Genetic Interaction of PGE2 and Wnt
Signaling Regulates Developmental
Wolfram Goessling,1,2,3,6,7Trista E. North,1,2,3,6,8Sabine Loewer,2,3Allegra M. Lord,2,3Sang Lee,2,4
Cristi L. Stoick-Cooper,5Gilbert Weidinger,5Mark Puder,2,4George Q. Daley,1,2,3Randall T. Moon,5
and Leonard I. Zon1,2,3,*
1Harvard Stem Cell Institute, Cambridge, MA 02138, USA
2Harvard Medical School, Boston, MA 02115, USA
3Stem Cell Program, Hematology/Oncology, Children’s Hospital, Howard Hughes Medical Institute, Boston, MA 02115, USA
4Department of Surgery, Children’s Hospital, Boston, MA 02115, USA
5Institute for Stem Cell and Regenerative Medicine, Howard Hughes Medical Institute, University of Washington School of Medicine,
Seattle, WA 98195, USA
6These authors contributed equally to this work
7Present address: Genetics and Gastroenterology Divisions, Brigham and Women’s Hospital, Medical Oncology,
Dana-Farber Cancer Institute, Boston, MA 02115, USA
8Present address: Department of Pathology, Beth Israel Deaconess Medical Center, Boston, MA 02115, USA
Interactions between developmental signaling path-
Prostaglandin (PG) E2 regulates vertebrate hemato-
poietic stem cells (HSC). Similarly, the Wnt signaling
marrow repopulation. Here, we show that wnt
reporter activity in zebrafish HSCs is responsive to
PGE2 modulation, demonstrating a direct interaction
in vivo. Inhibition of PGE2 synthesis blocked wnt-
the wnt signaling cascade at the level of b-catenin
degradation through cAMP/PKA-mediated stabi-
lizing phosphorylation events. The PGE2/Wnt inter-
action regulated murine stem and progenitorpopula-
tions in vitro in hematopoietic ES cell assays and
in vivo following transplantation. The relationship
between PGE2 and Wnt was also conserved during
regeneration of other organ systems. Our work
provides in vivo evidence that Wnt activation in
stem cells requires PGE2, and suggests the PGE2/
Wnt interaction is a master regulator of vertebrate
regeneration and recovery.
self-renewal and bone
Stem cells are characterized by their unique abilities to both self-
renew and differentiate to produce all mature cell lineages of
a given tissue type. In the adult vertebrate, HSCs reside in the
bone marrow (BM),while during embryonic development several
sites successively become competent to produce HSCs (Orkin
and Zon, 2008). An understanding of the complex network of
inductive signals regulating HSC development is of significant
therapeutic interest for HSC maintenance in the adult. The
aorta-gonad-mesonephros (AGM) region contains the first
adult-type long-term repopulating (LTR-) HSCs in the vertebrate
embryo; murine transplantation studies revealed that LTR-HSCs
can be found on the ventral wall of the dorsal aorta by e10.5. The
Runx1 protein, widely known for its involvement in leukemia, is
specifically expressed in the AGM and is required for the forma-
tion of functional HSCs (North et al., 2002). The expression of
Runx1 is highly conserved across vertebrate species (Orkin
and Zon, 2008).
We recently showed that PGE2 regulates vertebrate HSC
induction and engraftment (North et al., 2007). PGE2 was identi-
fied through a chemical genetic screen for modifiers of runx1
expression within the zebrafish AGM. A stabilized derivative,
16,16-dimethyl-PGE2 (dmPGE2), enhanced the formation of
stem cells and zebrafish marrow recovery following irradiation
injury. dmPGE2 significantly increased ES cell hematopoietic
colony formation and the frequency of both short (ST-) and
LTR-HSCs in the mouse BM. The exact mechanism by which
PGE2 exerts its effects on HSCs remains unknown. PGE2 has
a regulatory role during myeloid differentiation, erythropoiesis
and stromal cell homeostasis in murine BM (Fisher and Hagi-
wara, 1984; Nocka et al., 1989; Williams and Jackson, 1980).
Additionally, hematopoietic lineage regeneration is impaired in
cyclooxygenase (Cox) 2-deficient mice (Lorenz et al., 1999).
Together, these data indicate that PGE2 plays a critical role in
HSC induction as well as maintenance and function in the adult
Wnt signaling has been similarly implicated in HSC regulation
in the adult BM (Reya et al., 2003; Trowbridge et al., 2006). To
date, however, a role for wnt in HSC development has not
1136 Cell 136, 1136–1147, March 20, 2009 ª2009 Elsevier Inc.
been described. Wnt signaling regulates several aspects of
vertebrate embryogenesis, including gastrulation, somitogene-
sis and organogenesis (Goessling et al., 2008; Weidinger et al.,
2005). Wnt activation is required for liver and fin regeneration
(Goessling et al., 2008; Stoick-Cooper et al., 2007), as well as
the maintenance of hematopoietic, skin, and intestinal stemcells
regulator of stem cell induction during embryogenesis, and may
workinconjunction withPGE2inHSCformation andhematopoi-
Clinical evidence supported
lular regulator of wnt signaling, typically develop innumerable
colonic polyps and ultimately colon cancer. Treatment with
COX inhibitors significantly reduces polyp formation (Giardiello
et al., 1993). This observation was confirmed by chemical Cox
inhibition in APCMinmice (Boolbol et al., 1996) and genetic dele-
tion of Cox2 and PG synthase (Nakanishi et al., 2008; Oshima
et al., 1996). The connection between Wnt and PGE2 has been
mechanistically described in cellular proliferation and oncogen-
esis (Castellone et al., 2005; Shao et al., 2005). However, these
studies are limited to in vitro analyses using immortalized cell
lines, which often harbor mutations in the wnt pathway itself.
As such, they cannot address whether this interaction is func-
tionally relevant in vivo or if it is solely an aberrant regulatory
mechanism utilized in carcinogenesis (Buchanan and DuBois,
2006; Clevers, 2006).
Here we show that PGE2 can directly regulate wnt activity
in vivo during vertebrate development and organ regeneration.
This interaction occurs within HSCs and the hematopoietic
niche during embryogenesis and functions to regulate HSC
induction. PGE2 was required to mediate the effects of wnt acti-
vation and can act to further amplify wnt activity through cAMP/
PKA-mediated regulation of b-catenin protein stability in vivo.
Both in vitro, in murine ES cell hematopoietic assays, and
in vivo following BM transplantation, PGE2 modified wnt-medi-
ated regulation of hematopoietic stem and progenitor popula-
tions. Significantly, this role of PGE2 was conserved during
regeneration in several organ systems, indicating that the
PGE2/wnt interaction serves as a master regulator of vertebrate
the purported interaction
Prostaglandin Levels Directly Affect wnt/b-Catenin
Activity in HSCs In Vivo
Clinical evidence from colon cancer patients and cancer cell
lines suggested a close association between the wnt and
PGE2 pathways (Castellone et al., 2005; Giardiello et al., 2002;
Shao et al., 2005). To examine whether PGE2 regulates wnt
activity in vivo, we utilized TOP:dGFP b-catenin-responsive
reporter zebrafish embryos. 10 mM dmPGE2 caused a striking
increase in reporter activity throughout the embryo at 36 hr post-
fertilization (hpf; 99 increased [inc]/111 scored). Specifically, it
quadrupled the number of GFP+ cells in the AGM (12 ± 3.4
(dmPGE2) versus 3 ± 1.8 (control [con]) cells, p < 0.05; Figures
1A, 1B, and 1D). We previously showed by mass spectroscopy
that the non-selective cox inhibitor indomethacin (indo) sup-
pressed PGE2 production in zebrafish embryos (North et al.,
2007). 10 mM indo decreased GFP expression globally and abol-
ished wnt activity within the AGM (72 decreased [dec]/87, 0.7 ±
0.5 cells, p < 0.05; Figures 1C and 1D). qPCR analysis for GFP
confirmed that PGE2 significantly modulates wnt activity
in vivo (p < 0.05; Figure 1E).
To determine whether wnt activity was localized within the
HSCs, we examined TOP:dGFP
lmo2:dsRed, which label both HSCs and endothelial cells. wnt
activity in wild-type (WT) embryos was found in the endothelial
and subendothelial layers of the ventral wall of the dorsal aorta
(Figure 1F), which possess HSC potential in murine transplanta-
tion assays (North et al., 2002). dmPGE2 enhanced wnt activity
within the HSC/endothelial population and in the subendothelial
layer (Figure 1G). Indo decreased wnt-active cells in the AGM
(Figure 1H). FACS analysis of bigenic fish showed that dmPGE2
significantly enhanced lmo2+ (p < 0.05; Figures S2A and S2C
available online) and GFP+ wnt-active cells (Figures 1I, 1J, and
S2B); wnt activation was most pronounced within the lmo2+
fraction (Figure S2D). Indo significantly diminished wnt-active
and lmo2+ cells (p < 0.05; Figures 1K and S2A–S2E). Together,
these data demonstrate that PGE2 regulates wnt activity within
the HSC/endothelial cell population as well as the hematopoietic
niche during embryonic development.
PGE2 Modifies wnt-Mediated Regulation of HSC
Formation through Alterations in b-Catenin Availability
The role of wnt signaling in regulating embryonic HSC formation
has not been determined. Using inducible wnt8 transgenic
zebrafish, we previously studied the time-dependent effects of
wnt activation on organ formation (Goessling et al., 2008).
Here, we examined alterations in the expression of the
conserved HSC markers runx1/cmyb at 36 hpf (North et al.,
2007). Transplantation of FACS-fractionated murine AGM cells
demonstrated that all HSCs express Runx1 (North et al., 2002);
similarly, in zebrafish, AGM cells from subdissected tail regions,
encompassing the runx1+ population, possess LTR-HSC
activity, indicating HSC function with in the AGM is conserved
(T.E.N. and L.I.Z., unpublished data). We found that induction
of wnt8 during mid-somitogenesis enhanced runx1/cmyb+ cells
(47 inc/54; Figures 2A and 2C). To examine whether PGE2 was
required to mediate the effects of wnt activation on HSCs,
wnt8 induction was followed by indo exposure; this reduced
HSC number to WT levels (43 normal [norm]/46; Figures 2B
and 2D). These findings were confirmed by qPCR for runx1
and cmyb (Figures S3A and S3B) and demonstrate that PGE2
is required for wnt-mediated regulation of HSC formation
in vivo. Significantly, wnt activation synergized with PGE2 in
the majority of embryos (25 inc/41; Figures S3F–S3J) examined,
demonstrating that PGE2 can function to enhance wnt activity
To genetically localize the interaction between PGE2 and the
wnt pathway, transgenic fish expressing inducible negative
regulators of wnt signaling were exposed to dmPGE2. Dickkopf
(dkk), a membrane-level antagonist, inhibited runx1/cmyb
expression (34 dec/49; Figures 2E and 2G). This effect could
Cell 136, 1136–1147, March 20, 2009 ª2009 Elsevier Inc. 1137
2H). Axin, a central component of the b-catenin destruction
complex, and a dominant-negative form of the b-catenin tran-
scriptional coactivator T cell factor (TCF) also severely dimin-
ished HSC formation (47 dec/52 [axin]; 60 dec/62 [TCF]; Figures
2I and 2K). However, these effects could not be overcome by
dmPGE2 treatment (40 dec/45; 48 dec/51; Figures 2J and 2L).
These results were confirmed by qPCR for runx1 and cmyb
(Figures S3A and S3B). Expression of vascular markers flk1
and ephB2 (Figures S3C and S3D) and the wnt target cyclin D1
(Figures S3E) were coordinately affected by the PGE2/wnt inter-
action. These experiments indicate that PGE2 interacts with the
wnt pathway at the level of b-catenin destruction to control HSC
To demonstrate a direct effect of the PGE2/wnt interaction on
HSC number rather than gene expression alone, inducible wnt
transgenics were crossed to cmyb:GFP HSC reporter zebrafish
(North et al., 2007). In vivo confocal microscopy (Figures 2M
Figure 1. Prostaglandin Levels Directly Affect wnt
Activity in Zebrafish Embryos
(A–C) In situ hybridization for GFP in TOP:dGFP wnt reporter
embryos at 36 hpf shows widespread wnt activity; inset,
close-up of GFP+ (black arrowheads) cells in the AGM.
10 mM dmPGE2 enhanced GFP expression throughout the
embryo, while 10 mM indo decreased global wnt activity, and
in the AGM.
(D) Quantification (mean ± SD) of total GFP+ cells in the major
trunk vessels and (E) qPCR analysis for GFP in whole embryo
lysates following exposure to dmPGE2 or indo versus vehicle
con reveals significant alterations in wnt activity (*significant
(sig) across treatment groups, ANOVA, p < 0.05, n = 10/treat-
region of TOP:dGFP; lmo2:DsRed embryos following expo-
sure to con, dmPGE2, or indo are shown; differences can be
seen in the wnt-active (green, left column), HSC/endothelial
(red, middle), and colocalized (merged, right) populations.
(I–K) Representative FL1 (green)/ FL2 (red) FACS plots of indi-
vidual TOP:dGFP; lmo2:DsRed embryos after exposure to
con, dmPGE2 or indo confirm the confocal analyses (summa-
rized in Figure S2).
and 2N) revealed that indo caused a significant
decrease in HSC number (1.8 ± 0.8 versus 11.4 ±
3.2 [con], p < 0.001; Figures 2O and 2Z). wnt8
significantly increased HSCs (20.6 ± 3.2, p <
0.001; Figure 2P), while subsequent indo treatment
returned HSC numbers to near WT levels (11.8 ±
1.6; Figure 2Q). dmPGE2 enhanced HSC number,
as previously shown (North et al., 2007) (20.8 ±
1.9, p < 0.001; Figures 2R and 2S). dkk (2.6 ± 0.9,
p < 0.001; Figure 2T), axin (3.0 ± 1.0, p < 0.001,
Figure 2V), and dnTCF (1.4 ± 0.6, p < 0.001;
Figure 2X) diminished HSCs. dmPGE2 could only
rescue the effects of dkk, not of axin or dnTCF
(9.8 ± 0.8; 3.4 ± 1.3; 1.4 ± 0.9; Figures 2U, 2W,
and 2Y). These results demonstrate that PGE2
and wnt signaling interact at the level of the
b-catenin destruction complex and influence HSC number in
The PGE2/wnt Interaction Affects Cell Survival
and Proliferation in the AGM
To further delineate the impact of the PGE2/wnt interaction on
developing HSCs, we examined apoptosis and cellular prolifera-
tion. During embryonic development only a small fraction of
AGM cells appeared TUNEL+ (Figures 3A, 3B, S4A, and S4E);
cell counts in a defined anatomical section of the trunk revealed
5.6 ± 1.8 apoptotic cells in untreated WT embryos. wnt8 (3.6 ±
1.1; Figures S4C) or dmPGE2 (1.8 ± 1.5; Figures S4F) decreased
TUNEL+ cells. Indo induced wide-spread apoptosis (40.8 ± 4.6,
p < 0.001; Figures S4B) and counteracted the effect of wnt8
induction increased apoptosis in the AGM (43.2 ± 8.2; 33.2 ±
4.9; 34.6 ± 5.6; p < 0.001 versus con; Figures 3B, S4G, S4I,
and S4K), which was improved by dmPGE2 only in dkk embryos
1138 Cell 136, 1136–1147, March 20, 2009 ª2009 Elsevier Inc.
(9.2 ± 2.8, p < 0.001 versus dkk; Figures S4H, S4J, and S4L).
BrdU incorporation occurred globally during embryonic devel-
opment, including cells within the AGM region (10.4 ± 2.1;
Figures 3C, 3D, S4M, and S4Q); this effect was significantly
enhanced by wnt8 (24.4 ± 5.0; p < 0.001; Figures S4O) and
dmPGE2 (27.8 ± 3.0, p < 0.001; Figures S4R). Indo suppressed
Figure 2. PGE2 Regulates Wnt-Mediated
Effects on HSC Formation at the Level of
(A–D) wnt8 induction in hs:wnt8-GFP transgenic
embryosincreased runx1/cmyb+ HSCscompared
to WT; indo decreased HSCs in WT and wnt8
(E–L) Induction of dkk diminished runx1/cmyb
expression compared to WT. dmPGE2 enhanced
HSCs in WT embryos, and rescued runx1/cmyb
expression to approximately WT levels in dkk
(hs:dnTCF-GFP) reduced runx1/cmyb expression,
and dmPGE2 could not rescue those effects.
(M) Schematic of confocal microscopy. Imaging
was performed in the trunk/tail region of the
embryo, centered around the tip of the yolk sac
extension (YSE, blue arrowhead), encompassing
the dorsal aorta (red dots) and vein (blue dots),
as indicated by the pink bracket.
(N-Y) In vivo confocal microscopy of wnt pathway
inducible embryos crossed into the cmyb:GFP
HSC reporter line confirmed the in situ hybridiza-
tion analysis anddemonstrated
effects on cell number.
(Z) Cell counts (5 embryos/treatment, data repre-
sented as mean ± SD) were conducted in the
major vessels (pink bracket) in a 40x field centered
at the YSE; *sig versus con; **sig versus wnt8;
***sig versus dkk; ANOVA, p < 0.001.
BrdU incorporation (2.8 ± 1.5, p < 0.002;
Figures S4N) and blunted the proliferative
effect of wnt activation (7.2 ± 2.6, p <
0.001 versus wnt8; Figures S4P). dkk,
axin, or dnTCF similarly decreased BrdU
incorporation (4.6 ± 2.0; 5.8 ± 1.5; 3.6 ±
1.5, p < 0.002; Figures 3D, S4S, S4U,
and S4W); dmPGE2 only rescued dkk
(11.8 ± 3.4, p < 0.001 versus dkk; Figures
cellular proliferation and apoptosis may
be mechanisms by which PGE2 can
modify the effects of wnt signaling on
PGE2 Regulates the Effects of wnt
Signaling through cAMP/PKA
The genetic interaction studies sug-
gested that PGE2 may directly regulate
b-catenin destruction, and subsequent
protein available for transcriptional acti-
vation. To document whether PGE2 directly enhances b-cate-
nin levels in vivo, western blot analysis was performed. wnt8
decreased total b-catenin in WT and wnt8 pooled embryo
lysates. qPCR revealed that PGE2 did not affect b-catenin tran-
scription (Figures S3L). These data suggest that PGE2 directly
Cell 136, 1136–1147, March 20, 2009 ª2009 Elsevier Inc. 1139
influences the stability of b-catenin via non-transcriptional
b-catenin levels are tightly controlled within the cell by the
destruction complex, consisting of axin, GSK3b, CK1, and
ylated at N-terminal residues and targeted for ubiquitination/
degradation. C-terminal phosphorylation of b-catenin (S675)
stabilizes the protein by inhibiting destruction (Hino et al., 2005),
ylation of GSK3b at S9 (Fang et al., 2000). PGE2 regulates intra-
kinases PKA and PI3K/AKT (Fujino et al., 2002). To demonstrate
a functional role for cAMP in modulating wnt activity and HSC
development, TOP:dGFP embryos were exposed to forskolin,
a cAMP-activator. Forskolin (0.5 mM) increased wnt activity
(18 inc/21; 8.8 ± 1.3 TOP:dGFP + cells/tail [forskolin] versus 3.8 ±
0.8 [con], p < 0.001; Figures S5A, S5C, and S5E) and counter-
acted the wnt-inhibitory effects of indo (20 dec/25), confirming
that cAMP functions downstream of PGE2 to regulate wnt
signaling (17 norm/27; 6.4 ± 1.1 [indo + forskolin] versus 1.1 ±
0.7 [indo], p < 0.001; Figures S5B and S5D). We next sought to
determine if PKA or PI3K was the secondary effector of cAMP
in controlling wnt activity in the AGM in vivo. Exposure to the
PKA inhibitor H89 (0.5 mM) decreased wnt activity compared to
WT (19 dec/20; 0.6 ± 0.6; p < 0.001; Figures S5F, S5H, and
S5L). In contrast, the PI3K inhibitor LY294002 (1 mM, LY) did
not affect wnt activity appreciably (3 dec/27; 3.6 ± 1.1; Figures
S5J and S5K). Furthermore, H89, but not LY, blocked PGE2-
mediated elevation of wnt activity (23 inc/26 [PGE2]; 22 norm/
2.4 ± 1.1 [PGE2 + H89], p < 0.001; 9.8 ± 1.5 [PGE2 + LY]; Figures
S5G, S5I, and S5K). These results suggest that PGE2 directly
enhances wnt activity through cAMP/PKA-signaling.
We next tested whether cAMP and PKA are functionally rele-
vant in PGE2-mediated wnt regulation of HSCs in vivo. Exposure
to forskolin increased HSCs (Figures 4C and 4G), and rescued
the negative effect of indo treatment in WT (21 normal/33;
Figure 4D) and wnt8 embryos (18 inc/26, Figure 4H). PKA inhibi-
tion by H89 diminished HSCs in WT embryos and eliminated the
effects of dmPGE2 (13 norm/19; Figure 4L). H89 also inhibited
the dmPGE2-mediated rescue of dkk embryos (17 dec/26;
Figure 4P). The effects of cAMP/PKA modulation were quanti-
tated by qPCR for runx1 (Figures S6A and S6B). The conse-
quence of PKA inhibition was confirmed by exposure to 1 mM
KT5720 (19 dec/31 [KT]; 20 norm/29 [PGE2 + KT]; Figures
S6C–S6F). Treatment with the PI3K inhibitors LY (4 dec/39
[LY]; 34 inc/42 [PGE2 + LY]) and 1 mM Wortmannin (5 dec/48
[Wort]; 34 inc/46 [PGE2 + Wort]) did not considerably alter
HSC development (Figures S6G–S6J). These data indicate
PGE2 acts primarily via cAMP/PKA to affect wnt signaling and
regulate HSC formation in vivo.
The PGE2/wnt Interaction Is Functionally Conserved
in HSC Regeneration
Wnt signaling regulates adult HSC self-renewal, BM recovery
and repopulation (Congdon et al., 2008; Reya et al., 2003). To
determine whether the PGE2/wnt interaction affects hematopoi-
etic regeneration in adult fish, we examined kidney marrow
recovery following irradiation. In TOP:dGFP zebrafish, wnt
activity was significantly increased in the recovering marrow
compared to unirradiated controls (p < 0.001; Figure 5A). Wnt
activity was significantly enhanced by dmPGE2 and markedly
diminished by indo (p < 0.001; Figure 5A). FACS analysis of
recovering marrow identifies effects on all hematopoietic line-
ages, including the stem/progenitor population. wnt8 induction
significantly increased precursors (18.1 ± 3.5% [wnt8] versus
icantly decreased precursor recovery (4.2 ± 2.1%, p < 0.02) and
that PGE2 modulates the effects of wnt activation in the adult. In
support of this conclusion, the decrease in marrow recovery
following induction of dkk (3.4 ± 1.1% [dkk] versus 7.0 ± 1.1%
[WT]) was partially corrected by treatment with dmPGE2 (6.3 ±
1.0%, p < 0.001; Figure 5C).
The PGE2/wnt Interaction Is Conserved in Mammalian
Hematopoietic Stem and Progenitor Populations
To determine whether the Wnt and PGE2 pathways interact simi-
larly in the mammalian hematopoietic system, we examined
in vitro differentiation of murine embryonic stem cells (mESC).
Figure 3. PGE2-Mediated Modulation of wnt
(A and B) Indo treatment enhanced TUNEL+ cells
in the AGM in both WT and wnt8 embryos. dkk,
axin, or dnTCF enhanced apoptosis; dmPGE2
improved this effect only in dkk transgenics. Cell
counts (5 embryos/treatment) in the AGM showed
significant effects across treatment groups: *sig
versus con; **sig versus wnt8; ***sig versus dkk;
ANOVA; p < 0.05.
(C and D) wnt8 enhanced BrdU incorporation,
while indo diminished BrdU in both WT and wnt8
embryos. wnt inhibition by dkk, axin, or dnTCF
diminished BrdU incorporation; the effect of dkk
could be rescued by dmPGE2. *sig versus con;
**sig versus wnt8; ***sig versus dkk; ANOVA;
p < 0.05.
Data represented as mean ± SD.
1140 Cell 136, 1136–1147, March 20, 2009 ª2009 Elsevier Inc.
Wnt3A increased the total number of hematopoietic progenitors,
the Wnt3A-induced increase (p < 0.01), demonstrating a
conserved requirement for PGE2 in mediating the effects of
Wnt stimulation. Dkk1 exposure reduced total colony number,
formation and/or expansion (Figure S7B). This decrease in hema-
topoietic progenitors was reverted by dmPGE2 (p = 0.046) or for-
skolin, demonstrating that as in zebrafish, PGE2 modulates Wnt-
mediated effects on mammalian hematopoietic stem/progenitor
cells via the cAMP/PKA pathway. By western blot analysis,
Wnt3A and dmPGE2 treatment increased total b-catenin levels,
while exogenous Dkk1 or indo markedly decreased b-catenin
(Figure S7C). Indo diminished the effect of Wnt3A, and dmPGE2
alleled the effect on colony formation seen in the hematopoietic
differentiation assays and correspond to the zebrafish results.
We next tested whether the regulation of Wnt/b-catenin func-
tion by PGE2 extended to the mammalian hematopoietic system
in vivo. To assess the effects of PGE2/wnt modulation on func-
tional murine stem/progenitor cells, isolated cKit+Sca1+Line-
age- (KSL) BM cells were transplanted into lethally irradiated
mice. To modulate Wnt activity and PGE2 levels, the GSK3b
inhibitor 6-bromoindirubin-30-oxime (BIO, 50 mg/kg) (Meijer
et al., 2003), indo (2.5 mg/kg), or a combination of both, were
administered intraperitoneally to recipient mice every other day.
CFU-S12analysis revealed a significant increase in hematopoi-
etic progenitors after BIO treatment (p = 0.015; Figure S8A).
Concurrent administration of indo reduced CFU-S12numbers
Figure 4. PGE2 Regulates the Effects of wnt
Activity on HSCs via cAMP/PKA Signaling
(A, B, E, F, I, J, M, and N) The /PGE2/wnt interaction
affected runx1/cmyb+ HSC formation, as seen in Figure 2.
(C and G) cAMP enhancement by forskolin increased
runx1/cmyb expression in WT embryos and further
expanded HSCs in wnt8 embryos. (D and H) Forskolin
counteracted the inhibitory effect of indo on HSC forma-
tion in WT and wnt8 embryos. (K and O) PKA inhibition
by H89 decreased runx1/cmyb expression in WT embryos
and eradicated HSC formation in dkk embryos. (L and P)
The enhancing effect of dmPGE2 on HSCs was reduced
back to baseline levels by PKA inhibition; similarly, the
dmPGE2-induced rescue of HSCs in dkk embryos was
blocked by H89.
to baseline levels, implying that PGE2 in recip-
vation on hematopoietic progenitor cells. To
within hematopoietic progenitors or within the
niche, purified KSL cells were treated directly
ex vivo prior to transplantation. BIO significantly
was further enhanced by combined treatment
with dmPGE2(p <0.05). Indoreduced the effect
0.0001; Figure 6A). To confirm that these obser-
vations were due to specific interactions with the Wnt pathway
and not to off-target effects of BIO, we used KSL cells isolated
from APCMinmice with constitutively elevated b-catenin levels
(Figure 6B). APC loss and dmPGE2 synergistically enhanced
CFU-S12number (p < 0.0001), while indo inhibited this effect
(p = 0.0004). Similarly, in KSL cells treated with Dkk1, progenitor
number was significantly enhanced by dmPGE2 or forskolin
(Figure S8B). From these data, we conclude that the effect of
tions requires PGE2.
tionalsignificance in HSCs,weperformedlong-term competitive
BM repopulation experiments. ST-repopulation at 6 weeks, and
LTR-engraftment at 12 and 24 weeks showed identical results:
the average chimerism after BIO treatment was significantly
higher (15.3 ± 19.9%) than in untreated animals (3.8 ± 3.8%,
p = 0.017; Figure 6C), with 7/10 recipients (70%) showing PB
chimerism > 5% compared to WT (5/20 (25%), Fisher’s exact,
p = 0.045; Figures 6C and 6D). The effect of BIO was reduced
toward baseline after indo exposure (3.3 ± 3.3%), with only 3/9
(33.3%) mice showing repopulation from dual-treated test cells.
[22%, APC+indo]; 14.7 ± 15.0% versus 5.3 ± 5.6%, p = 0.004),
confirming that the effects were specifically due to alterations
in wnt signaling (Figure 6D). These data indicate that mammalian
LTR-HSCs are directly influenced by the PGE2/Wnt interaction.
Several biochemical interactions have been proposed for the
relationship between PGE2 and wnt in vitro (Castellone et al.,
2005; Fang et al., 2007; Fujino et al., 2002; Hino et al., 2005;
Cell 136, 1136–1147, March 20, 2009 ª2009 Elsevier Inc. 1141
Shao et al., 2005). To further characterize the mechanism of
PGE2-based wnt regulation in HSCs, we isolated WBM, treated
cells ex vivo with PGE2-modifiers, and performed a chemilumi-
nescence assay for cAMP activity. cAMP levels increased in
a dose-dependent manner following dmPGE2 exposure, similar
to controls exposed to forskolin (Figure S8C). Total b-catenin
levels in WBM increased following dmPGE2 treatment, while
indo diminished b-catenin (Figure S8D). These results confirm
a direct effect of PGE2 on b-catenin stability in murine hemato-
poietic cells. Time-course analysis after incubation with either
dmPGE2 or indo demonstrated that alterations in total b-catenin
were preceded by changes in the phosphorylation status of
b-catenin at S675 and GSK3b at S9; these modifications can
influence the stability and destruction of b-catenin in vitro (Cas-
tellone et al., 2005; Fujino et al., 2002; Hino et al., 2005). These
results show that in the hematopoietic compartment PGE2
actively regulates wnt activity through phosphorylation-based
modulation of b-catenin protein stability.
Figure 5. The PGE2/wnt Interaction Is Conserved in
HSC Regeneration after Injury
(A) wnt activity in the kidney marrow was enhanced in FACS
profiles on day 3 post irradiation (dpi) in TOP:dGFP fish; repre-
sentative FACS analyses are shown on the left, and summa-
rized (mean ± SD) on the right. dmPGE2 further increased
and indo inhibited wnt activity; *sig versus unirradiated
con, **sig versus irradiated con, ANOVA, p < 0.001, n = 5 fish/
(B) wnt8 (bottom left panel) enhanced the precursor popula-
tion (red), while indo suppressed that effect significantly; *sig
versus con, **sig versus wnt8, t test, p < 0.02, n = 10-15.
(C) dkk reduced progenitor recovery, which was rescued by
dmPGE2; *sig versus con, ***sig versus dkk, ANOVA, p <
0.05, n = 6.
The PGE2/wnt Interaction Is a Central
Regulator of Organ Regeneration
To explore whether the PGE2/wnt interaction func-
tions as a master regulator of organ regeneration/
recovery in non-hematopoietic tissues, we exam-
ined the process of liver regeneration, which is
known to be wnt dependent (Goessling et al.,
2008; Tan et al., 2006). APC+/?fish demonstrated
enhanced regenerative capacity compared to WT
controls at 3 days post resection (Goessling et al.,
2008). As in HSCs, indo significantly diminished
organ recovery in both WT and APC+/?fish (p <
0.001, Figures 7A–7E). Expression of cyclin D1
was coordinately affected by wnt activity and
PGE2 levels (Figure 7F). Murine liver resections
documented conservation of this interaction during
mammalian organ regeneration. Following 2/3
partial hepatectomy, nuclear b-catenin accumula-
tion increased in WT and APCMinmice, most strik-
ingly in the periportal region, the location of the
presumed hepatic stem cells. Indo diminished
b-catenin levels in both WT and APCMinmice,
with virtual exclusion of b-catenin from hepatocyte
nuclei following indo treatment, confirming that during liver
regeneration PGE2 is necessary for b-catenin-mediated wnt
signaling (Figures 7H and 7J).
To assess the functional consequences of wnt activity and its
suppression by indo, cell proliferation was examined. BrdU
incorporation was enhanced in livers of APCMinmice (64.7 ±
8.0 cells/periportal field [APC] versus 48.0 ± 5.7 [con], p <
periportal BrdU incorporation in WT (2.2 ± 1.4, p < 0.001) and
APCMinmice (4.0 ± 1.1; Figures 7G–7K). Similarly, significant
differences wereseen in thenumber of periportal cyclinD1+cells
(151.3 ± 13.6 [con]; 218.8 ± 15.7 [APC]; 64.0 ± 3.4 [indo]; 91.5 ±
10.9 [APC + indo]; Figure 7L). Indo also induced extensive hepa-
tocyte necrosis (Figure S8J). Both WT and APCMinmutant livers
exhibited b-catenin (S675) (Figures S9A and S9E) and GSK3b
(S9) stabilizing phosphorylation (Figures S9B and S9F), which
was absent following indo treatment (Figures S9C, S9D, and
S9G–S9I), consistent with our observations in HSCs. These
1142 Cell 136, 1136–1147, March 20, 2009 ª2009 Elsevier Inc.
results document that PGE2 regulates Wnt activity through alter-
ations in b-catenin transcriptional availability and subsequent
proliferation during solid organ regeneration.
This study reveals a previously uncharacterized genetic interac-
tion of PGE2 and the Wnt pathway in hematopoietic stem/
progenitor populations in vivo, which is conserved during devel-
opment and tissue regeneration across vertebrates. While
previous studies in cell culture have shown these two pathways
interact, our work demonstrates the relevant in vivo mechanisms
and functional consequences of this relationship in HSCs. An
intriguing finding of our work is that indo treatment significantly
suppresses Wnt activity, supporting a model in which PGE2 is
required to mediate the full effects of Wnt activation in vivo
(Figure S1). The conserved interaction of these pathways during
wnt-dependent regeneration in other organ systems not only
highlights its importance in regulating stem cell/progenitor
formation and function, but may expand potential therapeutic
interventions to modulate complex signaling networks in regen-
erative medicine and cancer treatment.
Wnt Signaling Influences HSC Formation
The requirement of wnt activity in HSC self-renewal and marrow
repopulation is controversial. Several studies have clearly indi-
cated a positive effect of wnt activation on HSC recovery after
injury and in transplantation assays (Congdon et al., 2008;
Reya et al., 2003; Trowbridge et al., 2006). Similarly, we found
that wnt activation is sufficient to stimulate HSCs. Conversely,
b- and g-catenin deficient animals show no impairment in either
hematopoiesis or lymphopoiesis; this was interpreted as
evidence that canonical wnt signaling could not play an essential
found that even in the absence of b- and g-catenin function,
canonical wnt activity was still present (Jeannet et al., 2008).
These data imply that the natural complexity and redundancy
of the wnt signaling pathway may subvert attempts to render it
non-functional, possibly by bypassing components typically
considered required. Nevertheless, wnt activation is sufficient
to stimulate HSCs. While we did not address the question of
a requirement for b-catenin directly, we found that TCF signaling
was necessary for optimal HSC formation. These findings are
consistent with an emerging role of wnt, not only as a crucial
factor affecting body axis and polarity during early development,
but as a central regulator of stem and progenitor populations in
multiple organs (Goessling et al., 2008; Hirabayashi et al., 2004).
PGE2 Regulates wnt Activity by Direct Phosphorylation
of b-catenin and GSK3b
Our work shows that PGE2 acts via cAMP/PKA signaling to
directly modify b-catenin stability and emphasizes the functional
importance and evolutionary conservation of these interactions
in organ development and regeneration. PGE2-mediated reg-
ulation of Wnt signaling was previously demonstrated in cell
lines. The proposed biochemical mechanisms mediating the
Figure 6. PGE2 Influenced wnt-Mediated
Repopulation of Murine HSC and Pro-
(A) Ex vivo treatment of purified C57Bl/6 KSL cells
with indo, BIO, and/or dmPGE2 prior to transplan-
tation into lethally irradiated mice revealed that
PGE2 modulation significantly impacts hemato-
poietic progenitors (mean ± SD). BIO significantly
enhanced colony formation, which could be sup-
pressed by indo. dmPGE2 further increased the
effect of BIO; *sig versus con, ANOVA, p < 0.05,
n R 7.
(B) Effect of ex vivo treatment with dmPGE2 or
indo on CFU-S12colony formation in KSL cells
from APCMinmice (mean ± SD). Genetic activation
of wnt signaling in APCMinhematopoietic progen-
itor cells has comparable effects on CFU-S12
colony formation to chemical stimulation by BIO,
and indo exposure blocks this enhancement; *sig
versus con, ANOVA, p < 0.05, n R 7.
(C and D) Wnt activation through the GSK3b inhib-
itor BIO or in APCMinmarrow enhanced chimerism
rates at 24 weeks; each effect could be inhibited
by indo. Test cell chimerism of individual mice is
shown (C), with the mean/group indicated by
a solid black line. The dashed black line demon-
strates the 5% cut-off value used to determine
engraftment frequencies (D); *sig versus con,
Fisher’s exact, p = 0.045; n R 8.
Cell 136, 1136–1147, March 20, 2009 ª2009 Elsevier Inc. 1143
interaction are remarkably diverse and may depend on the
particular cell line studied (Clevers, 2006). Castellone et al.
used colon cancer cell lines to demonstrate PGE2/PI3K-medi-
ated activation of Akt, leading to dissociation of Axin1 from the
destruction complex (Castellone et al., 2005). While we cannot
exclude the existence of this biochemical interaction in vivo,
our studies suggest that activation of PKA is the functionally
significant effector downstream of PGE2 in HSCs. Likewise,
both phosphorylation of GSK3b and b-catenin by PGE2-based
activation of PKA have been implicated in Wnt signaling regula-
tion in vitro. Fujino et al. used the transformed HEK293 cell line
and showed that both PKA and PI3K could function downstream
of PGE2 to phosphorylate GSK3b (Fujino et al., 2002). However,
PGE1-induced activation of PKA and phosphorylation of b-cate-
nin at S675 was cell-line dependent (Hino et al., 2005). While we
found both phosphorylation events occur in the presence of
PGE2 in vivo, it remains to be determined if phosphorylation of
Figure 7. The PGE2/wnt Interaction Is a Master Regu-
lator of Liver Regeneration
(A–D) Representative photomicrographs of en bloc dissec-
tions following liver resections at day 3 are shown; the liver
is highlighted by a yellow dotted line, the resection site by
a blue line, and the black arrow indicates the amount of liver
regrowth. Wnt activation in APC mutant zebrafish enhanced
liver regeneration compared to WT. Indo stymied liver regen-
(E) Quantification of zebrafish liver regeneration showed
significant differences across treatment groups; *sig versus
con, **sig versus APC+/?, ANOVA, p < 0.001, n R 6.
(F) qPCR for cyclin D1 gene expression; effects are coordi-
nately regulated by the PGE2/wnt interaction; *sig versus
con, **sig versus APC, ANOVA, p < 0.001, n = 7.
(G–J) wnt and PGE2 modulation has significant effects on
murine liver regeneration following 2/3 partial hepatectomy.
APCMinmice exhibit enhanced b-catenin staining (left panel),
particularly in the periportal areas (top middle panel), with
noticeablenuclearstaining (bottom middle panel). BrdUincor-
poration (top right panel) and cyclin D1 staining (bottom right
panel) indicated enhanced regenerative activity. Indo dimin-
ished global b-catenin staining (left and top middle panels),
excluded b-catenin from the nuclei (bottom middle panels),
and resulted in a corresponding decrease of both BrdU incor-
poration and cyclin D1.
(K and L) Quantification of BrdU incorporation and cyclinD1
*sig versus con, **sig versus APC, ANOVA, p < 0.05, n =
Data represented as mean ± SD.
of wnt activity by PGE2 in HSCs, especially given
a recently proposed functional redundancy of
GSK3b with GSK3a (Doble et al., 2007).
The Role of PGE2/wnt Interaction
in Regeneration and Carcinogenesis
The role of Wnt in organ regeneration has been
described (Goessling et al., 2008; Stoick-Cooper
et al., 2007). One universal response to tissue injury
is enhanced PGE2 production (Goldstein et al.,
1977). PGE2 may have coevolved with Wnt as a mechanism to
rapidly upregulate cellular proliferation to foster organ repair. In
this setting, PGE2—which is produced locally in response to
tissuedamage—is requiredforand canenhancethe proliferative
effects initiated by wnt activitation. Short-term exposure to
PGE2 has been shown to increase HSC engraftment and could
be useful for regeneration in many organs. In support of this
hypothesis, we found that wnt-mediated fin regeneration was
similarly regulated by PGE2 levels (Figures S9K and S9L). It is
also possible that chronic stimulation of PGE2 may exhaust
stem cells, as has been shown for the wnt pathway (Scheller
et al., 2006); thus, the therapeutic use of PGE2 to regulate wnt-
mediated processes will need to be investigated further.
The genetic interaction between PGE2 and wnt signaling may
provide insight into the basis of carcinogenesis occurring in
cases of chronic inflammation. Chronic overproduction of
PGE2 may lead to constitutive Wnt activation in vivo, resulting
1144 Cell 136, 1136–1147, March 20, 2009 ª2009 Elsevier Inc.
given to controlling inflammation during early therapeutic regi-
mens for colon cancer that led to the identification of COX inhib-
itors as potentregulators of tumor formation, long before the role
(Giardiello et al., 1993). Evolutionarily, what was greatly benefi-
cial for wound healing—the coordinated proproliferative, anti-
apoptotic effects of wnt and PGE2: may be detrimental in cases
of chronic inflammation or constitutive pathway activation and
lead to tumor initiation and growth. Targeting the wnt signaling
pathway as a cancer treatment option, such as through direct
inhibition of b-catenin, has proven problematic because of its
ubiquitous importance for tissue homeostasis (Barker and
Clevers, 2006). Our results indicate a mechanism whereby
chemical manipulation of PGE2 levels or signaling to regulate
Wnt activity in stem and progenitor cells may be therapeutically
beneficial for the controlled regulation of both tissue repair and
See also Supplemental Experimental Procedures.
genic lines lmo2:DsRed, TOP:dGFP, hs:wnt8-GFP, hs:dkk-GFP, hs:dnTCF-
GFP, hs:axin-GFP, and cmyb:GFP and the APC mutant were described previ-
ously (Dorsky et al.,2002; Hurlstone et al.,2003; Lewis et al.,2004; North etal.,
2007; Stoick-Cooper et al., 2007; Weidinger et al., 2005; Zhu et al., 2005).
Embryonic Zebrafish Experiments
All analysis was performed at 36 hpf. Embryos were exposed to compounds
from 10 somites to examination at the following doses, unless otherwise indi-
cated: dmPGE2, indo 10 mM; forskolin, H89 0.5 mM; LY294002, KT5720, Wort-
mannin 1 mM; DMSO carrier content was 0.1%. In situ hybridization with stan-
dard zebrafish protocols (zfin.org/ZFIN/Methods/ThisseProtocol.html) was
performed for runx1, cmyb, and GFP; qualitative changes from WT are
reported as the # altered/ # scored (median examples shown). Photomicro-
graphs were taken of representative examples with Nomarski optics at 43
and 203. FACS and confocal microscopy were conducted as described
(North et al., 2007). qPCR (primers shown in Table S1) and western blot anal-
ysis were performed on pooled whole embryo lysates. BrdU and TUNEL anal-
ysis was performed as published (Shepard et al., 2005).
Heatshock Modulation of wnt Signaling
Embryonic heatshock experiments were conducted as previously described
(Goessling et al., 2008); out-crossed embryos at the 10 somite stage were
shocked at 38?C for 20 min, and sorted by genotype on the basis of GFP
expression. Heatshock modulation in adult zebrafish was performed as
Adult Zebrafish Experiments
Adult zebrafish were exposed to 23 Gy of g-irradiation. KM recovery was
analyzed by FACS at days 3 or 10 as described (North et al., 2007); heatshock
occurred at 38?C from 36–48 hr postirradiation (hpi) and chemical treatment
from 48–60 hpi. KM from individual fish was manually dissected in 0.9%
PBS, dissociated and examined for alterations in GFP expression, or HSC
recovery by forward scatter/side scatter. Liver resections and regeneration
analysis were conducted as described (Goessling et al., 2008); heatshock
occurred from 6–16 hr postresection (hpr) and chemical treatment from
18–30 hpr. Liver regrowth was quantified in en bloc dissected specimen by
lobe to the original resection site.
cAMP Luminescence Assay
50,000 WBM cells were exposed to increasing doses of either dmPGE2 or for-
skolin for 15 min. The luminescence assay was performed according to stan-
dard manufacturer protocol (Promega, cAMP Glow).
ES Cell Culture and Differentiation
ES cells were cultured and differentiated as embryoid bodies (EBs) as
described (Kyba et al., 2002) with modifications: EBs were transferred to
differentiation media on day 2 and incubated for 4 days. On day 4, PGE2/
lin, 10 ng/ml Wnt3A, 400 ng/ml Dkk1, 10 mM dmPGE2. On day 6, EBs were
plated (105cells) into M3434 methylcellulose. CFU-Cs (CFU-E (erythroid),
CFU-M (monocyte), CFU-G (granulocyte), CFU-GM (granulocyte-monocyte),
and CFU-GEMM (granulocyte-erythroid-monocyte-megakaryocyte)) were
scored on day 8–10 by morphology. Averages +/?SEM in the fold changes
of total CFU-Cs relative to control were calculated.
WBM from 8-week old C57Bl/6 or APCMinmice were used for transplantation
studies. For CFU-S12analysis, KSL cells were FACS-sorted, treated as indi-
cated and transplanted at 500 cells/recipient as previously described (North
et al., 2007); spleen colonies were counted at day 12. For LTR low-dose
competitive transplantation using CD45.1/CD45.2 mice, 15,000 treated test
WBM cells were exposed ex vivo as indicated and injected with 200,000
untreated competitors into recipient mice; PB was obtained at 6, 12, and
Murine Liver Resections
Two-third partial hepatectomy was performed as described (Greene and
Puder, 2003); C57Bl/6 or APCMinmice were injected with DMSO or 2.5 mg/kg
indo i.p. every 12 hr, beginning 12 hr prior to resection. BrdU was injected 2 hr
before mice were sacrificed. BrdU and CD1 cell counts were performed on
corresponding sections of 203 microscopy fields focused on the periportal
regions (n = 10 sections).
Pooled data were calculated as mean ± SD, with number of repeats as indi-
cated. Pairwise comparison was performed by t test, multiple comparisons
by ANOVA, unless otherwise noted, using SigmaStat 3.5 software.
Supplemental Data include Supplemental Experimental Procedures, nine
figures, one table, and Supplemental References and can be found with this
article online at http://www.cell.com/supplemental/S0092-8674(09)00022-1.
We thank: D.Langenau and C.Ceol for critical reading of the manuscript;
C.R.Walkley and L.Purton for advice on HSC transplants; and J.Harris and
C.C.Cutting for technical assistance. This work was supported by the National
Institutes of Health (W.G., G.Q.D., R.T.M., and L.I.Z.), the American Gastroen-
terological Association (W.G.), the American Cancer Society (T.E.N.), and the
Human Frontier Science Program (S.L.). G.Q.D., R.T.M., L.I.Z. are Howard
Hughes Medical Institute investigators.
Received: August 26, 2008
Revised: November 7, 2008
Accepted: January 5, 2009
Published: March 19, 2009
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