HOXB4’s road map to stem cell expansion
Bernhard Schiedlmeier*†, Ana Cristina Santos‡§, Ana Ribeiro‡, Natalia Moncaut‡, Dietrich Lesinski*, Herbert Auer¶,
Karl Kornacker?, Wolfram Ostertag*, Christopher Baum*, Moises Mallo†‡, and Hannes Klump*
*Department of Experimental Hematology, Hannover Medical School, Carl-Neuberg-Strasse1, 30625 Hannover, Germany;‡Instituto Gulbenkian de Ciencia,
2780-156 Oeiras, Portugal;¶Columbus Children’s Research Institute, Columbus, OH 43210; and?Division of Sensory Biophysics, Ohio State University,
Columbus, OH 43205
Edited by Tasuku Honjo, Kyoto University, Kyoto, Japan, and approved September 10, 2007 (received for review April 3, 2007)
Homeodomain-containing transcription factors are important reg-
ulators of stem cell behavior. HOXB4 mediates expansion of adult
and embryo-derived hematopoietic stem cells (HSCs) when ex-
pressed ectopically. To define the underlying molecular mecha-
nisms, we performed gene expression profiling in combination
with subsequent functional analysis with enriched adult HSCs and
embryonic derivatives expressing inducible HOXB4. Thereby, we
identified a set of overlapping genes that likely represent ‘‘uni-
in signaling pathways important for controlling self-renewal,
maintenance, and differentiation of stem cells. Functional assays
performed on selected pathways confirmed the biological coher-
the detrimental effects mediated by the proinflammatory cytokine
TNF-?. This protection likely contributes to the competitive re-
population advantage of HOXB4-expressing HSCs observed in
vivo. The concept of TNF-? inhibition may also prove beneficial for
patients undergoing bone marrow transplantation. Furthermore,
we demonstrate that HOXB4 activity and FGF signaling are inter-
twined. HOXB4-mediated expansion of adult and ES cell-derived
HSCs was enhanced by specific and complete inhibition of FGF
receptors. In contrast, the expanding activity of HOXB4 on hema-
topoietic progenitors in day 4–6 embryoid bodies was blunted in
the presence of basic FGF (FGF2), indicating a dominant negative
effect of FGF signaling on the earliest hematopoietic cells. In
summary, our results strongly suggest that HOXB4 modulates the
response of HSCs to multiple extrinsic signals in a concerted manner,
thereby shifting the balance toward stem cell self-renewal.
hematopoiesis ? HOX genes ? microarray ? self-renewal ? embryoid bodies
and characteristic ability to undergo self-renewal divisions and/or
differentiate into mature blood cells, strategies to expand HSCs ex
become of key interest.
Expansion of HSCs naturally occurs in the fetal liver during
organism as a response to extrinsic cues. The signaling pathways
controlling this process and the molecular mechanisms specifying
whether the self-renewal potential is lost or retained during HSC
cell divisions remain largely ill-defined. However, accumulative
evidence shows that HSC fate decisions are controlled by a delicate
balance of the intrinsic genetic program of HSCs and extrinsic
signals provided by their microenvironment, the stem cell niche.
Some of the intrinsic regulatory genes (Bmi1, Cdkn1a, Pten, Etv6,
Mcl1, Hoxb4) that control stem cell maintenance, expansion, and
differentiation and some extrinsic signals (Wnt, Ang-1, Notch
ligands, Gp130 ligands) that are exchanged between HSCs and
niche cells have been identified recently (1).
Numerous members of the homeobox gene family are involved
in the regulation of normal and leukemic hematopoiesis at various
stages. In fact, the human homeodomain transcription factor
HOXB4 was the first gene shown to lead to profound HSC
expansion in vitro and in vivo when ectopically expressed in murine
he lifelong production of blood cells by the hematopoietic
system strictly depends on the maintenance of its central
BM cells. These HSCs fully replenish the stem cell pool of lethally
irradiated mice and maintain a normal supply of HSCs and mature
also enhances the in vitro development of definitive HSCs from
differentiating mouse ES cells that are capable of reconstituting
recipient mice (4, 5). However, ectopic expresssion of HOXB4 may
eventually transform hematopoietic cells. Our group and others
have described that the amount of ectopically expressed HOXB4
has an impact on myeloid, lymphoid, and erythroid differentiation
of adult HSCs of mice and humans in vitro and in vivo and on ES
cell-derived hematopoietic differentiation (6–8).
The molecular mechanism behind how this transcription factor
acts in immature blood cells is poorly understood. Homeodomain-
HOXB4-expressing HSCs (9). However, only a few HOXB4 target
genes have been identified so far. HOX proteins can form high-
affinity DNA-binding complexes with other homeodomain-
containing proteins such as PBX1 (10). However, direct HOXB4–
PBX1 protein interactions are not required for stem cell expansion
(9). In contrast, PBX1 limits HOXB4-induced HSC expansion in
To further characterize the molecular mechanisms underlying
the HOXB4-induced expansion of HSCs, we analyzed the gene
expression profiles of adult HSC/hematopoietic progenitor cells
(HPCs) and differentiating ES cells expressing inducible forms of
tests, in vivo, we demonstrate that HOXB4 changes the cellular
response of stem cells to conserved signaling pathways known to
affect cell fate decisions of HSCs in adult and differentiating ES
cells. These results may also reflect general pathways of homeobox
transcription factor activities that are relevant for developmental
Identification of HOXB4 Target Genes in Primary Murine HSCs and
HPCs. To understand the mechanisms of HOXB4 activity, we
wanted to identify target genes of HOXB4 in adult HSCs and
HPCs. We thus transduced murine HSC/HPCs with a retroviral
vector that coexpresses EGFP and a 4-hydroxytamoxifen (TMX)-
Author contributions: B.S., H.A., M.M., and H.K. designed research; B.S., A.C.S., A.R., N.M.,
D.L., H.A., and H.K. performed research; H.A. and K.K. contributed new reagents/analytic
tools; B.S., H.A., K.K., M.M., and H.K. analyzed data; and B.S., H.A., W.O., C.B., M.M., and
H.K. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Abbreviations: BM, bone marrow; HSC, hematopoietic stem cell; HPC, hematopoietic
progenitor cell; LSK, Lin?Sca1?c-kit?; EB, embryoid body; TMX, 4-hydroxytamoxifen;
hematopoietic cell; GEMM, granulocyte, erythroid, macrophage, megakaryocyte.
mh-hannover.de or email@example.com.
whomcorrespondencemaybe addressed. E-mail:schiedlmeier.bernhard@
§Present address: Molecular Hematology Laboratory, John Radcliffe Hospital, Oxford, OX3
9DS, United Kingdom.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2007 by The National Academy of Sciences of the USA
October 23, 2007 ?
vol. 104 ?
inducible form of HOXB4 (HOXB4-ER). Upon addition of TMX,
the HOXB4-ER fusion protein translocates from the cytoplasm to
the nucleus, consequently being capable of modulating gene ex-
the presence of TMX. Thereafter, HOXB4-ER?LSK (where LSK
is Lin?, Sca1?, c-kit?) cells were flow cytometrically isolated and
cultivated either with or without TMX for 1 or 4 h. Inactivation of
HOXB4 activity by TMX withdrawal was intended to mimic the
naturally occurring down-regulation of HOXB4 in differentiating
and the transcriptional profiles of HOXB4-ER?LSK ? TMX were
analyzed with the Affimetrix platform. As a control, profiling was
active HOXB4 (HOXB4const) ? TMX, to exclude changes in gene
expression caused by unknown effects of TMX itself [supporting
information (SI) Fig. 5].
Genes whose level of expression consistently and significantly (i)
changed ?2.0 fold 4 h after withdrawal of TMX in one of the two
independent experiments and (ii) changed ? 1.5-fold after with-
drawal of TMX in both HOXB4constLSK control group samples
were considered differentially and specifically regulated by
HOXB4. This analysis identified 156 HOXB4-regulated genes in
proliferating LSK cells, of which 23 were uncharacterized tran-
scripts (SI Table 1). All 23 uncharacterized target genes, and 103 of
the 133 characterized genes, were also differentially regulated
?1.5-fold in the second independent experiment. The 133 charac-
terized genes identified by the above criteria belonged to different
(in vivo) proliferating fetal liver HSCs and their quiescent adult
verify the observed expression differences, we selected 49 of the
identified HOXB4 target genes for qRT-PCR analysis (Fig. 1 and
SI Fig. 6). For this purpose, RNA was prepared from independent
samples of transduced and purified HOXB4-ER?LSK cells that
had been incubated with and without TMX for 4 h to induce
of HOXB4, samples of sorted HOXB4-ER?LSK cells were treated
with cycloheximide (CHX) 30 min before exposure to TMX. Thus,
any change of RNA levels in response to HOXB4 activation is
independent of new protein synthesis. The results of the qRT-PCR
generally showed that the majority of the genes found down-
(microarray results) are up-regulated when HOXB4 activity is
induced (36/49 ? 72%). With this approach, we identified 40 of the
50 tested genes as putative direct targets of HOXB4 activity (e.g.,
Bambi, Bre, Catnb, Cdkn1a, Cyclin G2, Dll-1, Dusp6, Foxo3a, Klf3,
Mad, Ptgs2, Socs2, Socs6, T1e1, Tnfrsf1b, and Zfx). Interestingly,
inhibiting translation before induction of HOXB4 activity evoked a
contrary gene expression pattern of 14 analyzed targets after 4 h.
This finding strongly suggests that the activating or repressing
activity of HOXB4-containing transcription factor complexes de-
pends on unstable protein cofactor(s).
HOXB4 Targets Similar Signaling Pathways in Differentiating ES Cells
and Adult HSCs. To uncover the genetic program underlying the
enhanced production of definitive HSCs from early embryonic
cells enforced by HOXB4 (4–6), we also analyzed gene expres-
sion changes in differentiating mouse ES cells containing a
tetracycline-inducible HOXB4 gene (4). We applied doxycycline
(Dox) for 48 h from day 4 to day 6 of embryoid body (EB)
differentiation, the period during which commitment to primi-
tive HSCs occurs and the very first HSCs are detectable (14, 15).
EBs were dissociated at day 6, and total RNA was isolated for
subsequent microarray hybridization.
We identified ?700 differentially regulated genes in HOXB4-
induced EBs whose level of expression consistently changed ?2.0-
of selected genes differentially regulated in EBs is provided in SI
Table 5. Remarkably, HOXB4 engaged in the same signal trans-
in part by targeting identical gene products. Fig. 2 summarizes the
results obtained with ES cell-derived cells and adult HSCs/HPCs
according to their affiliation to various signaling pathways already
implicated in HSC self-renewal (among others, TNF-?, FGF, Wnt,
in adult HSC/HPCs and EBs (SI Table 6). qRT-PCR analysis of
independent RNA populations from Dox-induced HOXB4EB
day-6 cells confirmed 19 of 30 common gene loci examined as
differentially expressed (SI Fig. 7). A similar analysis performed
with ES cell-derived hematopoietic cells (ES-HCs) (6) expressing
the TMX-inducible form of HOXB4 (SI Fig. 8) proved 31 of the 32
ray study. Gene expression changes were measured in triplicate analysis of RNA
samples from purified HOXB4ER?LSK cells that had been incubated with and
samples were calculated by using the 2???CTmethod (13) by normalizing the CT
shown as log2-fold induction (mean ? SD) in the absence (green bars) and
differential gene expression in the presence of CHX.
qRT-PCR confirms changes in gene expression observed in the microar-
Schiedlmeier et al.
October 23, 2007 ?
vol. 104 ?
no. 43 ?
turned out to be putative direct target genes. Some of the shared
direct target genes were regulated in opposite directions in adult
containing transcription factor complexes may either act as repres-
sors or activators of one and the same gene locus. The results
presented so far suggest that the overlapping genes represent
‘‘universal’’ targets of HOXB4 and affirm the idea that HOXB4
affects similar molecular circuits during the commitment of hema-
topoietic progenitors in differentiating ES cells (EBs) and during
the expansion of ES-HCs and adult HSCs.
HOXB4 Protects Long-Term Repopulating HSCs from the Negative
Effects of TNF-?. Gene profiling results per se are not sufficient to
on their known involvement in defined pathways and tested their
collective relevance for HSCs/HPCs’ self-renewal in functional
In adult HSCs/HPCs, HOXB4 mediated the down-regulation of
Tnfrsf1b (also known as p75/TNFR) and concomitantly up-
regulated Bre (brain and reproductive organ-expressed protein), an
inhibitor of TNF receptor 1 (p55/TNFR) and Fas signaling. TNF-?
negatively regulates self-renewal of cycling murine and human
HSCs by enforcing myeloid differentiation (16, 17). Our results
suggested that interference with this signaling pathway would
contribute to continued HSC self-renewal. To test this hypothesis,
LSK cells expressing the inducible HOXB4-ER vector or a trun-
cated form of human CD34 (tCD34) that does not affect multilin-
eage differentiation in vivo (18) were mixed in a 1:1 ratio and
cultivated for 7 days in serum-free medium with or without
TNF-? ? HOXB4 induction by TMX (Fig. 3A). Thereafter, we
determined the proportion of LSK cells in the HOXB4-ER/tCD34
cell mixes and tested their ability to reconstitute the hematopoietic
system of lethally irradiated mice. In the absence of TNF-? and
TMX, cultured HOXB4ER?and tCD34?cells both contained
of repopulation (Fig. 3B). However, in the presence of TNF-?
without HOXB4 induction, the reconstitution ability of both cell
and in the presence of TNF-?, multilineage reconstitution was
Hence, HOXB4 protects long-term repopulating HSCs from the
negative effects of TNF-?.
HOXB4 Modulates FGF Signaling. In EBs, HOXB4 regulated the
expression of FGFs (Fgf8, Fgf15), Fgf receptor 1 (Fgfr1), and Ets
player in the transcriptional activation of FGF target genes, which
is commonly phosphorylated in response to FGF signaling. In line,
qRT-PCR analysis of HOXB4-induced adult HSCs/HPCs showed
two members of the negative feedback loop of FGF signaling (19).
These findings prompted us to ask whether HOXB4 activity
functionally interferes with FGF signaling during hematopoiesis.
Thus, we measured colony formation by HOXB4-expressing dif-
ferentiating ES cells that had been exposed to an FGF receptor
tyrosine kinase inhibitor (SU5402) from day 4 to day 6 of EB
differentiation. In parallel, we also analyzed the expression of
Dusp6, Spry2, Etv5, and Fgf1 by qRT-PCR. Without HOXB4
formation of the most primitive hematopoietic colonies, CFU-
GEMM (granulocyte, erythroid, macrophage, megakaryocyte)
(Fig. 4A; P ? 0.093). The expression levels of the aforementioned
FGF targets (Fig. 4B) decreased. In contrast, induced ectopic
expression of HOXB4 without SU5402 stimulated an increase of
and EBs. HOXB4 influences the expression of genes involved in pivotal cell-
based on published reports in various mammalian systems. Arrows indicate
up-/down-regulation of gene expression as a consequence of HOXB4 induction.
Selected regulatory pathways affected by HOXB4 in adult HSCs/HPCs
expressing LSK cells in a competitive transplant setting. The progeny (expansion
equivalent) of 2,000 HOXB4ER-transduced and 2,000 control vector (tCD34)-
transduced LSK cells were mixed and cocultured, as indicated, in serum-free
cytokine-supplemented medium in the presence or absence of TNF-? or FCS ?
and tCD34-expressing LSK cells in the resulting cell populations was determined
irradiated recipient mice. (B) Reconstituting activity of HOXB4ER-expressing (■)
lines indicate the median percentage of HOXB4ER?or tCD34?cells in the pe-
ripheral blood of the mice 11 weeks after transplantation.
Induction of HOXB4 activity protects cultivated HSCs from the negative
www.pnas.org?cgi?doi?10.1073?pnas.0703082104Schiedlmeier et al.
CFU-GEMM numbers (Fig. 4A). HOXB4 activity down-regulated
expression of Etv5, Dusp6, and Fgfr1, providing evidence that
HOXB4 attenuates activation of these FGF target genes. HOXB4
uppermost level, the FGF receptor, significantly further increased
the frequency of CFU-GEMMs (P ? 0.0012). Consistently, we
observed further reduced expression levels of Dusp6 and Etv5 (Fig.
4 A and B). Addition of bFGF (FGF2) inhibited the HOXB4-
induced increase of CFU-GEMMs within differentiating EBs (P ?
0.0013; Fig. 4C) and congruently up-regulated the expression of
Dusp6 (Fig. 4B). These results suggest that the biological outcome
of HOXB4 activity is controlled by FGF signaling, which appears
up-regulation of T (Brachyury) and Kdr (Flk1) markers that are
hallmarks of mesoderm and hemangioblast development. Thus, to
further clarify whether FGF signaling affects the commitment to
definitive hematopoietic cells and/or their subsequent expansion/
differentiation, HOXB4-ER-expressing ES-HCs (21 days after EB
dissociation) were cultured with or without either bFGF or the
inhibitor SU5402 for 14 days and colony assays were performed.
HOXB4 induction plus inhibition of FGF signaling significantly
increased the frequency of CFU-GEMMs compared with those
treated with FGF2 (P ? 0.023). In contrast to EBs, addition of
bFGF did not apparently decrease the frequency of CFU-GEMMs
in cultured ES-HCs (SI Fig. 10). Thus, HOXB4 and FGF signaling
seem to act antagonistically during hematopoietic commitment
within EBs but not during expansion of ES-HCs, which is in
agreement with the known suppression of hematopoiesis by FGF
signaling during embryonic development (20).
Finally, we investigated the relationship between HOXB4 and
FGF signaling in the context of adult HSCs in vivo. HOXB4-ER/
irradiated mice after expanding them for 7 days in serum-free
medium with and without SU5402 in the presence of TMX. In the
exhibited long-term repopulating activity (SI Fig. 11). tCD34?cells
provided a slightly higher, but not significant (P ? 0.10), donor cell
chimerism than induced HOXB4-ER?cells did in the recipient
mice. However, in the presence of SU5402, the proportion of
HOXB4-ER?donor cells was significantly higher than the propor-
tion of tCD34?donor cells (1.6-fold; P ? 0.043). Particularly, the
proportion of HOXB4-ER?donor cells increased 1.95-fold,
whereas the proportion of tCD34?donor cells decreased 2.6-fold
compared with the chimerism generated without SU5402. Thus,
inhibition of FGF signaling stimulated the expansion of HOXB4-
ER?HSCs, whereas it suppressed the expansion of the tCD34?
control HSCs. Hence, as our inhibitor studies clearly indicate, FGF
signaling limits the extent of HSC expansion but only in the context
of HOXB4 activity, whereas it stimulates the expansion of normal
HSCs. This finding is congruent with the positive role of FGF
signaling in self-renewal of normal HSCs previously reported by de
Haan et al. (21).
To elucidate the molecular machinery responsible for HOXB4-
induced HSC expansion, we identified gene targets of
HOXB4 in adult HSCs/HPCs and ES cell-derived EB. We link
HOXB4 to genes that are known to be expressed in adult stem
cells. qRT-PCR assays confirmed the observed changes and
suggest that the majority are likely direct targets of HOXB4.
Some of these direct target genes like Cnkn1b, Mad, Foxo3a,
Ptgs2, and Zfx have recently been shown to be crucial for
self-renewal, survival, and maintenance of adult HSCs (22–28),
an observation that fortifies the validity of our results. Fifty-two
gene loci were regulated by this transcription factor in both adult
HSC/HPC and EBs. This set of overlapping genes, also validated
by real-time PCR, likely represent universal targets of HOXB4.
The target genes are involved in a wide range of cellular
processes, such as signal transduction, cell cycle, apoptosis, and
response to stress and transcription. Our results also demon-
strate that HOXB4 can act as a transcriptional activator or
repressor of a given responsive gene locus, depending on the
Interestingly, differentiating ES cells revealed far more genes
selectively regulated by HOXB4 than enriched adult HSCs. This
expression, suggesting that many of the targets are indirectly
regulated. Moreover, EBs contain a mixture of cells from all three
to the hematopoietic system.
A major finding of this study is that a large fraction of the
HOXB4 target genes are components or essential regulators of
signaling pathways, such as cytokine, Wnt/?-catenin, Notch, FGF,
TGF-?/Activin/BMP, and TNF-? signaling (Fig. 2). All of those
during embryonic development. Interestingly, HOXB4 orches-
trated the same signaling pathways in EBs. The results support our
proposed concept that HOXB4 modulates multiple inputs of
distinct extrinsic signaling pathways, consequently leading to dis-
crete biological outcomes (29).
HOXB4 Inhibits Negative Regulators of Adult HSC Self-Renewal.
HOXB4 mediated the down-regulation of Tnfrsf1b and up-
regulated the expression of Bre, which has been shown to inhibit
apoptosis induced by TNF-?, Fas ligand, and various other stress-
related stimuli (30). TNF-? negatively affects the self-renewal of
cycling murine and human HSCs (16, 17). In this study, we showed
that both Tnfrsf1b and Bre are directly regulated by HOXB4.
Moreover, we showed in a functional assay that HOXB4 activity
protected adult HSCs from the compromising effects of TNF-? on
HSC self-renewal. TNF-? levels quickly increase in irradiated or
fluorouracil–treated recipients (31, 32). Thus, it is likely that
were treated from days 4 to 6 of EB formation with and without Dox in the
absence or presence of either the FGF receptor inhibitor, SU5402, or bFGF
(FGF2) as indicated. EBs were collected on day 6 and processed for methylcel-
lulose-CFU assays or for RNA extraction and subsequent semiquantitative
RT-PCR analysis. (A) Inhibition of FGF signaling enhances HOXB4-mediated
Etv5, a key player in the activation of FGF target genes. (C) bFGF inhibits
HOXB4-mediated expansion of ES cell-derived early HPCs. Ery, definitive
erythroid; GM, granulocyte macrophage. Results are presented as mean ? SD
(n ? 3). P values were determined by Student’s t test.
HOXB4 and FGF signaling are intertwined. Inducible HOXB4 ES cells
Schiedlmeier et al.
October 23, 2007 ?
vol. 104 ?
no. 43 ?
vivo. This observation has potential clinical relevance, as licensed
drugs for targeted suppression of TNF-? signaling are available.
In vivo, HSC expansion is also promoted by Smad7 overex-
pression, which blocks all TGF-?/Activin and BMP signaling
(33). HOXB4 directly up-regulated the expression of Bambi, a
pseudoreceptor that also acts as a pan inhibitor of this signaling
pathway and therefore presumably facilitates in vivo HSC ex-
resistant to TGF-?-mediated growth inhibition of the parental
BaF3 cells (B.S., unpublished data). In contrast to adult HSCs/
HPCs, HOXB4 apparently modulated signaling of the TGF-?
family at multiple levels in differentiating ES cells. Increased
expression levels of the Smad-coactivator Dcp1a together with
elevated expression of attenuators of this signaling pathway
(Smurf2, Dach1, Etv1) suggest that HOXB4 presumably specifies
and fine-tunes the duration of the cellular responses elicited in
differentiating ES cells.
HOXB4 and FGF Signaling Are Intertwined. The role of the FGF
pathway in embryonic hematopoietic development and regula-
tion of adult HSCs is still controversial. Here, we showed that
FGF signaling is a target of HOXB4 activity and also negatively
regulates HOXB4-mediated expansion of adult and ES cell-
derived HSCs. Chemical blockage of FGF signaling augmented
the long-term repopulation activity of HOXB4-expressing
HSCs/HPCs expanded in vitro, whereas it suppressed the re-
population activity of control HSCs/HPCs. Similarly, inhibition
of FGF signaling in differentiating EBs between days 4 and 6
further expanded progenitor numbers, which were already in-
creased by ectopic HOXB4 expression. However, without
HOXB4 expression blocking, FGF signaling did not alter the
frequency of early progenitors. Between days 4 and 6 EBs
already contain mesodermal Brachyury?(Bry?) cells. Our re-
sults fit well to the fact that a conditional knockout of Fgfr1 or
Fgfr1/2 in already formed FLK1?mesodermal cells, in vivo, does
not affect the hematopoietic progenitor content of fetal livers
(34). Loss of Fgfr1 at earlier stages, however, allows differenti-
but they fail to express normal levels of FLK1 (35). Moreover,
inhibition of FGF signaling in chicken embryos has been shown
to promote blood cell differentiation and inhibit endothelial cell
differentiation, presuambly at the hemangioblast stage (20).
That finding supports our conclusions.
HOXB4 led to the down-regulation of Dusp6 in EBs, a
negative feedback regulator of FGF signaling, which also cross-
talks with other signaling pathways (36, 37). For example,
expansion of Flk1?/SCL?cells depends on VEGF-mediated
activation of Erk1/2 (38), a component of the MAP signaling
pathway, which can be inhibited by Dusp6. Down-regulation of
Dusp6 by HOXB4, thus, may enhance VEGF-mediated expan-
sion of Flk1?/SCL?precursor cells in EBs. Blockage of FGF
signaling in HOXB4?EBs would lead to a further down-
regulation of Dusp6, which, in turn, should boost VEGF-induced
progenitor expansion. Consistent with this interpretation, we
were able to abolish the synergistic HOXB4/SU5402 effect on
progenitor expansion by a complete chemical blockage of
ERK1/2 activation (SI Fig. 12).
Taken together, we demonstrate that modulation of FGF
signaling is an essential feature of HOXB4 activity in the context
showed that Hoxa2 blocks FGF-dependent gene activation dur-
ing mesenchymal patterning of the embryo, thus providing
supporting evidence for a general role of HOX genes in the
modulation of FGF signaling (39).
HOXB4 Modulates Wnt and Notch Signaling. The Wnt/?-catenin and
Notch pathway are potent regulators of HSC function (40, 41).
Continuous expression of stabilized ?-catenin in HSCs in vitro
and differentiation requires a fine-tuned control of Wnt signal-
ing. In adult HSCs/HPCs and EBs, HOXB4 induced the down-
regulation of Hbp1, which acts as a transcriptional repressor of
Wnt target genes and as a suppressor of cell cycle progression
(42). Furthermore, enhanced expression of Nrarp together with
down-regulation of Narf and Nlk indicate that TCF/LEF protein
stability is enhanced (43, 44). Moreover, HOXB4 also increased
in EBs the expression of Pitx2 and Sox17, two proteins that
mediate TCF/LEF-independent ?-catenin target gene expres-
sion (45, 46). The results suggest that HOXB4 influences Wnt
signaling at multiple stages. It may also govern which target
genes become activated in response to Wnt signaling.
The enhanced expression of Notch ligands (Dll1, Dll3,
idea that HOXB4 may stimulate Notch signaling and thereby
would prevent the loss of HSCs caused by accelerated differen-
cells capable of repopulating SCID mice (47).
Based on the presented results, we propose that HOXB4
governs pivotal cell-intrinsic pathways involved in the regulation
of cell cycle, differentiation, and apoptosis. Moreover, it mod-
ulates the response of adult HSC and EB cells to multiple
conserved extrinsic signals provided by the microenvironment.
which results in HSC expansion.
Mice and Transplantation. C57BL/6J (CD45.2) mice were used as
transplant donors and recipients. Details of the BM transplan-
tation procedure are described in SI Text.
Isolation of Lin?Sca1?cells. Mouse BM cells were depleted from
lineage-committed cells (Gr-1, CD11b, CD45R/B220, CD5,
TER-119; lineage depletion kit; Miltenyi Biotec, Bergisch-
Gladbach, Germany) according to the manufacturer’s recom-
mendation. The lineage-depleted cells were selected for Sca-1?
cells (Sca-1 selction kit; Miltenyi Biotech).
Flow Cytometry and Cell Sorting. Flow cytometry procedures and
sorting of LSK subpopulations are described in detail in SI Text.
Retroviral Constructs and Transduction of HSCs/HPCs. The retroviral
vectors, SF91-EGFP2AHAHOXB4-wPRE, expressing constitu-
tively active HOXB4 (HOXconst), and vector SF11-tCD34, ex-
pressing a truncated form of human CD34 (tCD34), have been
described (7, 48). The generation of the retroviral vector,
SF91-EGFP2AHAHOXB4ER-wPRE, allowing coexpression of
EGFP and a TMX-inducible form of HOXB4 (HOXB4ER), and
procedures for transduction of Lin?Sca1?and sorted LSK cells
are described in detail in SI Text.
Ex Vivo Expansion of HOXB4-Transduced HSCs/HPCs. Procedures and
culture conditions of short-term ex vivo expansion of HOXB4-
transduced HSCs/HPCs for microarray studies and in vivo re-
constitution experiments are described in detail in SI Text.
ES Cells and HOXB4 Induction.CultivationoftheHoxb4iEScellline
FGF receptor signaling are described in detail in SI Text. Details
of the cultivation of the CCE ES cell line (6) and the generation
and retroviral transduction of ES-HCs are also provided in SI
Microarray Expression Profiling. RNAs from EBs and adult LSK
cells were processed for use on Affymetrix GeneChips Mouse
Genome 430 2.0 (Affymetrix, Santa Clara, CA). All quality
www.pnas.org?cgi?doi?10.1073?pnas.0703082104Schiedlmeier et al.
parameters for the arrays were confirmed to be in the range Download full-text
recommended by the manufacturer. A more detailed description
of RNA isolation, target synthesis, and hybridization to Af-
fymetrix GeneChips is provided in SI Text.
GeneChip Data Analysis. For experiments with differentiated ES
cells, expression data were analyzed with dChip 1.3 by using a
perfect match-only model (49). Replicate data for the same
sample type were weighted genewise by using inverse squared
standard error as weights. All genes compared were considered
to be differentially expressed if the 90% lower confidence bound
of the fold change between experiment and baseline was ?1.4.
For experiments with adult HSCs/HPCs, log2 expression esti-
mates were calculated by RMAExpress using the RMA algo-
rithm (50). Probe sets are reported as differentially expressed if
at least one set of experiments showed absolute log2 difference
?1 between HOXB4ER?LSK minus TMX treatment and con-
trol cells (HOXB4ER?LSK plus TMX treatment).
Microarray Data. Microarray data are available at the GEO web
side (www.ncbi.nlm.nih.gov/geo) with GEO accession nos.,
GSE9010 (adult HSC/HPCs) and GSE9044 (differentiating ES
Gene Expression Analysis. qRT-PCR analysis of RNA from sorted
LSK cells and semiquantitative RT-PCR analysis of RNA from
EB cells is described in SI Text.
Statistical Analysis. Statistical significance was determined with
the two-tailed, paired Student’s t test with the ?-level set at 0.05.
We thank Michael Kyba (University of Texas Southwestern Medical
Center, Dallas) and George Daley (Children’s Hospital, Boston) for
sharing iHoxB4 cells; Arne Du ¨sedau and Mathias Ballmaier for expert
assistance with cell sorting; the animal facility team of the Hannover
Medical School for support; Thomas Neumann, Zhixiong Li, Axel
Schambach, Ute Modlich, and Martin Hapke (Hannover Medical
School) for technical assistance and animal care; and the Affymetrix
Core Facility of the Instituto Gulbenkian de Ciencia for microarray
hybridization and analysis. This study was supported by Deutsche
Krebshilfe Mildred Scheel Stiftung Grants 106334 and 10-1763-OS5,
Deutsche Forschungsgemeinschaft Grants KL 1311/41 and 1311/2-4, the
European Commission (European Union CONSERT Grant LHSB-CT-
2004-005242), and Excellence Cluster REBIRTH (Deutsche For-
schungsgemeinschaft). Work in M.M.’s laboratory was supported by
Fundac ¸a ˜o para a Cie ˆncia e a Tecnologia Grants POCI/SAU-MMO/
60419/2004 and FEDER/POCI 2010.
1. Akala O, Clark MF (2006) Curr Opin Genet Dev 16:1–6.
2. Antonchuck J, Sauvageau G, Humphries RK (2002) Cell 10:39–45.
3. Antonchuck J, Sauvageau G, Humphries RK (2001) Exp Hematol 29:1125–
4. Kyba M, Perlingeiro RC, Daley GQ (2002) Cell 109:29–37.
5. Rideout WM, Hochedlinger K, Kyba M, Daley GQ, Jaenisch R (2002) Cell
6. Pilat S, Carotta S, Schiedlmeier B, Kamino K, Mairhofer A, Will E, Modlich
U, Steinlein P, Ostertag W, Baum C, et al. (2005) Proc Natl Acad Sci USA
7. Schiedlmeier B, Klump H, Will E, Arman-Kalcek G, Li Z, Wang Z, Rimek A,
Friel J, Baum C, Ostertag W, et al. (2003) Blood 101:1759–1768.
8. Brun AC, Fan X, Bjornssons JM, Humphries RK, Karlssons S (2003) Mol Ther
9. Beslu N, Krosl J, Laurin M, Mayotte N, Humphries RK, Sauvageau G (2004)
10. Chang CP, Brocchieri L, Shen WF, Largman C, Cleary ML (1996) Mol Cell Biol
11. Krosl J, Beslu N, Mayotte N, Humphries RK, Sauvageau G (2003) Immunity
12. Pineault N, Helgason CD, Lawrence HJ, Humphries RK (2002) Exp Hematol
13. Livak KJ, Schmittgen TD (2001) Methods 25:402–408.
14. Perlingeiro RC, Kyba M, Dayley GQ (2001) Development (Cambridge, UK)
15. Choi K, Kennedy M, Kazarov A, Papadimitriou JC, Keller G (1998) Develop-
ment (Cambridge, UK) 125:725–732.
16. Bryder D, Ramsfjell V, Dybedal I, Theilgaard-Monch K, Hogerkorp CM,
Adolfsson J, Borge OJ, Jacobsen SE (2001) J Exp Med 194:941–952.
17. Dybedal I, Bryder D, Fossum A, Rusten LS, Jacobsen SE (2001) Blood
18. Li Z, Fehse B, Schiedlmeier B, Dullmann J, Frank O, Zander AR, Ostertag W,
Baum C (2002) Leukemia 16:1655–1663.
19. Tsang M, Dawid IB (2004) Sci STKE 228:pe17.
20. Nakazawa F, Nagai H, Shin M, Sheng G (2006) Blood 108:3335–3343.
21. deHann G, Weersing E, Dontje B, van Os R, Bystrykh LV, Vellenga E, Miller
G (2003) Dev Cell 4:241–251.
22. Chen T, Rodrigues N, Shen H, Yang Y, Dombkowski D, Sykes M, Scadden DT
(2000) Science 287:1804–1808.
23. Walkley CR, Fero ML, Chien WM, Purton LE, McArthur GA (2005) Nat Cell
24. North TE, Goessling W, Walkley CR, Lengerke C, Kopani KR, Lord AM,
Weber GJ, Bowman TV, Jang IH, Grosser T, et al. (2007) Nature 447:1007–
25. Yamazaki S, Iwama A, Takayanagi SI, Morita Y, Eto K, Ema H, Nakauchi H
(2006) EMBO J 25:3515–3523.
26. Tothova Z, Kollipari R, Huntly BJ, Lee BH, Castrillon DH, Cullen DE,
McDowell EP, Lazo-Kallanian S, Williams IR, Sears C, et al. (2007) Cell
27. Miyamoto K, Araki KY, Naka K, Arai F, Takubo K, Yamazaki S, Matsuoka
S, Miyamoto T, Ito K, Ohmura M, et al. (2007) Cell Stem Cell 1:101–
28. Galan-Caridad JM, Harel S, Arenzana TL, Hou ZE, Doetsch F, Mirny LA,
Reizis B (2007) Cell 129:345–357.
29. Will E, Speidel D, Wang Z, Ghiaur G, Rimek A, Schiedlmeier B, Williams DA,
Baum C, Ostertag W, Klump H (2006) Cell Cycle 5:14–22.
30. Li Q, Ching AK, Chan BC, Chow SK, Lim PL, Ho TC, Ip WK, Wong CK, Lam
CW, Lee KK, et al. (2004) J Biol Chem 279:52106–52116.
31. Xun CQ, Thompson JS, Jennings CD, Brown SA, Widmer MB (1994) Blood
32. Okamoto M, Ohe G, Oshikawa T, Nishikawa H, Furuichi S, Yoshida H, Sato
M (2000) Anticancer Drugs 11:165–173.
33. Blank U, Karlsson G, Moody JL, Utsugisawa T, Magnusson M, Singbrant S,
Larsson J, Karlsson S (2006) Blood 108:4246–4254.
34. Park C, Lavine K, Mishina Y, Deng D-C, Ornitz DM, Choi K (2006)
Development (Cambridge, UK) 133:3473–3484.
35. Faloon P, Arentson E, Kazarov A, Deng D-C, Porcher C, Orkin S, Choi K
(2000) Development (Cambridge, UK) 127:1931–1941.
36. Mason JM, Morrison DJ, Basson MA, Licht JD (2006) Trends Cell Biol
37. Cela C, Llimargas M (2006) Development (Cambridge, UK) 133:3115–3125.
38. Park C, Afrikanova I, Cung YS, Chang WJ, Arentson E, Fong GH, Rosendahl
A, Choi K (2004) Development (Cambridge, UK) 131:2749–2762.
39. Bobola N, Carapuco M, Ohnemus S, Kanzler B, Leibbrandt A, Neubuser A,
Drouin J, Mallo M (2003) Development (Cambridge, UK) 130:3403–3414.
40. Trowbridge JJ, Moon RT, Bhatia M (2006) Nat Immunol 7:1021–1023.
41. Duncan AW, Rattis FM, DiMascio LN, Congdon KL, Pazianos G, Zhao C,
Yoon K, Cook JM, Willert K, Gaiano N, et al. (2005) Nat Immunol
42. Sampson EM, Haque ZK, Ku M-C, Tevosian SG, Albanese C, Pestell RG,
Paulson KW, Yee AS (2001) EMBO J 20:4500–4511.
43. Ishitani T, Matsumoto K, Chitnis AB, Ito M (2005) Nat Cell Biol 7:1106–
44. Yamada M, Ohnishi J, Ohkawara BO, Iemura S, Satoh K, Hyodo-Mirua J,
Kawachi K, Natsume T, Shibuya H (2006) J Biol Chem 281:20749–20760.
45. Sinner D, Rankin S, Lee M, Zorn AM (2004) Development (Cambridge, UK)
46. Kioussi C, Briata P, Baek SH, Rose DW, Hamblet NS, Herman T, Ohgi KA,
Lin C, Gleiberman A, Wang J, et al. (2002) Cell 111:673–685.
47. Delaney C, Varnum-Finney B, Aoyama K, Brashem-Stein C, Bernstein ID
(2005) Blood 106:2693–2699.
48. Fehse B, Richters A, Putimtseva-Scharf K, Klump H, Li Z, Ostertag W, Zander
AR, Baum C (2000) Mol Ther 1:448–456.
49. Li C, Wong WH (2001) Proc Natl Acad Sci USA 98:31–36.
50. Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ, Scherf U,
Speed TP (2003) Biostatistics 2:249–264.
Schiedlmeier et al.
October 23, 2007 ?
vol. 104 ?
no. 43 ?