tm2amc; Mx1-Cre, C57BL/6-Smotm2amc/⫹; Mx1-Cre or control
C57BL/6-Smotm2amc/⫹ mice.
Immunofluoresence analysis of BMP4 expression by donor
bone marrow cells in the spleen of transplanted mice
Spleens were harvested 1 week after 2 ⫻ 106 CD45.1 spleen cells were
transplanted into WBB6F1 KitW/Wv mice. The spleens were processed for
paraffin sections as previously described.2,10 The sections were stained with
anti-BMP4 antibodies (Novocastra, Newcastle, United Kingdom; and
Vector Laboratories, Burlingame, CA) using an Alexa Fluor 660 (Invitrogen, Carlsbad, CA) secondary antibody as previously described2,10 and with
fluorescein isothiocyanate–conjugated anti-CD45.1 (BD Biosciences PharMingen, San Diego, CA) at a 1:100 dilution. Slides were analyzed by confocal
microscopy using an Olympus FV300 confocal microscope (Tokyo, Japan).
Analysis of BMP4 and Hedgehog expression in the spleen
RNA was isolated from the indicated cells or tissues using TRIzol reagent
(Invitrogen) according to the manufacturer’s instructions. Expression of
BMP4 mRNA was analyzed by real-time PCR using Taqman gene
expression assay for murine BMP4 (Applied Biosystems, Foster City, CA).
Results
BMP4-dependent stress progenitors completely differentiate
during the recovery from acute anemia
Our previous analysis of the recovery from phenylhydrazine
(PHZ)–induced acute anemia showed that BMP4-responsive stress
progenitors, BMP4R cells, which are resident in the spleen, rapidly
proliferate in response to BMP4, SCF, and hypoxia-dependent
signals to expand the population of stress BFU-E. By 36 hours after
treatment, these progenitors expand 45-fold, which leads to recovery in approximately 6 to 7 days.2,3 We would predict that mice
would be able to activate this stress erythropoiesis pathway
multiple times in response to future anemic conditions. However,
the mechanisms by which this pathway is maintained are not well
understood. To address this question, we tested whether the number
of BMP4R cells is maintained during the recovery period. C57BL/6
control mice were treated with PHZ, and the increase in stress
BFU-E after treatment with BMP4 was used as a measure of
BMP4R cells. The data in Figure 1A show that, in the first 12 hours
after treatment, BMP4R cells were present in the spleen as
demonstrated by the 5- to 7-fold increase in stress BFU-E with the
inclusion of BMP4 in the media. Our previous work showed that
BMP4 expression is induced in the spleen starting at 24 hours after
PHZ treatment.2 At this point, we no longer observed BMP4R cells
in the spleen, and it appeared that all BMP4R cells had differentiated into stress BFU-E. This lack of response was not simply the
result of the high expression of BMP4 in the spleen causing the
differentiation of all BMP4R cells because no response was also
observed at later time points (days 4-8) when expression of BMP4
is no longer observed.2 These data indicate that excess BMP4R cells
are not maintained in the spleen, and PHZ-induced acute anemia
results in the complete activation of the BMP4-dependent stress
erythropoiesis pathway.
The complete mobilization of BMP4R cells and stress BFU-E
suggest that there must be mechanisms by which this response is
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
BLOOD, 22 JANUARY 2009 䡠 VOLUME 113, NUMBER 4
Total BFU-E
(Epo+IL-3)
Total BFU-E
(Epo+IL-3)
40
20
0
0 7 14 21
Days After
Bone marrow Transplant
40
20
0
0 7 14 21
Days After
Spleen cell transplantation
Stress BFU-E
20
60
Spleen
40
20
0
0
0
7
14
21
Days After
Spleen cell Transplantation
0
7
14
21
Days After
Bone marrow Transplant
Epo + BMP4
30
Total BFU-E
(Epo+IL-3)
Total BFU-E
(Epo+IL-3)
B
20
10
0
30
20
10
0
0
7
14 21
Days After
Spleen cell Transplantation
0
7 14 21
Days After
Bone Marrow Transplant
Stress BFU-E
P < .001
Stress BFU-E
The complete differentiation of BMP4R cells during the recovery
from acute anemia and their gradual replenishment during the
2 weeks after recovery suggest that immature progenitors, which
are either present in the spleen or migrate to the spleen, give rise to
new BMP4R cells. To distinguish between these possibilities, we
established a transplantation assay that took advantage of our
previous observation that mice mutant for the Kit receptor,
WBB6F1 KitW/Wv (W/Wv), lacked BMP4R cells in their spleens.3 In
addition, these mice have been used extensively in transplantation
studies because transplantations into W/Wv mice do not require
myeloablative preconditioning before transplantation.12 We transplanted bone marrow or spleen cells from C57BL/6 control mice
into W/Wv mice. We then analyzed the spleens of recipient mice 7,
14, and 21 days after transplantation for BMP4R cells and stress
BFU-E. Stress BFU-E are able to form colonies in media containing only erythropoietin (Epo), in contrast to steady-state BFU-E,
which require a burst-promoting factor such as interleukin-3 (IL-3)
in addition to Epo. BMP4R cells differentiate into stress BFU-E
after exposure to BMP4. The increase in stress BFU-E when BMP4
is included in the media is indicative of BMP4R cells. Transplantation of spleen or bone marrow cells resulted in an increase in the
total number of BFU-E (stress ⫹ steady-state BFU-E) as measured
by plating cells in Epo plus IL-3 on each of the days analyzed. The
number of stress BFU-E in the spleen also increased. In mice
transplanted with bone marrow cells, the number of stress BFU-E
steadily increased on days 7, 14, and 21 (Figure 2A). In contrast,
when W/Wv mice were transplanted with spleen cells, we observed
that the number of stress BFU-E did not significantly change from
7 to 21 days after transplantation (Figure 2A). The situation was
quite different when we examined BMP4R cells by assaying the
increase in stress BFU-E when BMP4 was added to the cultures.
Only transplantations using bone marrow cells were able to give
rise to BMP4R cells. We observed a significant increase in stress
BFU-E when BMP4 was included in the media 14 days after
transplanting bone marrow cells (Figure 2A). Fourteen days after
transplantation is functionally equivalent to reappearance of BMP4R
cells 21 days after the initial treatment of control mice with PHZ.
BMP4R cells were not observed at 21 days after bone marrow cell
transplantation. This difference may be caused by the mobilization
of new donor-derived BMP4R cells to alleviate the anemia of the
W/Wv mice. Spleen cell transplantations did not result in BMP4R
cells in the recipient spleens at any of the time points. These data
suggest that the bone marrow, but not the spleen, contains an
immature progenitor that gives rise to BMP4R cells.
60
P < .01
40
Epo
Bone marrow cells can home to the spleen and differentiate
into BMP4R cells
913
A
Stress BFU-E
replenished. We next tested how long a control mouse takes to
recover after an initial treatment with PHZ before it can respond
like an untreated mouse. C57BL/6 mice were treated with PHZ,
and 7, 14, and 21 days after the first treatment, the mice were
rechallenged with a second dose of PHZ. We measured stress
BFU-E 36 hours after the second treatment with PHZ because our
previous work demonstrated that at this time point the maximal
expansion of stress BFU-E was observed.2 We observed that mice
treated with a second dose of PHZ 7 days after the initial dose were
unable to expand stress BFU-E in their spleens. However, by
21 days after the initial treatment, mice were able to expand stress
BFU-E similarly to control untreated mice. Interestingly, at 14 days,
there was an intermediate response suggesting that BMP4R cells
gradually repopulate the spleen after recovery from acute anemia.
HEDGEHOG IS REQUIRED FOR STRESS ERYTHROPOIESIS
30
20
10
0
30
20
10
0
0
7
14
21
Days After
Bone Marrow Transplant
Epo
0
7
14
21
Days After
Spleen Cell Transplantation
Epo + BMP4
Figure 2. Transplanted bone marrow cells give rise to BMP4R cells in the
recipient spleen. WBB6F1 KitW/Wv mice were transplanted with C57BL/6 donor bone
marrow cells or spleen cells, and the development of stress BFU-E was assayed.
(A) Analysis of stress BFU-E in the spleens of transplanted mice after transplantation
with bone marrow cells (left) or spleen cells (right). (Top graphs) Total BFU-E
observed after spleen cells were plated on indicated days in media containing
Epo ⫹ IL-3. (Bottom graphs) Stress BFU-E observed on the indicated days when
spleen cells were plated in media containing Epo or Epo ⫹ BMP4 as indicated.
(B) Analysis of stress BFU-E in the bone marrow of transplanted mice after
transplantation of bone marrow cells (left) or spleen cells (right). (Top graphs) Total
BFU-E observed after spleen cells were plated on indicated days in media containing
Epo ⫹ IL-3. (Bottom graphs) Stress BFU-E observed on the indicated days when
spleen cells were plated in media containing Epo or Epo ⫹ BMP4 as indicated. For
each time point, 3 recipient mice were analyzed. Significant differences are indicated
in the figure.
We also tested whether the transplanted cells could give rise to
BMP4R cells and stress BFU-E in the bone marrow of recipient
mice. Analysis of bone marrow cells from recipient mice showed
that transplanted either bone marrow or spleen resulted in an
increase in the total number of BFU-E (Figure 2B). However, when
we examined stress BFU-E and BMP4R cells, the situation was
different. As expected, transplanting bone marrow cells did not
result in an increase in stress BFU-E or BMP4R cells in the bone
marrow of recipients (Figure 2B). Spleen cell transplantations,
however, did result in BMP4R cells in the bone marrow 21 days
after transplantation (Figure 2B). These data suggest that the spleen
contains BMP4R cells, which expand in the bone marrow microenvironment. It also suggests that, once a cell adopts the splenic
BMP4R cell potential, it maintains that potential even if it finds
itself in a different microenvironment.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
914
BLOOD, 22 JANUARY 2009 䡠 VOLUME 113, NUMBER 4
PERRY et al
A
B
P < .05
BMP4 expression
(relative to Gapdh)
BFU-E
30
Epo
20
Epo + BMP4
10
35
30
25
20
15
10
5
0
0
Control
C
BFU-E
30
0hr
2hrs
6hrs
24hrs
+ Shh
D
P < .05
BMP4
Epo
Epo + Noggin
HPRT
0
7
14
21
Days After Transplantation
20
10
0
Control
+Shh
E
SA
RP
CD45.1
Overlay
RP
WP
WP
BMP4
Figure 3. Treatment of bone marrow cells with Sonic Hedgehog induces stress BFU-E formation. (A) Bone marrow cells
were preincubated overnight in Iscove modified Dulbecco medium plus 5% fetal calf serum supplemented with (⫹Shh) or without
(Control) Sonic Hedgehog. Afterward, the cells were plated in
methylcellulose media containing either Epo (3 U/mL) alone or
Epo ⫹ BMP4 (15 ng/mL), and BFU-E were scored. (B) Real-time
RT-PCR analysis of the expression of BMP4 by bone marrow cells
cultured in the presence or absence of Shh. (C) Bone marrow cells
were preincubated for 24 hours with Shh as described in panel A
and then plated in methylcellulose media containing Epo or
Epo ⫹ Noggin (200 ␮g/mL), and BFU-E were scored. (D) RT-PCR
analysis of BMP4 expression in the spleen of WBB6F1 KitW/Wv
recipient mice transplanted with CD45.1 donor bone marrow cells.
The vertical white line between time 0 and 7 days has been
inserted to indicate a repositioned gel lane. (E) Analysis of BMP4
expression by CD45.1⫹ donor cells in the spleen of KitW/Wv mice
after bone marrow transplantation. (Left) BMP4 is shown in red
and CD45.1 in green in a low power (original magnification ⫻20)
analysis of BMP4 expression. Overlap between the 2 signals is
shown in yellow. SA indicates splenic artery; RP, red pulp; and WP,
white pulp. (Right) Spleen sections from additional mice at higher
power (original magnification ⫻40). BMP4 is shown in red, CD45.1
in green, and the overlap in signals as yellow. All assays were done
in triplicate and are representative of 2 independent experiments.
Significant differences are indicated in the figure. Slides were
viewed with an Olympus BX61Epi Fluorescence microscope (Olympus, Center Valley, PA) using a UPlanF1 lens at 40⫻/0.75 NA and
Slow Fade Gold antifade agent (Invitrogen, Carlsbad, CA). Images
were acquired using an Olympus DP71 camera (Olympus) and
were processed with DP-BSW Basic software for the DP71
camera and Adobe Photoshop imaging software (Adobe, San
Jose, CA).
WP
RP
Treatment of bone marrow cells with Hedgehog results in the
development of BMP4R cells and stress BFU-E
Our previous work showed that bone marrow cells do not respond
to BMP4 like spleen BMP4R cells.2 However, when transplanted
bone marrow cells migrate to the spleen, they acquire the ability to
respond to BMP4. Taken together, these observations suggest that
there is a signal in the spleen microenvironment that promotes the
differentiation of bone marrow progenitors into BMP4R cells. To
identify this signal, we relied on earlier observations about the role
of BMP4 signaling in chondrocyte development. In this system, it
was proposed that somatic mesoderm cells develop into chondrocytes in response to BMP4.13-15 However, when presomitic mesoderm cells were cultured in the presence of BMP4, they failed to
differentiate into chondrocytes unless they were first treated with
Shh.13 This analysis showed that Shh made the cells competent to
respond to BMP4; together, these 2 factors promote the differentiation of chondrocytes. Using this paradigm, we proposed that
hedgehog signals promote the differentiation of bone marrow
progenitors into BMP4R cells. If Hedgehog is the signal in the
spleen microenvironment that promotes the development of BMP4R
cells, we would predict that culturing bone marrow cells with
Hedgehog would result in the development of BMP4R cells in vitro.
Treatment of bone marrow cells with Shh resulted in a significant
increase in the number of stress BFU-E when cells were grown in
methylcelluolose media containing only Epo (Figure 3A). However, when we added BMP4 to the media, no increase in stress
BFU-E was observed. In the chondrocyte system, treatment of
presomitic mesoderm cells with Shh induces the expression of
BMP4.13 We tested whether bone marrow cells treated with Shh
also expressed BMP4. The data in Figure 3B show that, similar to
presomitic mesoderm, bone marrow cells express BMP4 in response to Shh treatment. Furthermore, when we sorted bone
marrow hematopoietic cells from nonhematopoietic stromal cells,
the Kit plus hematopoietic cells express BMP4 when cultured in
media containing Shh (data not shown). Therefore, the expression
of endogenous BMP4 by bone marrow progenitor cells would
induce the expansion of stress BFU-E and mask the effect of
exogenously added BMP4. To demonstrate whether this possibility
were true, we treated bone marrow cells with Shh in the presence or
absence of the BMP4 antagonist, Noggin.16-18 In Figure 3C, we
show that inclusion of Noggin in the culture blocks the increase in
stress BFU-E induced by Shh. These data show that hedgehogdependent signaling induces BMP4 expression, and these 2 signals
act in concert to promote the differentiation of bone marrow
progenitor cells into stress BFU-E.
The ability of Shh to induce BMP4 expression in vitro
suggested that transplanted bone marrow cells homing to the spleen
would also express BMP4. We tested this hypothesis by examining
BMP4 expression in W/Wv mice that had been transplanted with
bone marrow cells. We used donor bone marrow cells from
B6.SJL-Ptprca Pep3b/BoyJ, which carry the CD45.1 allele, which
can be distinguished from recipient WBB6F1-W/Wv cells, which
carry the CD45.2 allele. Our previous work showed that W/Wv
mice do not express BMP4 in the spleen.3 However, when we
examined the expression of BMP4 in the spleen by RT-PCR on
days 7, 14, and 21 after transplantation, we observed BMP4
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
BLOOD, 22 JANUARY 2009 䡠 VOLUME 113, NUMBER 4
HEDGEHOG IS REQUIRED FOR STRESS ERYTHROPOIESIS
Time after PHZ injection
Hours
Days
0
12 24 36 48
4
6
8
C
Dhh
Ihh
Shh
Time after PHZ injection
Hours
Days
0 12 24 36 48 4
6
8 C1 C2
Beta actin
Figure 4. Expression of Hedgehog family members in the spleen. (Top) RT-PCR
analysis of Ihh, Shh, and Dhh in the spleen at the indicated times after induction of
acute anemia by PHZ injection. C indicates positive control RT-PCR analysis for Ihh,
Dhh, and Shh. Dhh control used spleen RNA, and Ihh and Shh controls used thymus
RNA. (Bottom) ␤-Actin control RT-PCR was used as a loading control. C1 indicates
Dhh control RNA; C2, control RNA for Ihh and Shh.
expression at all 3 time points (Figure 3D). This observation
supports the idea that bone marrow cells have migrated into the
spleens of the W/Wv recipient mice and now express BMP4. We
tested whether donor bone marrow cells homing to the spleen
would express BMP4. We stained spleen sections derived from
W/Wv mice 7 days after transplantation with anti-BMP4 and
anti-CD45.1 to distinguish donor-derived cells. The data in Figure
3E show that BMP4 is expressed by donor bone marrow cells.
These data are consistent with the hypothesis that hedgehog
signaling in the spleen induces the expression of BMP4 by bone marrow
cells homing to the spleen. In addition, we also observed BMP4
expression by recipient spleen cells, which before transplantation do not
express BMP4. These data suggest the possibility that donor-derived
BMP4R cells may in part regulate BMP4 expression in the spleen and
the expression of BMP4 by recipient cells may represent an attempt by
donor cells to induce recovery from anemia in W/Wv mice. Further
analysis will be needed to investigate this question.
Dhh and Ihh are expressed in the spleen
There are 3 vertebrate hedgehog genes, Shh, Dhh, and Ihh.19 Shh
and Ihh have been implicated in hematopoiesis previously.20-25 We
examined the expression of the 3 hedgehog family members in the
spleen during the recovery from PHZ-induced anemia by RT-PCR.
In Figure 4, we show that Dhh and Ihh are expressed in the spleen.
Ihh is expressed in untreated animals and continuously throughout
the recovery period. Dhh is expressed at low levels in untreated
animals, but after PHZ treatment Dhh expression is up-regulated
and maintained during the recovery from PHZ-induced acute
anemia. Our previous work showed that, during the recovery from
acute anemia, BMP4 expression is induced in nonhematopoietic
stromal cells in the red pulp of the spleen. Similarly, nonhematopoietic cells in the fetal liver express BMP4 during embryogeneiss.2,10
We wanted to determine which cells express Dhh in the spleen, so
we examined spleen sections for BMP4 and Dhh expression by
immunofluorescence. We observed that Dhh and BMP4 are coexpressed, which suggests that nonhematopoietic stromal cells express both BMP4 and Dhh (Figure S1, available on the Blood
website; see the Supplemental Materials link at the top of the online
article). Further studies will need to be done to localize Ihh
expression in the spleen during the recovery from acute anemia.
Mutation of the hedgehog-signaling pathway prevents
development of BMP4R cells in the spleen
Our in vitro data show that Shh can induce the BMP4-dependent
expansion of stress BFU-E, so we next tested whether donor bone
marrow cells that were mutant in the hedgehog-signaling pathway
could generate new BMP4R cells when transplanted into W/Wv
mice. Hedgehog signaling uses 2 receptors, Ptc and Smo.26 Ptc is
the negative receptor, which inhibits Smo signaling. On binding
hedgehog, Ptc inhibition of Smo is released and Smo transmits the
signal. Mutation of Smo blocks hedgehog signaling. Because
Smo⫺/⫺ mice are embryonic lethal, the role of Smo-dependent
signaling in adult stress erythropoiesis has not been investigated.27
We used a conditional allele of Smo (Smotm2amc), which is a floxed
allele of Smo and the interferon inducible Mx1-Cre.4,6 We deleted
Smo in the bone marrow by repeated injections of poly(I)poly(C).9
Deleted bone marrow expressed very little Smo mRNA (Figure
5A). The deletion of Smo did not adversely affect hematopoiesis in
the injected mice, which is consistent with previous work28 (data
not shown). Smo⌬/⌬, Smo⌬/⫹, and control bone marrow cells were
transplanted into W/Wv mice. The ability of mutant and control cell
to home to the spleen was no different from wild-type control cells
(data not shown). Fourteen days later, we examined the ability of
spleen cells from the transplanted mice to respond to BMP4 in
BFU-E colony assays. As shown in Figure 5B, Smo⌬/⌬ cells were
unable to respond to BMP4, whereas control and Smo⌬/⫹ cells did
respond to BMP4. These data demonstrate that donor bone marrow
cells require hedgehog signaling to develop into BMP4-responsive
splenic stress BFU-E.
A
B
Smo∆/+
P < .05
P < .05
40
Smo∆/∆
BFU-E
HPRT
Smo
30
Epo
Epo + Bmp4
20
10
0
C
BFU-E
Figure 5. Mutation of the Hedgehog receptor Smoothened blocks
the ability of bone marrow cells to generate new BMP4R cells in the
spleen. (A) RT-PCR analysis of Smo expression in Smo⌬/⫹ and Smo⌬/⌬
after poly(I)poly(C) treatment to delete the conditional allele. Hypoxanthine-guanine phosphoribosyl transferase expression is used as a
loading control. (B) Smo⌬/⫹, Smo⌬/⌬, and control bone marrow cells were
transplanted into WBB6F1 KitW/Wv recipient mice. Fourteen days after
transplantation, cells were plated in methylcellulose media containing
Epo or Epo ⫹ BMP4, and BFU-E were scored. Significant differences are
indicated in the figure. At least 3 recipient mice were analyzed for each
donor cell genotype. (C) Smo⌬/⫹, Smo⌬/⌬, and control mice were treated
with PHZ to induce acute anemia and allowed to recover for 7 days. The
mice were challenged with PHZ a second time on the indicated days after
the initial treatment. At 36 hours after the second treatment, spleen cells
were isolated and plated in methylcellulose media containing Epo alone,
and BFU-E were scored. Significant differences are indicated in the
figure. At least 3 mice per genotype were analyzed at each time point.
915
Smo∆/+
P < .005
Control
Smo∆/+
70
Smo∆/∆
60
50
P < .002
40
30 P < .03
P < .02
20
10
0
7
14
21
Days after initial PHZ injection
Smo∆/∆
Control
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
80
70
60
50
40
30
20
10
0
B
p<0.05
20
10
5
0
Ptc∆/∆
Ptcfx/+
15
Ptcfx/+ Ptc∆/∆
Spleen
Bone marrow
C
BMP4 expression
(relative to Gapdh)
18
16
60
50
40
30
20
10
0
Ptcfx/+ Ptc∆/ ∆
Ptcfx/fx
Ptcfx/fx;
Ptcfx/fx
CreERTM
Figure 6. Mutation of Ptc induces stress BFU-E formation in the
bone marrow in vivo and in vitro. (A, top) Bone marrow or spleen cells
as indicated were isolated from Ptc⌬/⫹ and Ptc⌬/⌬ mice and plated in
methylcellulose media containing Epo alone, and BFU-E were scored.
(A, bottom) PCR analysis of Ptc deletion by 4-hydroxytamoxifen.
(B) Bone marrow cells were isolated from Ptcfloxed/⫹ and Ptcfloxed/floxed;
CreER, and deletion of Ptc was induced in vitro by incubating the cells
overnight with 4-hydroxytamoxifen. (Top) The cells were washed and
plated in methylcellulose media containing Epo alone, and BFU-E were
scored. (bottom) PCR analysis of Ptc deletion by 4-hydroxytamoxifen.
(C) Real-time RT-PCR analysis of BMP4 expression by in vitro deleted
Ptc⌬/⫹ and Ptc⌬/⌬ bone marrow cells. Significant differences are indicated in the figure. All assays were done in triplicate and are representative of 2 independent experiments.
P < .05
Stress BFU-E
Stress BFU-E
A
BLOOD, 22 JANUARY 2009 䡠 VOLUME 113, NUMBER 4
PERRY et al
Stress BFU-E
916
Ptcfx/fx;
CreERTM
-- Ptc∆ 490bp
-- Ptc∆ 490bp
-- Ptcfx 307bp
-- Ptcfx 307bp
P < .01
14
12
10
8
6
4
2
0
Ptcfx/+
Ptc∆/∆
Using this transplantation model, we have established a role for
hedgehog signaling in development of BMP4R cells in the spleen.
In Figure 1B, we show that, after PHZ induced acute anemia, the
recovery of the BMP4-dependent stress erythropoiesis pathway
occurs 21 days after the initial PHZ treatment. We next investigated
the ability of Smo⌬/⌬ mice to recover BMP4R cells in the spleen
after PHZ-induced anemia. Smotm2amc/tm2amc;Mx1-cre, Smotm2amc/⫹;
Mx1-cre, and control Smotm2amc/⫹ mice were treated with poly(I)poly(C) to induce interferon and delete the Smo gene. The mice
were injected with PHZ to induce acute anemia. Seven, 14, and
21 days after the initial PHZ treatment, we examined the ability of
these mice to expand stress BFU-E in response to a second PHZ
treatment. All genotypes recovered from the initial PHZ treatment
similarly (data not shown). In Figure 5C, we show that control mice
exhibit an expansion of stress BFU-E at 21 days, which is similar to
that observed in untreated mice (Figure 1B). In contrast, Smo⌬/⌬
mice were unable to recover BMP4R cells in the spleen, even at
21 days after the initial anemia. Furthermore, none of these Smo⌬/⌬
mice survived longer than 48 hours after the second PHZ treatment
(data not shown). These data show that in vivo hedgehog signaling
is required for the recovery and maintenance of the BMP4dependent stress erythropoiesis pathway in the spleen; and in the
absence of Hedgehog signaling, mice are unable to recover from
repeated PHZ treatments.
Surprisingly, the Smo⌬/⫹ mice also exhibited a phenotype in this
assay. At 14 days, the recovery of the heterozygous mice was
significantly greater than the homozygous mutant mice, but less
than control mice, which is consistent with the analysis of the
transplantations of Smo⫹/⫺ and Smo⫺/⫺ transplantations into W/Wv
mice in Figure 5B. However, by 21 days, the heterozygotes did not
fully recover their ability to respond to a second dose of PHZ.
Haplo-insufficient phenotypes for Smo have not been reported
previously in mice, but in humans heterozygous mutations in the
hedgehog-signaling pathway can cause pathology.29-32
Mutation of Patched circumvents the need for Hedgehog and
leads to stress BFU-E in the bone marrow
Hedgehog signaling is tightly regulated by the negative receptor
Ptc, which inhibits the signaling activity of Smo.33 We hypothesized that mutation of Ptc in the bone marrow could lead to the
expansion of stress BFU-E in the bone marrow where few stress
BFU-E are normally observed. We tested this idea by crossing a
conditional allele of Ptc (Ptcfx) with the tamoxifen-inducible Cre
recombinase strain Cre-ERT. We treated Ptcfx/fx;CreERT and Ptcfx/⫹
control mice with repeated injections of 4-hydroxy tamoxifen
(4OHT),8 after which bone marrow cells were isolated and plated
in methylcellulose media containing only Epo to score stress
BFU-E. In Figure 6A, we show that Ptc⌬/⌬ mice exhibited a 4-fold
increase in the number of stress BFU-E in the bone marrow
compared with control mice. Although these data suggest that
deleting Ptc in bone marrow releases Smo-dependent signaling
resulting in the development of stress BFU-E, it is possible that
deleting Ptc could increase the number of stress BFU-E in the
spleen, which could then migrate to the bone marrow. We
addressed this possibility in 2 ways. When we examined the
spleens of Ptc⌬/⌬ mice, we found that the number of stress BFU-E
was unchanged, which is consistent with the constitutive expression of Ihh and Dhh in the spleen. Second, we isolated bone
marrow cells from Ptcfx/fx;CreERT and Ptcfx/⫹ control mice and
deleted Ptc with 4OHT in vitro. We plated the treated bone marrow
cells in methylcellulose to assay for stress BFU-E. Similar to what
we observed when Ptc was deleted in vivo, Ptc⌬/⌬ cells exhibited a
5-fold increase in the number of stress BFU-E (Figure 6B). Indeed,
Ptc⌬/⌬ cells generated significantly larger colonies (data not shown).
We also observed that deletion of Ptc induces the expression of
BMP4, which mimics the effects of Shh treatment of bone marrow
cells (Figure 6C). Taken together, these data suggest that hedgehog
signaling promotes the development of stress BFU-E and hedgehog
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
BLOOD, 22 JANUARY 2009 䡠 VOLUME 113, NUMBER 4
Bone marrow
Spleen
Dhh/Ihh
HEDGEHOG IS REQUIRED FOR STRESS ERYTHROPOIESIS
BMP4
917
BMP4R progenitors are
replenished in the spleen
Dhh/Ihh
Bone marrow cells
migrate into the
spleen where they
encounter Dhh
Dhh induces BMP4
expression and together
these signals promote
development of BMP4R
progenitors.
BMP4
BMP4
BMP4
BMP4
Acute anemia
induces BMP4
expression which
acts in concert with
SCF and hypoxia to
promote the
expansion of stress
BFU-E
Figure 7. Model for the role of Hedgehog in the recovery of the BMP4-dependent stress erythropoiesis pathway in the spleen.
signaling is tightly regulated so that access to Hedgehog is
compartmentalized, which promotes the development of BMP4responsive stress BFU-E only in the spleen.
Discussion
Here we show that Hedgehog-dependent signaling is required for
the maintenance of the BMP4-dependent stress erythropoiesis
pathway in the murine spleen. These data suggest a model (Figure
7) where bone marrow cells migrate to the spleen after recovery
from acute anemia. Although our previous work demonstrated that
bone marrow cells are unable to respond to BMP4 like spleen stress
BFU-E2, once in the spleen, the bone marrow cells encounter Dhh
or Ihh. Hedgehog signaling induces the expression of BMP4 in the
bone marrow cells, and these 2 signals are both required to promote
the differentiation of bone marrow progenitors into BMP4responsive spleen stress erythroid progenitors. Our analysis of
Smo⌬/⌬ mice showed that deletion of Smo affected the recovery of
spleen stress progenitors to such an extent that the mice were
unable to recover from the anemia induced by a second dose of
PHZ. These data demonstrate the key role played by Hedgehog
signaling in the maintenance of the BMP4-dependent stress
erythropoiesis pathway in the spleen. These data also suggest a
wider role for BMP4-dependent signals. Our observation that
Noggin can completely inhibit the ability of Shh to induce the
differentiation of stress BFU-E (Figure 3C) shows that BMP4 plays
an early role in the development of stress erythroid progenitors
where it acts in concert with Hedgehog. This role is in addition to
the role that we described previously where BMP4, SCF, and
hypoxia are required for the rapid expansion of stress BFU-E
during the recovery from acute anemia.2,3
Hedgehog- and BMP4-signaling pathways are required in a
wide range of developmental systems34-37 and during hematopoiesis,20-25,27,28 they have been shown to play role in the expansion of
stem cells.20,25 However, the situation that is most similar to stress
erythropoiesis is somitic chondrocyte development. In this system,
Shh initiates a chondrocyte development program, which allows
somitic mesoderm cells to differentiate into chondrocytes in
response to BMP4.13-15 Similar to what we observe in stress
progenitors, Shh induces BMP4 in somitic mesoderm cells. The 2
signals cooperate to establish a chondrocyte gene expression
pattern that is subsequently activated and extended by BMP4
signaling during differentiation. Based on our observations, we
propose that hedgehog signaling induces a pro-stress erythropoiesis
fate that allows bone marrow cells to respond to BMP4. We
hypothesize that Hedgehog (Ihh or Dhh) and BMP4 in the spleen
act to promote the development of BMP4-responsive stress erythroid progenitors, which in turn, at times of acute erythropoietic
stress rapidly respond to BMP4, SCF, and hypoxia to generate new
erythrocytes.
Our data also suggest that spleen BMP4-responsive stress
erythroid progenitors represent a lineage committed progenitor,
which does not rely on spleen-specific signals to maintain this cell
fate. Our transplantation experiments showed that BMP4responsive stress progenitors from the spleen maintained their
potential to respond to BMP4, even though they had lodged in the
bone marrow. Furthermore, the compartmentalization of the stress
erythropoiesis pathway to the spleen appears to be maintained by
regulating access to hedgehog. Although others have reported that
Shh and Ihh are expressed in the bone marrow,20,38 progenitors
destined to become BMP4 response stress progenitors do not
appear to have access to this source of Hedgehog. However, when
we deleted Ptc, BMP4-responsive cells were readily observed in
the bone marrow. These data, coupled with observation that in vitro
deletion of Ptc also leads to the development of stress BFU-E,
suggests that activation of hedgehog-signaling pathways by Ptc
mutation and the subsequent induction of BMP4 expression are
sufficient to promote the differentiation of stress erythroid progenitors in the absence of hedgehog-secreting cells.
The role hedgehog and BMP4 in regulating the development of
stress erythroid progenitors may not be limited to the adult spleen.
Recent work from our laboratory has shown that the BMP4dependent stress erythropoiesis pathway is required to rapidly
generate new erythrocytes at a critical juncture in fetal development.10 BMP4 expression is first observed in the fetal liver at
embryonic day 14.5 (E14.5). Stress BFU-E rapidly expand at E15.5
where they make up the majority of BFU-E in the fetal liver. Before
the expansion of stress BFU-E at E15.5, we detected BMP4R cells
in the fetal liver. Because hematopoietic progenitor cells do not
develop de novo in the fetal liver,39-41 we proposed that definitive
progenitors from the yolk sac may seed the fetal liver at earlier
times and develop into BMP4-responsive stress progenitors. In
support of this idea, we showed that yolk sac contains progenitor
cells that are capable of differentiating into stress BFU-E when
cocultured on AFT024 fetal liver stromal cells.42 This cell line
expresses BMP4 at high levels, but BMP4 alone is unable to cause
yolk sac progenitor cells to differentiate into stress BFU-E. It is
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
918
BLOOD, 22 JANUARY 2009 䡠 VOLUME 113, NUMBER 4
PERRY et al
tempting to speculate that, similar to the adult spleen, hedgehog
signals may be involved in the development of BMP4-responsive
stress progenitors in the fetal liver. New experiments will be
needed to test this hypothesis.
In conclusion, we have shown that the BMP4-responsive stress
erythropoiesis pathway is maintained by bone marrow cells that
migrate to the spleen. Once in the spleen, they encounter Hedgehog, which induces BMP4 expression. These 2 signals together
promote the differentiation of bone marrow progenitor cells into
BMP4-responsive stress erythroid progenitors, which maintains the
BMP4-dependent stress erythropoiesis pathway in the spleen.
Acknowledgments
The authors thank the members of the Paulson Laboratory and the
Center for Molecular Immunology and Infectious Disease for their
comments on the paper, Brandon Wainwright and C. C. Hui for
providing the Ptcfloxed mice, and Bennie Luscher for providing the
CreER mice.
This work was supported by National Institutes of Health grant
HL70720 (R.F.P.) and, in part, by a grant with the Pennsylvania
Department of Health using Tobacco Settlement Funds.
The Pennsylvania Department of Health specifically disclaims
responsibility for any analyses, interpretations, or conclusions.
Authorship
Contribution: J.M.P., O.F.H., and P.P. performed experiments and
analyzed data; S.H. and A.K.K. performed experiments; and R.F.P.
designed experiments, analyzed data, and wrote the paper.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Robert F. Paulson, Center for Molecular
Immunology and infectious Disease, Department of Veterinary and
Biomedical Sciences, 115 Henning Building, Pennsylvania State
University, University Park, PA 16802; e-mail [email protected].
References
1. Socolovsky M. Molecular insights into stress
erythropoiesis. Curr Opin Hematol. 2007;14:215224.
2. Lenox L, Perry J, Paulson R. BMP4 and Madh5
regulate the erythroid response to acute anemia.
Blood. 2005;105:2741-2748.
3. Perry J, Harandi O, Paulson R. BMP4, SCF and
Hypoxia cooperatively regulate the expansion of
murine stress erythroid progenitors. Blood. 2007;
109:4494-4502.
4. Long F, Zhang XM, Karp S, Yang Y, McMahon AP.
Genetic manipulation of hedgehog signaling in
the endochondral skeleton reveals a direct role in
the regulation of chondrocyte proliferation. Development. 2001;128:5099-5108.
5. Ellis T, Smyth I, Riley E, et al. Patched 1 conditional
null allele in mice. Genesis. 2003;36:158-161.
6. Kuhn R, Schwenk F, Aguet M, Rajewsky K. Inducible gene targeting in mice. Science. 1995;269:
1427-1429.
7. Hayashi S, McMahon AP. Efficient recombination
in diverse tissues by a tamoxifen-inducible form
of Cre: a tool for temporally regulated gene activation/inactivation in the mouse. Dev Biol. 2002;
244:305-318.
8. Indra AK, Warot X, Brocard J, et al. Temporally
controlled site-specific mutagenesis in the basal
layer of the epidermis: comparison of the recombinase activity of the tamoxifen-inducible CreER(T) and Cre-ER(T2) recombinases. Nucleic
Acids Res. 1999;27:4324-4327.
9. Mikkola HK, Klintman J, Yang H, et al. Haematopoietic stem cells retain long-term repopulating
activity and multipotency in the absence of stemcell leukaemia SCL/tal-1 gene. Nature. 2003;421:
547-551.
10. Porayette P, Paulson R. BMP4/Smad5 dependent
stress erythropoiesis is required for the expansion of erythroid progenitors during fetal development. Developmental Biology. 2008;317 24-35.
11. Lewis MT, Ross S, Strickland PA, et al. Defects in
mouse mammary gland development caused by
conditional haploinsufficiency of Patched-1. Development. 1999;126:5181-5193.
12. Russell ES, Bernstein SE, Lawson FA, Smith LJ.
Long-continued function of normal blood-forming
tissue transplanted into genetically anemic hosts.
J Natl Cancer Inst. 1959;23:557-566.
13. Murtaugh LC, Chyung JH, Lassar AB. Sonic
hedgehog promotes somitic chondrogenesis by
altering the cellular response to BMP signaling.
Genes Dev. 1999;13:225-237.
14. Murtaugh LC, Zeng L, Chyung JH, Lassar AB.
The chick transcriptional repressor Nkx3.2 acts
downstream of Shh to promote BMP-dependent
axial chondrogenesis. Dev Cell. 2001;1:411-422.
15. Zeng L, Kempf H, Murtaugh LC, Sato ME, Lassar
AB. Shh establishes an Nkx3.2/Sox9 autoregulatory loop that is maintained by BMP signals to
induce somitic chondrogenesis. Genes Dev.
2002;16:1990-2005.
16. Groppe J, Greenwald J, Wiater E, et al. Structural
basis of BMP signalling inhibition by the cystine
knot protein Noggin. Nature. 2002;420:636-642.
17. Smith WC, Harland RM. Expression cloning of
noggin, a new dorsalizing factor localized to the
Spemann organizer in Xenopus embryos. Cell.
1992;70:829-840.
quired for murine yolk sac angiogenesis. Development. 2002;129:361-372.
28. El Andaloussi A, Graves S, Meng F, Mandal M,
Mashayekhi M, Aifantis I. Hedgehog signaling
controls thymocyte progenitor homeostasis and
differentiation in the thymus. Nat Immunol. 2006;
7:418-426.
29. Bale AE. Hedgehog signaling and human disease. Annu Rev Genomics Hum Genet. 2002;3:
47-65.
30. Mullor JL, Sanchez P, Altaba AR. Pathways and
consequences: Hedgehog signaling in human
disease. Trends Cell Biol. 2002;12:562-569.
31. Nieuwenhuis E, Hui CC. Hedgehog signaling and
congenital malformations. Clin Genet. 2005;67:
193-208.
18. Yanagita M. BMP antagonists: their roles in development and involvement in pathophysiology.
Cytokine Growth Factor Rev. 2005;16:309-317.
32. Pasca di Magliano M, Hebrok M. Hedgehog signalling in cancer formation and maintenance. Nat
Rev Cancer. 2003;3:903-911.
19. Echelard Y, Epstein DJ, St-Jacques B, et al.
Sonic hedgehog, a member of a family of putative
signaling molecules, is implicated in the regulation of CNS polarity. Cell. 1993;75:1417-1430.
33. Stone DM, Hynes M, Armanini M, et al. The tumour-suppressor gene patched encodes a candidate receptor for Sonic hedgehog. Nature. 1996;
384:129-134.
20. Bhardwaj G, Murdoch B, Wu D, et al. Sonic
hedgehog induces the proliferation of primitive
human hematopoietic cells via BMP regulation.
Nat Immunol. 2001;2:172-180.
34. Chari NS, McDonnell TJ. The sonic hedgehog
signaling network in development and neoplasia.
Adv Anat Pathol. 2007;14:344-352.
21. Crompton T, Outram SV, Hager-Theodorides AL.
Sonic hedgehog signalling in T-cell development and
activation. Nat Rev Immunol. 2007;7:726-735.
22. Detmer K, Thompson AJ, Garner RE, Walker AN,
Gaffield W, Dannawi H. Hedgehog signaling and
cell cycle control in differentiating erythroid progenitors. Blood Cells Mol Dis. 2005;34:60-70.
23. Detmer K, Walker AN, Jenkins TM, Steele TA,
Dannawi H. Erythroid differentiation in vitro is
blocked by cyclopamine, an inhibitor of hedgehog
signaling. Blood Cells Mol Dis. 2000;26:360-372.
35. Charron F, Tessier-Lavigne M. The Hedgehog,
TGF-beta/BMP and Wnt families of morphogens
in axon guidance. Adv Exp Med Biol. 2007;621:
116-133.
36. Miura T. Modeling lung branching morphogenesis. Curr Top Dev Biol. 2008;81:291-310.
37. van den Brink GR. Hedgehog signaling in development and homeostasis of the gastrointestinal
tract. Physiol Rev. 2007;87:1343-1375.
38. Dierks C, Grbic J, Zirlik K, et al. Essential role of
stromally induced hedgehog signaling in B-cell
malignancies. Nat Med. 2007;13:944-951.
24. Dyer MA, Farrington SM, Mohn D, Munday JR,
Baron MH. Indian hedgehog activates hematopoiesis and vasculogenesis and can respecify
prospective neurectodermal cell fate in the
mouse embryo. Development. 2001;128:17171730.
39. Dzierzak E, Speck NA. Of lineage and legacy: the
development of mammalian hematopoietic stem
cells. Nat Immunol. 2008;9:129-136.
25. Trowbridge JJ, Scott MP, Bhatia M. Hedgehog
modulates cell cycle regulators in stem cells to
control hematopoietic regeneration. Proc Natl
Acad Sci U S A 2006;103:14134-14139.
41. Palis J, Robertson S, Kennedy M, Wall C, Keller
G. Development of erythroid and myeloid progenitors in the yolk sac and embryo proper of the
mouse. Development. 1999;126:5073-5084.
26. Cohen MM, Jr. The hedgehog signaling network.
Am J Med Genet A 2003;123:5-28.
42. Moore KA, Ema H, Lemischka IR. In vitro maintenance of highly purified, transplantable hematopoietic stem cells. Blood. 1997;89:4337-4347.
27. Byrd N, Becker S, Maye P, et al. Hedgehog is re-
40. McGrath KE, Palis J. Hematopoiesis in the yolk
sac: more than meets the eye. Exp Hematol.
2005;33:1021-1028.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
2009 113: 911-918
doi:10.1182/blood-2008-03-147892 originally published
online October 16, 2008
Maintenance of the BMP4-dependent stress erythropoiesis pathway in
the murine spleen requires hedgehog signaling
John M. Perry, Omid F. Harandi, Prashanth Porayette, Shailaja Hegde, Arun K. Kannan and Robert
F. Paulson
Updated information and services can be found at:
http://www.bloodjournal.org/content/113/4/911.full.html
Articles on similar topics can be found in the following Blood collections
Red Cells, Iron, and Erythropoiesis (796 articles)
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society
of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.