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Developmental Biology 288 (2005) 113 – 125
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Bmp signaling promotes intermediate mesoderm gene expression in a
dose-dependent, cell-autonomous and translation-dependent manner
Richard G. James, Thomas M. Schultheiss *
Molecular and Vascular Medicine Beth Israel Deaconess Medical Center, Boston, MA 02215, USA
Harvard Medical School, Boston, MA 02215, USA
Received for publication 16 June 2005, revised 8 September 2005, accepted 8 September 2005
Available online 21 October 2005
Abstract
The intermediate mesoderm lies between the somites and the lateral plate and is the source of all kidney tissue in the developing vertebrate
embryo. While bone morphogenetic protein (Bmp) signaling is known to regulate mesodermal cell type determination along the medio-lateral
axis, its role in intermediate mesoderm formation has not been well characterized. The current study finds that low and high levels of Bmp ligand
are both necessary and sufficient to activate intermediate and lateral mesodermal gene expression, respectively, both in vivo and in vitro. Dosedependent activation of intermediate and lateral mesodermal genes by Bmp signaling is cell-autonomous, as demonstrated by electroporation of
the avian embryo with constitutively active Bmp receptors driven by promoters of varying strengths. In explant cultures, Bmp activation of Oddskipped related 1 (Odd-1), the earliest known gene expressed in the intermediate mesoderm, is blocked by cyclohexamide, indicating that the
activation of Odd-1 by Bmp signaling is translation-dependent. The data from this study are integrated with that of other studies to generate a
model for the role of Bmp signaling in trunk mesodermal patterning in which low levels of Bmp activate intermediate mesoderm gene expression
by inhibition of repressors present in medial mesoderm, whereas high levels of Bmp repress both medial and intermediate mesoderm gene
expression and activate lateral plate genes.
D 2005 Elsevier Inc. All rights reserved.
Keywords: Kidney; Chick embryo; Bmp; Embryonic patterning; Mesoderm
Introduction
Secreted growth factors of the bone morphogenic protein
(Bmp) family establish concentration gradients that contribute
to patterning along the embryonic medio-lateral axis (in other
embryos, such as Drosophila and Xenopus, this axis is ‘‘dorsoventral’’). In several contexts in both Drosophila (Holley and
Ferguson, 1997; Podos and Ferguson, 1999; Rusch and Levine,
1996) and vertebrates (Harland and Gerhart, 1997; Hogan,
1996; Niehrs et al., 2000), the level of Bmp signaling along the
medio-lateral axis correlates with cell fate. In vertebrate,
mesoderm high levels of Bmp signal have been found to
promote formation of lateral structures such as blood;
intermediate levels to promote intermediate structures such as
* Corresponding author. Molecular and Vascular Medicine Beth Israel
Deaconess Medical Center, 330 Brookline Avenue, RW-663, Boston, MA
02215, USA. Fax: +1 617 667 2913.
E-mail address: [email protected] (T.M. Schultheiss).
0012-1606/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.ydbio.2005.09.025
kidney; and lower levels are associated with muscle and
notochord development (Dosch et al., 1997; Jones et al., 1996).
Two specific mechanisms have been proposed to explain
dose-dependent activation of target genes by Bmp. One
proposed mechanism is that components of the Bmp pathway
interact with regulatory regions of Bmp-responsive genes in a
concentration-dependent manner. In the case of Drosophila
Decapentaplegic (Dpp, the Drosophila Bmp-2/4 homolog),
enhancer elements can have differential binding affinity for the
Dpp transducer Mothers against dpp (Mad) (Wharton et al.,
2004): high-dose responders have low affinity Mad binding
sites and are transcribed only in response to high concentrations of Mad, whereas low-dose responders have high affinity
Mad binding sites and are transcribed in response to both low
and high concentrations of Mad. Alternatively, in the Drosophila wing and ectoderm, autonomous expression of some
low-dose Bmp responders does not require Mad. Instead, in the
absence of Dpp signal, the protein Brinker represses transcription of these genes (Campbell and Tomlinson, 1999; Jazwinska
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R.G. James, T.M. Schultheiss / Developmental Biology 288 (2005) 113 – 125
et al., 1999a; Minami et al., 1999). Low doses of Dpp facilitate
Smad-dependent silencing of Brinker (Jazwinska et al.,
1999a,b; Pyrowolakis et al., 2004) and indirect activation of
the low-dose responders.
In vertebrates, Bmp responders such as Msx-1/2, Id-1/2/3
and others can be directly activated by Bmp signaling in vitro
(Hollnagel et al., 1999), and Smad binding sites in the Msx-2
enhancer are necessary for its expression (Brugger et al., 2004).
However, it is unclear how differential doses of Bmp signaling
activate tissue-specific genes during vertebrate mesodermal
patterning in vivo. We sought to address this issue by studying
the formation of the avian intermediate mesoderm (IM).
The IM is a strip of tissue, located between the developing
somites and lateral plate, and which is the source of all kidney
tissue in the body (Sainio and Raatikainen-Ahokas, 1999).
Shortly after gastrulation, the transcription factors Odd-1
(previously known as Osr-1) (So and Danielian, 1999) (Fig.
1A), Pax-2 (Dressler et al., 1990) (Fig. 1B) and Lim-1 (Fujii et
al., 1994) (Fig. 1C) are expressed in the developing IM.
Reports in Xenopus and zebrafish have shown that Bmp
signaling is necessary for the expression of Lim-1 and Pax-2
(Kishimoto et al., 1997; Mullins et al., 1996), and the presence
of nuclear localized phosphorylated Smad-1 protein in the IM
demonstrates that it is an area of active Bmp signaling (Faure et
al., 2002).
In this report, we demonstrate that low, but not high doses of
Bmp signal activate transcription of Lim-1, Pax-2 and Odd-1 in
a cell-autonomous, but translation-dependent manner. Combining our findings with those of others, we propose a two-part
model to explain how low-dose Bmp response genes are
activated specifically in the intermediate mesoderm. First, lowdoses of Bmp signal promote transcription of Odd-1, Lim-1
and Pax-2 in the IM by inhibiting a repressive activity that is
present in somitic mesoderm. Second, high doses of Bmp
signal activate additional inhibitors in the lateral plate, which
restrict the expression of Odd-1, Pax-2 and Lim-1 to the
intermediate mesoderm.
Materials and methods
Cloning of chick Odd-1
A portion of the mouse Odd-skipped related 1 gene (mOdd-1) (So and
Danielian, 1999) corresponding to the coding part of the three zinc finger
motifs was used to probe 5 105 plaques from an HH stage 11 – 14 chick
embryo lambda ZapII Phage library (Nieto et al., 1994). Three plaques were
identified, one of which contained a full-length coding sequence for a gene
81% identical to mOdd-1 at the amino acid level. This clone, which is named
chick Odd-1 (cOdd-1), will be described in detail in a separate publication
(R.G.J. and T.M.S., in preparation).
Fig. 1. Exogenous Bmp protein can induce IM gene expression in the paraxial
mesoderm. (A – C) Expression of Odd-1 (A), Pax-2 (B) and Lim-1 (C) in stages
10 – 11 control embryos. (D – F) Effects of a heparin-acrylic bead soaked in
recombinant human Bmp-2 (b) placed in the embryo at stages 5 – 6 and cultured
until stages 10 – 11. A control bead (c) was placed on the opposite side. On the
Bmp-treated side, expressions of Odd-1 (D), Pax-2 (E) and Lim-1 (F) are all
moved towards the midline (arrows, D, E, F, G, K), into the region where
paraxial genes are normally expressed. Note that in some regions expression of
IM genes is also decreased on the treated side. (G – I) Bmp-2 affects the identity
of paraxial mesoderm cells and not their migration. After labeling prospective
paraxial mesoderm with DiI at HH stage 5, when it resided in the primitive
streak (H), a Bmp-2 bead was placed in lateral plate, and embryos were
cultured through stage 10 (I) when they were processed for expression of Odd-1
by in situ hybridization (G). Comparison of treated and control sides at stage 10
reveals that Odd-1 was expressed more medially on the Bmp-treated side (G),
while migration patterns of the prospective paraxial mesoderm were unchanged
(H, I). (J – K) Sections of Bmp-treated embryos. In Bmp-treated embryos,
lateral plate (J, Cytokeratin) and intermediate mesoderm (K, Lim-1) gene
expression moved towards the midline. np, neural plate; s, somite.
Expression plasmids
In situ hybridization
Constitutively active Alk3 and Alk6 (caALK3 and caAlk6) were obtained
from L. Niswander (Zou et al., 1997) and subcloned into pCIG (Faure et al., 2002),
which drives gene expression from a combination of chick beta actin enhancer and
CMV promoter and which contains an Internal Ribosomal Entry Sequence (IRES)
driving nuclear Green Fluorescence Protein. They were also subcloned into
pCS2+, which drives gene expression from a CMV promoter/enhancer.
pCAGGSdsRed was obtained from C. Cepko (Matsuda and Cepko, 2004).
RNA probes were generated for chicken Odd-1 (this manuscript), Pax-2
(Burrill et al., 1997; Herbrand et al., 1998), Lim-1 (Tsuchida et al., 1994),
Paraxis (Barnes et al., 1997), Tbx-6L (Knezevic et al., 1997) and cytokeratin
(Tonegawa et al., 1997) using standard methods, as previously described
(Schultheiss et al., 1995). Whole mount in situ hybridization was performed as
previously described (Schultheiss et al., 1995).
R.G. James, T.M. Schultheiss / Developmental Biology 288 (2005) 113 – 125
Immunohistochemistry
A peptide consisting of the predicted amino acids 125 – 139 of the chicken
ODD-1 protein was synthesized, linked to KLH (Pierce) and injected into
guinea pigs (Covance). Bleeds were tested by immunofluorescence microscopy
for their ability to detect a nuclear signal specifically in cells where Odd-1
message is expressed (in the IM and medial part of the lateral plate). High titer
bleeds were affinity-purified against the immunizing peptide using a Sulfolink
Kit (Pierce).
The following primary antibodies were used: guinea pig – anti-Odd-1
(1:750), mouse – anti-Lim-1/2 (1:10) (Developmental Studies Hybridoma
Bank), rabbit – anti-Pax-2 (1:250) BAbCo, mouse – anti-Pax7 (1:10) DSHB,
rabbit – anti-Scl/Tal (1:50) (Drake et al., 1997), anti-Quail nuclear antigen
QCPN (1:20) DSHB and mouse and rabbit anti-GFP (Molecular Probes).
Affinity-purified secondary antibodies were purchased from Jackson Immunoresearch and used at 1:250. DAPI (Sigma) was included in the penultimate wash
at 1 Ag/ml to label DNA.
Embryo culture and electroporation
Embryos were grown in modified New culture as previously described
(James and Schultheiss, 2003). Heparin acrylamide beads were soaked in 33
ng/Al human recombinant Bmp-2 (R&D) and implanted into embryos as
previously described (Schultheiss et al., 1997). Noggin-expressing fibroblasts
(Smith and Harland, 1992) were cultured as previously described (Schultheiss
et al., 1997), aggregated by brief centrifugation followed by 1 h at 37- in a
humidified incubator, following which pellets of aggregated cells were cut
and placed in the embryo using a sharpened tungsten microneedle.
Electroporation was carried out as previously described (Wilm et al., 2004).
In some embryos, DiI was injected into the primitive streak at the same time
as electroporation, as previously described (James and Schultheiss, 2003). For
some experiments, electroporation was performed in quail embryos, using
similar methods, and electroporated portions of the primitive streak were
transplanted into chicken host embryos, as previously described (James and
Schultheiss, 2003).
Explant culture
Regions of the primitive streak or mesoderm were dissected and cultured in
collagen gels as previously described (James and Schultheiss, 2003). In some
cases, recombinant Bmp-2 or noggin (R&D Systems) was added to the
medium. To block protein synthesis, cyclohexamide (Sigma) was added to the
medium at a concentration of 10 Ag/ml. At this concentration, greater than 95%
of protein synthesis is blocked as measured by 35S-methionine incorporation
into proteins (T.M.S., unpublished data). Explants were processed for in situ
hybridization in the collagen gels, as previously described (James and
Schultheiss, 2003).
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al., 2002). We hypothesize that Bmp signaling promotes IM
gene expression as this tissue differentiates. This paper is an
investigation of that general hypothesis and the specific
mechanism by which Bmp signaling activates dose-responsive
gene expression in the avian IM.
Exogenous Bmp-2 promotes lateral and intermediate
mesoderm fates at the expense of paraxial fates
To gain initial insight into the in vivo role of Bmp signaling
on IM patterning, we investigated the effects of manipulating
Bmp levels during the time period when IM was being
determined. Bmp-2-soaked heparin-coated beads were implanted into the lateral plate of HH4 – 8 embryos, cultured for
15– 20 h and then assayed for IM gene expression. We
observed that the lateral plate was expanded (see Cytokeratin
in Fig. 1J) while somites were reduced in size or absent
(arrows, Figs. 1D –F, G, K). The IM was shifted medially and
was often compressed (Odd-1: Figs. 1A, D; Pax-2: Figs. 1B, E;
and Lim-1: Figs. 1C, F). Note that the medial shift of IM
markers is actually comprised of two effects: repression of IM
genes in the normal IM position accompanied by ectopic
expression of IM genes in a portion of the paraxial region.
The ectopic expression of IM genes in more medial positions
could be explained by two mechanisms: (1) re-patterning of the
trunk resulting in a compressed somite/IM compartment or (2)
death of somite cells and migration of the endogenous IM into a
paraxial position. To differentiate between these two possibilities, the migration of primitive streak cells was mapped in the
presence of Bmp-2 beads (Figs. 1G – I). Lipophilic fluorescent
dye was injected into the primitive streak of stages 4– 8 embryos
after implantation of the beads and was tracked for 15 – 20 h. In
all cases (10/10), the migration pattern of the labeled cells was
normal and consistent with previous fate maps (Garcia-Martinez
and Schoenwolf, 1992; James and Schultheiss, 2003; Psychoyos
and Stern, 1996), while expression of Odd-1 was shifted
medially (Fig. 1G). This result implies that the somite and IM
compartments are being re-patterned and compressed upon
treatment with exogenous Bmp-2.
Results
Exogenous Bmp-2 promotes dose-dependent activation of IM
gene expression in somitic explants
The precursors of the anterior IM migrate through the
primitive streak starting at Hamburger Hamilton stage (HH) 4
(Hamburger and Hamilton, 1951) and reach their final destination lateral to the somites at HH9 (Garcia-Martinez and
Schoenwolf, 1992; James and Schultheiss, 2003; Psychoyos
and Stern, 1996). Previously, we have shown that during this
time period the fates of neither IM nor other trunk mesodermal
tissues are determined and that co-culture of somitic mesoderm
with lateral plate causes activation of Lim-1 and Pax-2 within
the somitic tissue (James and Schultheiss, 2003). Several Bmp
family members are expressed in the lateral plate (Schultheiss
et al., 1997), and phosphorylated Smad1, an indicator of active
Bmp signaling, is detectable in IM precursor cells between
HH5 and HH8 but not in somites or their precursors (Faure et
The previous results suggest that Bmp-2 can re-pattern the
somite region of the embryo and cause it to express IM
markers. This effect could be the result of the action of Bmp
itself or of interaction between Bmp-2 and signals derived from
other tissues in the embryo such as Hensen’s node, notochord
or neural tube. Additionally, the previous experiments did not
examine the dose dependence of the Bmp response. In order to
investigate whether specific levels of Bmp signaling can repattern somite tissue to IM in the absence of embryonic
signaling centers, paraxial mesoderm or its precursors were
dissected from chick embryos and cultured in serum-free
medium containing Bmp-2 at concentrations ranging from
10 7 to 5 10 7 g/ml. Explants were cultured for 15– 20 h and
then processed for in situ hybridization.
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R.G. James, T.M. Schultheiss / Developmental Biology 288 (2005) 113 – 125
In the absence of Bmp-2, explant cultures of HH5 anterior
primitive streak express the somite marker Paraxis (Fig. 2A,
top panel), but not the lateral plate marker Cytokeratin (Fig.
2D, top panel; the small foci of expression seen in these
explants are in the ectoderm included in the explant) or the IM
markers Odd-1 (Fig. 2B, top panel) or Lim-1 (Fig. 2E, top
panel). Paraxis is downregulated (Fig. 2A, middle and bottom
panels) and Cytokeratin is upregulated (Fig. 2D, middle and
bottom panels) in anterior primitive streak cultured with low or
high concentrations of Bmp-2. Intriguingly, Odd-1 (Fig. 2B,
compare middle and bottom panels) and Lim-1 (Fig. 2E,
compare middle and bottom panels) are expressed more
efficiently in anterior primitive streaks cultured with low
concentrations of Bmp-2. These results indicate that the
anterior primitive streak is malleable with respect to its
response to Bmp signaling and that IM genes are preferentially
activated by low concentrations of Bmp. Note that the normal
pattern of Odd-1 expression is broader than that of Lim-1
(compare Figs. 1A, C). Consistent with this, Odd-1 is capable
of being expressed in a broader range of Bmp concentrations,
as evidenced by the expression of Odd-1 in some explants
treated with high doses of Bmp (Fig. 2B, bottom panel).
Similar effects were seen when older HH8 paraxial
mesoderm was treated with Bmp-2 (Figs. 2C, F), indicating
that low doses of Bmp-2 can re-pattern the somite to express
IM markers several stages after the IM is initially specified. By
HH10, somites do not express IM markers in response to Bmp2 treatment (data not shown), indicating that they are
determined with respect to Bmp signals at this time.
Expression of high levels of activated Alk3 and Alk6 inhibits
differentiation of somite and promotes lateral plate fates
The previous experiments demonstrate that Bmp signaling
of low and high levels is sufficient to promote IM and lateral
plate formation, respectively, in somitic tissue. One important
Fig. 2. Dose-dependent activation of intermediate mesoderm genes by Bmp-2 in explant culture. Treatment of anterior primitive streak with recombinant human
Bmp-2 resulted in downregulation of the paraxial marker Paraxis (A) and upregulation of the IM markers Odd-1 (B) and Lim-1 (E) and the lateral plate marker
cytokeratin (D). The IM markers Odd-1 and Lim-1 were more strongly activated at lower doses of Bmp-2 (B, E), while cytokeratin was also activated strongly at
higher doses (D). Stage 8 somite tissue shows the same dose-dependent activation of IM genes in response to Bmp-2 (C, F). Doses of Bmp-2 are given in g/ml.
R.G. James, T.M. Schultheiss / Developmental Biology 288 (2005) 113 – 125
question that has not been addressed in this or earlier research
is whether the Bmp signals that pattern the IM are received
and translated by individual cells. To begin to address this
question, plasmids were constructed that express constitutively active forms of the Bmp-2/4 type I receptors Alk3 and
Alk6 (caAlk3 and caAlk6; also called caBmpRIa and
caBmpRIb) (Zou et al., 1997) under the control of a strong
avian promoter, CAGGS (Niwa et al., 1991, see also
Supplementary Fig. 1). Both plasmids contain an IRESNuclear-GFP cassette so that GFP can be visualized in the
nucleus of every cell that expresses the activated Alk gene.
Chick embryos were transfected by electroporation into the
primitive streak at HH stages 4 – 6 and assayed for gene
expression after culture for 15 –20 h.
Embryos in which most of the trunk cells were transformed
lost somite morphology and contained patches of paraxial cells
which did not express Paraxis (Fig. 3A, section Fig. 3G) or the
presomitic mesoderm marker Tbx-6L (Fig. 3B, section Fig.
3H). Similar to what was observed in the Bmp bead studies, the
lateral plate marker Cytokeratin is upregulated in electroporated embryos (arrows, Fig. 3C). In sections, Cytokeratin
expression can be seen adjacent to the notochord in clumps of
cells that are not associated with the other somite cells (Figs.
3F, I). Similar results were obtained with both caAlk3 and
caAlk6 constructs, while these effects were not seen with
control empty CAGGS electroporations.
117
In order to characterize the effects of high levels of activated
Alk3/6 on trunk gene expression in individual cells, the
electroporation experiments were analyzed by immunofluorescence. Several insights emerged from these studies. First, GFP
cells never co-stained with the somite marker Pax-7, even if they
were isolated within an aggregate of cells that looked
superficially like a somite (‘‘x’’ in Figs. 4A, D, G, J). Second,
GFP-positive cells were rarely, if at all, co-stained with IM
markers. If a GFP-positive cell was isolated in the IM
(arrowhead, Fig. 4D, G, and insets), it did not express Pax-2
(arrowhead, Fig. 4E and inset) or Odd-1 (arrowhead, Fig. 4H
and inset). On occasion, ectopic Pax-2 (star, Fig. 4E) and Odd-1
(star, Fig. 4H)-positive cells were found in or near the somite. In
most of these cells, GFP could not be detected, while in others
GFP expression was barely detectable (see star, Figs. 4D,G).
Either these cells express low levels of activated Alk3 or they are
induced to express Pax-2 and Odd-1 in a non-cell-autonomous
manner (see Discussion). Lastly, GFP cells often formed clumps
adjacent to the notochord (‘‘x’’ in Figs. 4A, D, G, J).
Previous studies have shown that Bmp-4 promotes vasculogenesis in paraxial mesoderm (Nimmagadda et al., 2005) and
that, in the absence of Noggin, putative vascular cells
inappropriately migrate to the midline and form accumulations
(Reese et al., 2004), similar to what we observed in cells
expressing activated Alk3. In order to test whether the GFPpositive cells that were observed near the midline are
Fig. 3. Effects on mesodermal gene expression of constitutively active Alk3 (BmpRIa) expressed under control of a strong promoter. Images of embryos
electroporated with pCAGGS-caAlk3-IRES-GFP (A – C, G – I) or control embryos (D – F) stained by in situ hybridization for Paraxis (A, D, G), Tbx-6L (B, E, H) or
Cytokeratin (C, F, I). In regions where many cells were electroporated, somite morphology is interrupted (arrows in A, B, G, H, I). caAlk3 leads to downregulation of
the paraxial markers Paraxis (A, D, G) and Tbx-6 (B, E, H) and ectopic expression of cytokeratin in paraxial mesoderm (C, F, I). Arrows in panels G – I indicate
affected areas. N, notochord; np, neural plate; s, somite.
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R.G. James, T.M. Schultheiss / Developmental Biology 288 (2005) 113 – 125
Fig. 4. Examination of the effects of high level expression of caAlk3 at single cell resolution. Sections of embryos electroporated with pCAGGS-caAlk3 (A, B, D, E,
G, H, J, K) or control embryos (C, F, I, L). pCAGGS-caAlk3 generates clumps of cells which accumulate adjacent to the notochord (x in A, D, G, J). These clumps
express the blood/vascular marker Scl/Tal (J, K) but do not express Pax-7 (A, B), Pax-2 (D, E) or Odd-1 (G, H). caAlk3-expressing cells (as indicated by GFP
expression) are in general not seen in well-formed somites, although they can be seen in poorly formed paraxial regions (D). GFP-expressing cells in the endogenous
IM typically do not express Odd-1 or Pax-2 (arrows in D, E and G, H, higher magnification shown in inset). A small number of ectopic Pax-2 and Odd-1-expressing
cells are seen (asterisks in E, H), which typically do not express detectable levels of GFP (D, G). im, intermediate mesoderm; lp, lateral plate; n, notochord; np, neural
plate; s, somite. The vertical line marks the border between the somite and the intermediate mesoderm.
vasculogenic, electroporated embryos were stained for the
vascular/hematopoietic marker SCL/TAL (Drake et al., 1997).
Strikingly, more than 50% (counted in 11/11 embryos) of all
GFP-expressing cells located in the somitic or IM regions
expressed SCL/TAL (arrowhead, Figs. 4J, K). All ectopic SCL/
TAL cells observed in the paraxial region also express GFP. In
summary, the most significant effect of expressing high levels
of activated Alk-3/6 constructs was to convert somite and IM
cells into lateral plate and vascular/hematopoietic cells.
Expression of low levels of activated Alk3 in somites promotes
an IM fate cell-autonomously
In order to examine the cell-autonomous effects of low
levels of Bmp signaling, we generated plasmids that express
activated Alk3 or Alk6 driven off the CS2 promoter (CS2caAlk3 or CS2-caAlk6) and transformed them into chicken
embryos. The CS2 promoter expresses activated Alk constructs
at much lower levels than CAGGS in chicken embryos (Zou et
al., 1997, see Supplementary Fig. 1).
Unlike the effect seen with CAGGS-caAlk3/6, electroporation of CS2-caAlk3 or CS2-caAlk6 did not noticeably affect
the morphology of the embryos. Upon sectioning and staining
for Pax-7, most of the GFP-expressing cells in the somite were
Pax-7-positive (Figs. 5A, B). Ectopic Odd-1 expression was
widespread in these embryos in GFP-positive cells (arrowhead
Figs. 5E, F), and it is likely that Odd-1 and Pax-7 are
coexpressed in many somitic cells. Pax-2 expression was
observed in the somites of every embryo that was electroporated
with CS2-caAlk3 (11/11), although Pax-2 was activated in a
much lower percentage of GFP-expressing cells than Odd-1.
Ectopic Pax-2-positive cells were all GFP-positive and occasionally appeared to be migrating dorsally with a morphology
resembling the nephric duct (arrowhead, Figs. 5C, D). Lastly,
SCL/TAL expression was occasionally seen in some extremely
bright GFP somitic cells (arrowhead, Figs. 5G, H), consistent
with it requiring high levels of Bmp signal to be expressed.
These data demonstrate that low levels of Bmp signaling are
sufficient to induce IM gene expression in a cell-autonomous
manner, in at least a subset of paraxial cells.
R.G. James, T.M. Schultheiss / Developmental Biology 288 (2005) 113 – 125
Fig. 5. Effects of low-level expression of caAlk3 on mesodermal gene
expression. Alk3, expressed under the CMV promoter, was electroporated into
chick embryos together with a GFP reporter plasmid. Expression of caAlk3 did
not inhibit paraxial Pax-7 expression (A, B). Many caAlk3-expressing cells in
the somite expressed ectopic Odd-1 (E, F), and some expressed ectopic Pax-2
(C, D arrow). The ectopic Pax-2-positive cells tended to express GFP at higher
levels and were often seen appearing to be separating from the somite dorsally.
Most GFP-expressing cells in the somite did not express Scl/Tal, although some
cells outside of the somite coexpressed GFP and Scl/Tal (G, H, arrow).
Abbreviations as in Fig. 4.
Translation is required for Bmp-dependent activation of Odd-1
The above findings indicate that Bmp signals are capable
of inhibiting somite differentiation and promoting expression
of Odd-1, Lim-1 and Pax-2 (Figs. 1 –5) in paraxial tissue.
This effect is cell-autonomous (Figs. 4, 5) and dosedependent (Figs. 2, 5).
To further examine the response of mesodermal genes to
Bmp, the exact time course of transcription factor response to
Bmp-2 was detailed. Presomitic mesoderm from Hamburger
Hamilton stage 8 embryos was cultured in media alone or in
media supplemented with Bmp-2 and harvested after 1.5, 3 or 5
h. We found that expression of the somite markers Paraxis (Fig.
6A, compare 1st and 2nd panel) and Foxc2 (data not shown)
were not decreased in response to Bmp-2 within 5 h of culture
(contrast with Fig. 2A, bottom panel). In contrast, Tbx-6L and
Odd-1 responded quickly: after 3 h of culture with Bmp-2,
119
Tbx-6 expression decreased (Fig. 6C, compare 1st and 2nd
panels) and Odd-1 transcription was activated (Fig. 6D,
compare 1st and 2nd panel). Interestingly, Odd-1 was also
upregulated in explants treated with high concentrations of
Bmp-2 starting at 1.5 h (Fig. 6B, 1st panel), and expression was
maintained for at least 5 h in culture (Fig. 6B, 2nd panel). In
contrast, Odd-1 expression was not detectable after 15 h of
culture with high concentrations of Bmp-2 (Fig. 2C, bottom
panel). Combined, these results suggest that Bmp-2 signals
activate expression of Odd-1 in somitic tissue, but at high
concentrations they also activate a slower acting repressor of
Odd-1 transcription (see Discussion).
To test whether the modifications of Tbx-6L or Odd-1
transcription due to Bmp-2 were direct responses to activation
of the signaling pathway, we tested if the changes still
occurred in the presence of the translation inhibitor cyclohexamide. In each case, presomitic mesoderm was cultured for 3
h under four conditions: (1) in media alone, (2) in media
containing 5 10 7 g/ml Bmp-2, (3) in media containing
cyclohexamide and (4) in media containing 5 10 7 g/ml
Bmp-2 and cyclohexamide.
First, we found that the repression of Tbx-6L expression
mediated by Bmp-2 was translation-dependent. Although
Bmp-2 alone potently downregulates Tbx-6L (Fig. 6C,
compare 1st and 2nd panels), it did not do so in the presence
of cyclohexamide (Fig. 6C, compare 3rd and 4th panels),
indicating that the Tbx-6L downregulation is likely indirect.
Interestingly, Odd-1 expression was somewhat upregulated in
explants cultured in cyclohexamide alone (Fig. 6D, compare
1st and 3rd panels). This observation suggests that Odd-1
transcription may be repressed by a factor present in the
developing somite that requires active translation. Expression
of Odd-1 in explants treated with both Bmp-2 and cyclohexamide was not appreciably greater than in explants treated with
cyclohexamide alone (Fig. 6D, compare 3rd and 4th panels),
implying that Bmp-mediated activation of Odd-1 expression is
also translation-dependent. As a positive control for the
cyclohexamide experiments, we also assayed explants for
expression of the direct Bmp effectors Msx-1 and Msx-2
(Hollnagel et al., 1999). As expected, the expression of both
was upregulated in the presence of Bmp-2 with or without
cyclohexamide (Msx-2 is shown in Fig. 6E). Thus, transcription of the earliest known gene to be expressed in the IM – Odd1– does not appear to be activated directly by transducers of the
Bmp signaling pathway.
Bmp signals are required during IM specification
The previous set of experiments examined the ability of
Bmp signaling to regulate IM gene expression. In order to
investigate whether Bmp signaling is required for IM differentiation, loss of function experiments were performed in
which explanted tissue was cultured in the presence of the
extracellular Bmp inhibitor Noggin. We first examined whether
Noggin could re-pattern the posterior primitive streak, which is
a zone of high Bmp signaling that normally gives rise to lateral
plate tissue (data not shown). When grown in the highest
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Fig. 6. Does regulation of IM and somite genes by Bmp require protein synthesis? Paraxial tissue from stage 8 embryos was cultured either alone or in the presence
of 10 7 g/ml (A) or 5 10 7 g/ml (B – E) of Bmp-2. (A) Paraxial explants did not downregulate Paraxis after 5 h of exposure to Bmp-2 (compare with Fig. 2A,
where explants were cultured at the same dose for 16 h). (B) Explants upregulated Odd-1 within 1.5 h after being exposed to Bmp-2. (C – E). Paraxial explants were
cultured for 3 h either untreated (top panels), in the presence of Bmp-2 (second panels), in the presence of cyclohexamide (third panels) or in the presence of both
Bmp-2 and cyclohexamide (bottom panels). In the presence of cyclohexamide, Bmp does not upregulate Odd-1 beyond the mild upregulation seen with
cyclohexamide alone (D), indicating that Odd-1 is not an immediate response gene to Bmp signaling. Bmp-induced repression of Tbx-6L expression is blocked by
cyclohexamide (C), indicating that downregulation of Tbx-6L expression is also not an immediate response gene to Bmp signaling. As a positive control, Msx-2 was
activated by Bmp-2 even in the presence of cyclohexamide (E), indicating that it is an immediate response gene to Bmp signaling.
concentration of Noggin, posterior streak explants upregulated
expression of Paraxis (Fig. 7A, 3/3) and lost expression of
Cytokeratin (Fig. 7B, 0/5). Weak expression of Lim-1 was
observed at intermediate (Fig. 7C, middle panel, 3/4), but not at
the highest concentration (Fig. 7C, bottom panel, 0/4) of
Noggin. These results are consistent with a model in which low
levels of Bmp signaling promote IM gene expression.
To test whether Bmp is necessary for IM gene expression
after stage 5, middle primitive streak, which is specified to
express IM factors (Figs. 7D, E, top panel), was explanted and
cultured in Noggin-containing medium. Lim-1 was potently
downregulated by Noggin at 10 6 g/ml (Fig. 7E, bottom panel,
0/6) and mildly downregulated at 10 7 g/ml (Fig. 7E, middle
panel, 3/5) relative to control explants (Fig. 7E, top panel, 6/6).
In contrast, Odd-1 expression in developing middle primitive
streak explants was unaffected by Noggin concentration (Fig.
7D), which implies that Bmp signals are not required for Odd-1
expression after HH5. HH8 prospective IM explanted in media
containing Noggin maintained expression of Lim-1 even at the
highest Noggin concentration (Fig. 7F). These results imply
that Odd-1 expression does not require Bmp signals after HH5,
whereas Lim-1 expression requires Bmp signaling between
HH5 and HH8.
To investigate whether Bmp signaling is necessary in vivo
for IM formation, pelleted rat fibroblast cells stably transfected with Noggin cDNA (Smith et al., 1993) were
implanted into the presumptive lateral plate or IM of HH4 –
7 embryos. These were grown for 15– 20 h and analyzed for
morphological changes and modification of kidney gene
expression. When noggin-secreting cells were implanted prior
to HH6, ectopic somites were often formed adjacent to the
endogenous somitic mesoderm (data not shown), consistent
with previous results (Tonegawa et al., 1997). Two different
results were obtained upon visualization of IM markers. First,
the domain of Odd-1 (Fig. 7G) was expanded into the lateral
plate mesoderm near the graft (star, Fig. 7G) but inhibited in
tissue immediately adjacent to the graft. This result is
consistent with the hypothesis that Odd-1 is activated in
response to low doses of Bmp signaling. In contrast, Pax-2
(Fig. 7H) and Lim-1 (Fig. 7I) gene expression was inhibited
in response to the noggin grafts. The observed inhibition
occurs both near the graft (carat, Figs. 7H, I) and at some
distance away (star, Figs. 7H, I). Neither Pax-2 nor Lim-1
was expressed in secondary locations in the lateral plate as
would be expected if their expression was simply responding
to Bmp levels. Coupled with the fact that Pax-2 is activated at
a much lower rate than Odd-1 by CS2-caAlk3/6 (Fig. 5), this
finding suggests that, within the context of the embryo in
vivo, expression of Pax-2 and Lim-1 may require other
signaling in addition to that from Bmps.
Discussion
Bmp signaling promotes IM gene expression in a
dose-dependent and cell-autonomous manner
Genetic manipulation of Bmp pathway components in
Xenopus, zebrafish and mouse embryos has demonstrated that
R.G. James, T.M. Schultheiss / Developmental Biology 288 (2005) 113 – 125
121
Fig. 7. Effects of Bmp inhibition on mesodermal gene expression. Explants from the indicated region of the primitive streak (A – E) or mesoderm (F) were cultured
for 16 h in the presence of the indicated concentration of the Bmp antagonist noggin (g/ml). Posterior primitive streak activates Paraxis expression and represses
Cytokeratin expression at the highest dose (A, B), while Lim-1 is expressed most strongly at intermediate levels (C). Treatment of mid-streak cultures with noggin
results in repression of Lim-1 expression (E), but Odd-1 expression is not significantly affected (D). By stage 8 (F), Lim-1 expression in the IM can no longer be
repressed by noggin. (G – I) Pellets of rat fibroblasts expressing noggin (circled regions) were implanted in stages 5 – 6 embryos, which were cultured until stages
11 – 12. Control fibroblasts were implanted on the opposite side. Noggin caused a broadening of Odd-1 expression into more lateral regions of the embryo (*) but
also a repression of Odd-1 expression in the area immediately surrounding the cell pellet (G). In contrast, Pax-2 (H) and Lim-1 (I) were repressed by the noggin cells
(* and <).
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R.G. James, T.M. Schultheiss / Developmental Biology 288 (2005) 113 – 125
Bmp signals can act in a dose-responsive manner in the
patterning of vertebrate trunk mesoderm (reviewed in Harland
and Gerhart, 1997; Hogan, 1996; Munoz-Sanjuan and Brivanlou, 2001; Niehrs et al., 2000; Schier, 2001; Sive, 1993). In
most of these studies, Bmp signaling was manipulated at very
early stages in development. Since early alterations in Bmp
signaling could have multiple complex effects on embryonic
development, one could not determine from these experiments
the time during embryogenesis at which Bmp signaling was
actually acting to pattern the mesoderm. In addition, most of
these studies altered Bmp signaling throughout the embryo.
Thus, the question of whether individual cells can modulate
mesodermal gene expression in response to variations in Bmp
signaling strength could not be directly addressed. In the
present study, we have taken advantage of the experimental
strengths of the avian embryo model system to manipulate
Bmp signaling levels in a time-specific manner in tissues and
individual cells, in vivo and in vitro, in order to study the
mechanism of Bmp-dependent gene activation in the trunk
mesoderm.
Timing: Bmp signaling patterns trunk mesoderm during and
after gastrulation
Treatment of prospective somites explanted from HH5 or
HH8 embryos with low and high doses of Bmp promotes IM
and lateral plate fate, respectively (Fig. 2), while at stage 10
somitic mesoderm is determined relative to Bmp-2 levels. In
addition, Lim-1 expression is inhibited by treatment of
prospective IM with the Bmp antagonist noggin at HH stage
5 but not at HH stage 8, while Odd-1 expression was not
inhibited by noggin even at HH stage 5. These results imply
that Bmp patterning of the IM is already underway by midgastrulation (HH5) and continues through early somite stages
(HH8) but is completed by HH10, by which time a battery of
IM transcription factors has already begun to be expressed
(James and Schultheiss, 2003; Mauch et al., 2000; ObaraIshihara et al., 1999).
Cells fated to develop into anterior paraxial, intermediate
and lateral plate mesoderm initially gastrulate through the
primitive streak at stages 4 and 5 (Garcia-Martinez and
Schoenwolf, 1992; James and Schultheiss, 2003; Psychoyos
and Stern, 1996). At these stages, the anterior (300 – 600 Am
posterior to Henson’s node), middle (600 – 900 Am) and
posterior (900 –1200 Am) regions within the primitive streak
are already specified to differentiate as paraxial, intermediate
and lateral plate mesoderm (Figs. 2, 7). The spatio-temporal
localization of active Bmp signaling in the mesoderm has been
mapped in chick embryos by distribution of phosphorylated
Smad-1/5/8 (Faure et al., 2002). At stages 4 and 5, phosphorylated Smad-1/5/8 is localized in the middle and posterior, but
not anterior primitive streak, while at stage 8 phosphorylated
Smad-1/5/8 is detectable within the intermediate and lateral
plate mesoderm and not in presomitic mesoderm. Thus, the
pattern and timing of Bmp signaling in the embryo, as revealed
by phosphorylated Smad-1/5/8 activity, are consistent with the
current finding that low doses of Bmp signaling pattern the IM
between stages 5 and 8.
Bmp signals act cell-autonomously
Expression of constitutively activated Alk-3 or Alk-6 under
the control of weak and strong promoters cell-autonomously
activates expression of IM and lateral plate genes, respectively
(Figs. 3 –5). These results indicate that, during mesodermal
patterning, individual mesodermal cells can respond to local
Bmp levels and activate mesodermal genes in a dose-specific
cell-autonomous manner. Several elegant reports of the
Drosophila wing and ectoderm have also identified genes that
respond cell-autonomously to low or high doses of Bmp
signals (Lecuit et al., 1996; Nellen et al., 1996).
While the vast majority of cells expressing ectopic IM
markers in response to intracellular expression of CaAlk3/6
also expressed GFP (see Figs. 3– 5), there were a few cells in
which this was not clear. To examine more rigorously the
possibility of non-cell-autonomous activation of IM target
genes by Bmp, we transplanted quail primitive streak cells that
were electroporated with activated Alk3 into chicken anterior
primitive streak (Supplementary Fig. 2). Pax-2 and Odd-1 were
occasionally activated ectopically in isolated chicken cells that
were immediately adjacent to quail grafts. These isolated cells
represent a small minority of the ectopic Pax-2 and Odd-1 gene
expression as most of these cells co-stained with the quail
epitope. However, because they exist, we cannot rule out the
possibility that some indirect activation of Pax-2 and Odd-1 is
caused by other signals activated by Bmp. This result is only
observed in cells adjacent to those electroporated with activated
Alk3 under the control of the strong CAGGS promoter.
Additionally, non-quail ectopic Pax-2-positive cells were
observed only within one cell diameter of a quail cell,
indicating that any non-cell-autonomous effect is very short
range. In the zebrafish neural tube, misexpression of activated
Alk3 was shown to activate epidermal gene expression strictly
in cells that expressed a co-transfected tracer dye (Nikaido et
al., 1999). Taken together, our data suggest that signals from
ectopic Bmp receptors activate transcription of mesodermal
target genes in a dose-dependent and largely cell-autonomous
manner.
The role of Bmp signaling in a mesodermal transcription factor
network
The findings in this report suggest that Bmp signals activate
transcription of intermediate mesodermal markers in a dosedependent, cell-autonomous and translation-dependent manner.
We have combined these findings with those of previous
studies to generate a model (Fig. 8) in which Bmp signals
regulate IM gene expression through a three-fold mechanism:
(1) cell-autonomous factors expressed in the paraxial mesoderm negatively regulate IM gene expression; (2) Bmp
signaling (at high or low levels) inhibits paraxial gene
expression, thereby de-repressing IM gene expression; and
(3) high levels of Bmp signaling repress IM gene expression,
thereby preventing IM genes from being expressed in the
lateral plate. Through this mechanism, IM gene expression is
restricted to a strip of tissue between the somites and the lateral
plate. The components of the model are discussed below.
R.G. James, T.M. Schultheiss / Developmental Biology 288 (2005) 113 – 125
123
and expanded lateral plate mesoderm. The data presented in
this study indicate that activation of Bmp signaling can inhibit
somite differentiation in a cell-autonomous manner (Figs. 4, 5).
The repression of Tbx-6 (Fig. 6C) was dependent on protein
translation, suggesting that Bmp-mediated repression of Tbx-6
is indirect. The immediate Bmp responder Msx-1 has been
shown to physically associate with Histone H1b, specifically
repress transcription of MyoD and prevent muscle differentiation (Lee et al., 2004). One possibility is that Msx-1, or other
transcription factors that are directly activated by Bmp
signaling, represses transcription of somitic genes such as
Tbx-6, Paraxis and Foxc1/2.
Fig. 8. Model of the regulation of trunk mesodermal gene expression by Bmp
signaling. In paraxial (somite) regions, Bmp signaling is very low to absent.
Paraxial transcription factors, such as Foxc1/2, activate somite genes and
repress IM gene expression. In the IM, low levels of Bmp signaling repress
somite gene expression and as a result de-repress IM gene expression, leading
to activation of IM genes. In the lateral plate, high levels of Bmp signaling
activate genes such as cytokeratin and repress IM genes. See text for details.
IM gene expression is under negative regulation by
cell-autonomous factors expressed in the paraxial mesoderm
The findings in Fig. 6D (compare 1st and 3rd panels)
demonstrate that Odd-1 transcription can be activated in
presomitic mesoderm upon treatment with cyclohexamide
alone. This suggests that some molecule(s) present in the somite
may be necessary to actively inhibit Odd-1 transcription. In the
absence of translation of this/these molecule(s), all signals
necessary for Odd-1 transcription are present in somitic
mesectoderm. Previous studies have shown that the forkhead
transcription factors Foxc1 and Foxc2 are necessary and
sufficient to inhibit the transcription of IM factors (Wilm et al.,
2004). Mice and zebrafish lacking Foxc1 and Foxc2 do not
develop somites (Topczewska et al., 2001; Wilm et al., 2004).
Instead, expression of Odd-1 and Lim-1 is expanded medially so
that they are present in paraxial and intermediate mesoderm
(Wilm et al., 2004). Additionally, when Foxc2 or Foxc1 is
transiently transfected into the IM of chick embryos, expression
of Pax-2 and Lim-1 is repressed in the treated cells (Wilm et al.,
2004). It is unclear if Foxc1/2 directly repress transcription of IM
genes or if they act indirectly via other molecules.
Bmp signaling represses paraxial gene expression
Previous studies have found that Bmp pathway activation
inhibits formation of somitic mesoderm and promotes more
lateral fates (reviewed in Dale and Jones, 1999; Harland and
Gerhart, 1997; Hogan, 1996; Schier, 2001). Exposure of chick
presomitic mesoderm to high levels of ectopic Bmp ligand
either in culture (Fig. 2; Reshef et al., 1998) or in vivo (Fig. 1;
Pourquie et al., 1996; Tonegawa et al., 1997) leads to inhibition
of somite differentiation and activation of lateral plate fate.
Consistent with this, loss of function of the Bmp antagonists
Noggin (McMahon et al., 1998) and Chordin (Dick et al.,
2000; Wagner and Mullins, 2002) results in reduced paraxial
An activity within the lateral plate inhibits IM differentiation
Several reports indicate that there is an activity within the
lateral plate that can inhibit IM differentiation. If specified IM
(HH8 or younger) is transplanted into the lateral plate
mesoderm (James and Schultheiss, 2003) or cultured in vitro
in the presence of lateral plate (Mauch et al., 2000), it will not
express Lim-1 or Pax-2 as it would if cultured alone (Fig. 7,
James and Schultheiss, 2003). Additionally, experimental
expansion of the neural plate leads to expansion of somitic
mesoderm into the lateral plate (Mariani et al., 2001). Rather
than causing lateral displacement of the IM, this manipulation
results in an absence of IM gene expression, indicating that the
lateral plate environment is not permissive for IM differentiation. In the current study, culture of presomitic mesoderm
treated with high concentrations of Bmp-2 for 15 h results in
upregulation of the lateral plate marker Cytokeratin and absence
of Odd-1, Lim-1 and Pax-2 (Fig. 2). Conversely, if the same
tissue is cultured for only 5 h, Odd-1 transcription is
upregulated (Fig. 6). These results indicate that Odd-1 is
initially activated by the presence of Bmp but subsequently
inhibited. This is likely due to the indirect activation of an
inhibitor that responds to high concentrations of Bmp signaling.
An alternative to the model presented here is that low doses
of Bmp signaling activate transcriptional activators present in
the intermediate mesoderm. These activators could in turn
promote transcription of Odd-1, Pax-2 and Lim-1. One or all of
these could then actively repress expression of somite
transcription factors such as Foxc genes. However, mice
mutant for both Foxc1 and Foxc2 exhibit medial expansion
of IM expression in the absence of changes in the distribution
of phosphorylated Smad-1 (Wilm et al., 2004). Thus, IM gene
expression can be activated by the inhibition of paraxial gene
expression, in the absence of alterations in Bmp signaling.
These data are consistent with a model in which Bmp signaling
activates IM genes by negatively regulating inhibitors of IM
gene expression such as Foxc1 and Foxc2 (as proposed in Fig.
8), rather than by inducing activators of intermediate mesodermal gene transcription.
Acknowledgments
The authors would like to thank the following for providing
probes: P. Danielian (mouse Odd-1), R. Maas and E.
Matsunaga (chick Pax-2), T. Jessell (chick Lim-1), R. Tuan
124
R.G. James, T.M. Schultheiss / Developmental Biology 288 (2005) 113 – 125
(chick Paraxis), S. Mackem (chick Tbx-6L) and Y. Takahashi
(chick cytokeratin). L. Niswander and M. Whitman kindly
provided Alk3 and Alk6 expression constructs. S. Brandt
generously provided the anti-chicken Scl/Tal antibody. The
authors would especially like to thank Mozhgan Afrakhte and
Devin Powell for helping with the cloning of the chick Odd-1
cDNA and to all members of the Schultheiss laboratory for
very helpful discussion and comments. R.G.J. was supported
by a predoctoral grant from the Howard Hughes Medical
Institute. This work was supported by grant R01-DK59980 to
T.M.S.
Appendix A. Supplementary data
Supplementary data associated with this article can be found
in the online version at doi:10.1016/j.ydbio.2005.09.025.
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