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First published online 23 October 2008
doi: 10.1242/dev.029264
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Department of Biology, Emory University, Atlanta, GA 30322, USA.
* Author for correspondence (e-mail: afritz{at}emory.edu)
Accepted 28 September 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Bmp4, Chordamesoderm, Notochord, Zebrafish
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Schier and Talbot, 2005;
Yamamoto and Oelgeschlager,
2004). SMAD1/5/8 activity on the presumptive ventral side of the
blastula activates the expression of ventral genes, whereas vent1,
ved and vox repress the expression of genes that confer dorsal
identity such as the BMP antagonist chordin
(Imai et al., 2001;
Kawahara et al., 2000;
Melby et al., 2000).
Establishment of the DV axis is followed by a second phase of BMP signaling
during mid to late gastrulation, when the interaction of BMP antagonists and
agonists is thought to establish a BMP activity gradient
(Kimelman, 2006;
Schier and Talbot, 2005).
According to this model, high, intermediate and low levels of BMP activity in
the mesoderm specify ventral, intermediate and dorsal fate, respectively.
Low levels of BMP activity direct cells surrounding the dorsal organizer to
become axial mesoderm, whereas their positions within this tissue influence
their exposure to other activity gradients
(Schier and Talbot, 2005;
Stemple, 2005). For example,
Nodal activity is crucial for the process of further specification of axial
mesoderm. High levels of Nodal, received by cells deep in the organizer,
specify cells to become prechordal plate
(Gritsman et al., 2000;
Saude et al., 2000). Low
levels of Nodal activity found in the superficial organizer specify cells to
become chordamesoderm, the antecedent of the notochord
(Gritsman et al., 2000;
Saude et al., 2000).
The molecular events surrounding the transition of chordamesoderm into
mature notochord are not fully understood, although there are several defining
features of the process (Stemple,
2005). Differentiated notochord cells acquire large vacuoles that
allow the tissue to provide structural support to the embryo. Coupled with
this, genes expressed in chordamesoderm are extinguished as the tissue
matures. These include the sonic hedgehog and indian
hedgehog homologs, shha and ihhb, respectively, as well
as the extracellular matrix (ECM) gene collagen 2a (col2a1a)
(Currie and Ingham, 1996;
Krauss et al., 1993;
Yan et al., 1995). Mutagenesis
screens and studies examining ECM members have begun to elucidate the
chordamesodermal transition into mature notochord. Embryos lacking Laminin 1
subunits (bashful, grumpy and sleepy)
(Parsons et al., 2002),
Laminin 
4/5 (Pollard et al.,
2006) and Collagen15a1
(Pagnon-Minot et al., 2008)
continue to express chordamesodermal markers after expression has been
extinguished in wild-type embryos, suggesting that notochord differentiation
is impaired. However, given their nature, these proteins are unlikely to play
instructive roles in notochord differentiation. The signal responsible for
promoting notochord development has yet to be defined.
Although it is clear that initial specification of chordamesoderm is
reliant upon the absence of BMP from the dorsal organizer, subsequent
proliferation and differentiation of this tissue may depend upon it. Posterior
to the notochord lies the chordoneural hinge (CNH), a stem cell pool that
contributes to notochord, floor plate and tailbud mesoderm
(Cambray and Wilson, 2002;
Cambray and Wilson, 2007;
Charrier et al., 1999;
Davis and Kirschner, 2000;
Kanki and Ho, 1997). Several
agonists of BMP activity, including the BMP ligand ADMP, are expressed in
axial territories during gastrulation and in the CNH during segmentation
stages (Dickmeis et al., 2001;
Lele et al., 2001). Thus, BMP
signaling may play an important role in the development of axial mesoderm
after specification of the DV axis. However, the role of BMPs in DV
establishment masks their later functions. For example, manipulation of ADMP
levels within the embryo causes DV patterning phenotypes that are difficult to
resolve from tissue-specific defects (Lele
et al., 2001; Willot et al.,
2002).
In contrast to the known role of BMP activity in DV establishment, prior
studies have not addressed the role of the BMP activity gradient in relation
to axial mesoderm. In this study, we have identified both a requirement for
BMP activity in the development of this tissue, as well as the signaling
molecule responsible for notochord differentiation. We have been able to
address these issues through the inactivation of two BMP antagonists,
fstl1 and fstl2 (Dal-Pra
et al., 2006), that act redundantly to inhibit BMP activity
beginning at late gastrulation. Inactivation of these genes, as well as of
Bmp4 and BMP signaling, reveals that Bmp4 promotes the proliferative capacity
of notochord and CNH cells. In the absence of Bmp4, chordamesoderm fails to
proliferate and the notochord differentiates prematurely. Our results
illustrate the requirement of fstl1 and fstl2 in late
gastrulation to maintain proper BMP activity levels, which are necessary for
the development and timely differentiation of dorsal structures.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). tBR embryos were heat-shocked at 37°C for 1
hour at the time indicated according to Pyati et al.
(Pyati et al., 2005). Where
appropriate, wild-type embryos were heat-shocked under the same conditions to
serve as controls.
In situ hybridization
The following probes were used: admp
(Lele et al., 2001),
bmp4 (Nikaido et al.,
1997), col2a1a (Yan
et al., 1995), eve1
(Joly et al., 1993),
flh (Talbot et al.,
1995), fstl1 (Dal-Pra
et al., 2006), fstl2
(Dal-Pra et al., 2006),
gsc (Schulte-Merker et al.,
1994), ihhb (Currie
and Ingham, 1996), ntl
(Schulte-Merker et al., 1992),
shha (Krauss et al.,
1993) and spt
(Griffin et al., 1998).
mRNA synthesis
Full-length fstl1/2 cDNA was cloned into pCS2+. Capped mRNAs were
transcribed using RNA polymerase in vitro transcription kits (mMESSAGE
mMACHINE; Ambion). Approximately 100 pg of fstl1 or fstl2
mRNA was injected into one- and two-cell stage embryos.
Morpholino injection
Translation-blocking morpholinos are overlap with 22 nucleotides at the
5' end of the previously published MO sequence
(Dal-Pra et al., 2006).
fstl1 and fstl2 translation-blocking MOs were as follows:
fstl1, 5'-GCAGCTGCATGGACAGAGTGAAAAC-3'; fstl2,
5'-CACGGGTAAACACCGAAACATCATT-3'. fstl splice-blocking
morpholinos were as follows: fstl1,
5'-CAGACTTACCTTCACATTGTCCGTC-3'; fstl2,
5'-AAATTAAAGCTCACCATCACAAGTC-3'. To ensure MO function, RNA was
isolated from injected embryos and RT-PCR was performed using primers designed
around intron-exon boundaries. Those sequences are as follows: fstl1,
5'-TAATCATCCAGTCTGTGGCAGTAAT-3' and
5'-CTTGGGCTGTTGATGAT-3'; fstl2,
5'-GGTCTGCACCGCCATGACTTGT-3' and
5'-ACACGGCGGGTCCACTCCTC-3'. For single morpholino injections, 8 ng
of MO were injected into one- and two-cell stage embryos. For double
morpholino injections, 
6 ng of each MO was injected. The control
morpholino sequence used was 5'-CCTCTTACCTCAGTTACAATTTATA-3'.
Where appropriate, control MO was injected into embryos that were subsequently
heat-shocked at 37°C for 1 hour. The bmp4 splice-blocking
morpholino used has been previously characterized
(Chocron et al., 2007).
Antibody staining
Labeling with PSMAD1/5/8 was as previously described
(Rentzsch et al., 2006).
Labeling with Myf5 and F59 were as previously described
(Hammond et al., 2007;
Topczewska et al., 2001). The
primary antibodies were against Myf5 (anti-Myf5 recognizes Myod1 in zebrafish;
Santa Cruz, C-20) at 1:50, F59 (anti-MyHC; University of Iowa Developmental
Studies Hybridoma Bank) at 1:10, 4D9 (anti-Engrailed; University of Iowa
Developmental Studies Hybridoma Bank) at 1:20, Prox1 (Angiobio) at 1:500 and
P-SMAD1/5/8 (Chemicon International) at 1:100. Alexa-conjugated Phalloidin
(Molecular Probes) was used at a 1:50 dilution to label F-actin. Appropriate
Alexa Fluor (Molecular Probes) secondary antibodies were used.
Western blots
Protein extracts were prepared using standard procedures
(Westerfield, 1994). Anti-P
SMAD1/5/8 and Anti-P SMAD2 (Cell Signaling Technology) were used at a
concentration of 1:1000.
BrdU labeling
Twelve-somite embryos were incubated with a solution of 425 µl BrdU
labeling reagent (Roche Applied Science) and 75 µl DMSO for 45 minutes at
6°C. They were then washed in embryo medium for 30 minutes at 28.5°C
and fixed with 4% PFA in PBS. BrdU incorporated cells were detected using
Alexa Fluor 594 goat anti-mouse IgG at 1:500.
Cell transplantation and margin extirpation
Surgical transplantation and extirpation experiments were performed as
described (Saude et al.,
2000). For ventral margin extirpation, 100 cells were removed
by suction from the ventral margin of 80% epiboly embryos. For cell
transplantation, donor embryos were injected with a 5% solution of fluorescein
dextran (10K MW; Molecular Probes) at one- and two-cell stage embryos.
Approximately 30 ventral margin cells taken from shield-stage donors were
transplanted into the dorsal organizer of an equivalently staged host.
tBR embryos were obtained from an incross of homozygous tBR
parents. Both donor and host embryo were then heat-shocked after
transplantation to maximize BMP signaling inhibition according to previous
transplantation protocols (Pyati et al.,
2005).
Cell cycle inhibition
Published protocols were followed with slight modifications
(Stern et al., 2005). Embryos
were treated with a combination of 150 µM aphidicolin and 20 mM hydroxyurea
in 4% DMSO between 80% epiboly and bud stage. Cell proliferation was assayed
through BrdU incorporation at stages during and after application.
Bead implantation
Human Bmp2/7 and zebrafish Bmp4 protein-coated beads (R&D Systems) were
prepared by overnight incubation of 45 µm polystyrene beads (Polysciences)
in a 500 µg/ml solution of recombinant Bmp2/7 or Bmp4 protein (R&D
Systems) in PBS. Before implantation, beads were rinsed three times for 10
minutes in PBS. Control beads were loaded with 500 µg/ml BSA.
Cell counting
DAPI-positive cells were quantified and compared by one-way ANOVA, followed
by a two-tailed, equal variance t-test.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). These
genes are structurally and functionally related to Follistatin (Fst), a known
inhibitor of Bmp4 and ADMP (Dal-Pra et
al., 2006; Dosch and Niehrs,
2000; Fainsod et al.,
1997; Iemura et al.,
1998). Although fstl2 mRNA can be detected in
cleavage-stage embryos, fstl1 expression is initiated during late
gastrulation in dorsal structures. Unlike fst, which is expressed
only in anterior paraxial mesoderm (Bauer
et al., 1998), fstl1/2 share largely overlapping
expression patterns throughout axial and paraxial mesoderm during late
gastrulation and segmentation stages (see Fig. S1 in the supplementary
material) (see also Dal-Pra et al.,
2006). Fstl2 plays a minor role in BMP antagonism during DV axis
specification, as depletion of Fstl2 in a Chordin/Noggin1-deficient embryo
slightly increases the severity of the ventralization phenotype observed in
Chordin/Noggin1 double morphants. Loss of Fstl2 in either Chordin or Noggin1
single morphant embryos does not enhance the ventralization phenotype
(Dal-Pra et al., 2006). As
shown by these authors, we observed no DV patterning defects when either Fstl
protein was reduced by itself or in combination with each other. The most
pronounced morphological defect observed in Fstl1/2 morphants was an enlarged
undulating notochord that became less prominent as somitogenesis continued
(e.g. see Fig. S2 in the supplementary material; Figs
3 and
6). In addition, we found an
increase in the levels of molecular markers that indicated an increase in BMP
activity (Figs 1 and
2).
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;
Szeto and Kimelman, 2006), was
increased in Fstl morphant embryos and enlarged to a greater degree when Fstl1
and Fstl2 were knocked down in concert. The expression domain of
spadetail (spt), a T-box domain transcription factor
required for trunk segmental identity
(Griffin et al., 1998), was
increased in Fstl1/2 morphant embryos (Fig.
1A,B; see Fig. S2 in the supplementary material). The domain of
expression of BMP agonists, which are positively regulated in response to BMP
activity, was expanded in the tailbud in a similar fashion
(Yamamoto and Oelgeschlager,
2004). This included the expression domains of bmp4 and
the transcriptional target of BMP activity, eve1
(Fig. 1E,F,M,N; see Fig. S2 in
the supplementary material) (Pyati et al.,
2005), as well as admp in the posterior notochord
(Fig. 1Q,R). We also observed
an increase in phosphorylated (P) SMAD 1/5/8 in the tailbud, which is
activated in response to BMP signaling
(Fig. 1I,J)
(Yamamoto and Oelgeschlager,
2004). Western blots against PSMAD1/5/8 indicated that BMP
activity is increased in Fstl1/2 morphants beginning at late gastrulation.
Prior to 80% epiboly, we observed no change in PSMAD1/5/8 levels (not shown).
At the end of gastrulation, we observed an increase in BMP activity that was
present at every stage assayed, through 18 somites
(Fig. 1U). Follistatin also
binds Activin ligands with high affinity, which initiates intracellular Nodal
cascades (Harrington et al.,
2006; Nakamura et al.,
1991). We observed no change in PSMAD2 levels, a target of Nodal
signaling (reviewed by Kitisin et al.,
2007), in Fstl1/2 morphants at any stage assayed
(Fig. 1U). To assess morpholino specificity, we compared the phenotypes generated using non-overlapping translation- and splice-blocking morpholino sequences. The tailbud and notochord phenotypes observed using translation-blocking morpholinos were indistinguishable from those observed using splice-blocking morpholinos (see Fig. S2 in the supplementary material). In general, morphant phenotypes were reliable and were observed in greater than 90% of embryos (n>500). Furthermore, we attempted to rescue the tailbud phenotype observed in Fstl1/2 morphants. Injection of either fstl1 or fstl2 mRNA caused a reduction in the tailbud domain, consistent with a decrease in BMP activity and a role for Fstl1 and Fstl2 as BMP antagonists (Fig. 1C,G,K,O,S) (also data not shown). Co-injection of either fstl1 or fstl2 mRNA and fstl1/2 morpholinos was sufficient to rescue the Fstl1/2 double morphant phenotype, demonstrating the specificity of our morpholinos and a functional redundancy between Fstl1 and Fstl2 (Fig. 1D,H,L,P,T) (also data not shown). As all of the morphant phenotypes were comparable with each other, the Fstl1/2 morphant phenotypes illustrated here are of embryos injected with fstl1/2 splice-blocking morpholinos.
Bmp4 activity during late gastrulation is required for chordamesoderm patterning
The expression of fstl1/2 along the dorsal midline and the
notochord defects observed in fstl1/2 morphants suggest a function in
axial development. We observed no changes in the expression of axial markers
in Fstl1/2 morphants until late gastrulation, consistent with the temporal
rise in PSMAD1/5/8 levels. At 70% epiboly, goosecoid (gsc)
expression in prechordal mesoderm
(Schulte-Merker et al., 1994)
and floating head (flh), no tail (ntl) and
admp expression in chordamesoderm
(Dickmeis et al., 2001;
Schulte-Merker et al., 1992;
Talbot et al., 1995) of
Fstl1/2 morphants were comparable with controls
(Fig. 2A,B,H,I,O,P,V,W). At 90%
epiboly, the domain of prechordal mesoderm was unchanged in Fstl1/2 morphant
embryos (Fig. 2C,D), but the
domain of chordamesoderm was expanded anteriorly
(Fig. 2J,K,Q,R,X,Y).
As changes in chordamesodermal gene expression in Fstl1/2 morphants
appeared to be a result of increased BMP activity, we wished to determine the
effects of lowering BMP activity on this tissue. We were able to conditionally
inactivate BMP activity through the use of a transgenic zebrafish line
carrying a truncated BMP-type I receptor fused to GFP under the control of a
heat-shock promoter (abbreviated tBR)
(Pyati et al., 2005). Heat
shocking these embryos for 1 hour at 37°C blocks BMP effector expression
for at least 2 hours and is capable of inducing severe dorsalization
phenotypes depending on the timing of heat shock
(Pyati et al., 2005). To
determine the temporal effectiveness of this transgene, tBR embryos
were heat-shocked during late gastrulation in hourly intervals and stained
with PSMAD1/5/8 antibodies (see Fig. S3 in the supplementary material).
Between 1 and 3 hours post heatshock, PSMAD1/5/8-positive cells were not
detectable in tBR embryos, suggesting that heatshock attenuates BMP
activity over this time range during early somitogenesis.
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A crucial window of Bmp4 activity establishes the proliferative state and size of the CNH and notochord
Using riboprobes against shha and ntl, we examined the
effect of manipulating BMP activity on the size of the notochord and CNH. The
size of the CNH in Fstl1/2 morphant embryos was expanded when compared with
controls (Fig. 3A,B,K,L).
Sections through the trunk between somites 5-10 revealed that the diameter of
the notochord was also increased in these embryos; the mean number of cells
populating the notochord was increased by 63% when compared with controls
(Fig. 3F,G) (4.1 cells versus
6.5 cells, n=10; P<0.001).
In contrast to the expansion of the notochord and CNH observed in Fstl1/2
morphant embryos, attenuation of Bmp4 activity caused a reduction in both the
diameter of the notochord as well as the size of the CNH. The axial phenotype
observed when Bmp4 was knocked down largely resembled that of tBR
embryos that were heat-shocked at 80% epiboly
(Fig. 3C,E,M,O), with the mean
number of cells populating the notochord decreased by 41%
(Fig. 3H,J) (4.1 cells versus
2.4 cells and 2.9 cells, n=10; P<0.001). The difference
in notochord cells of tBR embryos and Bmp4 morphants was not
significant (P>0.05). In both tBR embryos heat-shocked at
80% epiboly and Bmp4 morphants, we observed the formation of ectopic tail
structures. These observations are consistent with roles assigned to BMP
activity in patterning ventroposterior tissues during late gastrulation
(Pyati et al., 2005;
Stickney et al., 2007).
To determine the temporal requirements of axial cells for BMP activity, we heat-shocked tBR embryos at hourly intervals beginning at 80% gastrulation, corresponding to the time at which increased PSMAD1/5/8 levels were observed in Fstl1/2 morphants. The size of the notochord and CNH in embryos heat-shocked at bud stage was comparable to those of embryos heat-shocked at 80% epiboly (not shown). Embryos heat-shocked at the three-somite stage yielded a notochord diameter and CNH domain that were indistinguishable from heat-shocked controls (Fig. 3D,I,N) (4.1 cells versus 4.3 cells, n=10; P>0.05).
We wished to address the underlying cause of the increase in
chordamesoderm. Because chordamesoderm transfates to paraxial mesoderm in
flh mutants (Halpern et al.,
1995), we reasoned that an expanse in chordamesoderm may occur at
the expense of paraxial mesoderm. However, the analysis of deltaC
(dlC), myod1 and paraxial protocadherin
(papc) expression revealed no changes in paraxial territory of
Fstl1/2 morphants or in tBR embryos heat-shocked at 80% epiboly (see
Fig. S7 in the supplementary material) (also data not shown).
|
). Nevertheless, reduction of Bmp4 levels was sufficient to
inhibit axial proliferation; cell proliferation in Bmp4 morphants resembled
that of tBR embryos heat-shocked at 80% epiboly. This suggests that
BMP activity, particularly Bmp4, acts prior to the 3-somite stage to promote
chordamesodermal proliferation.
Ventral margin as the source of axially required BMP ligands
The ventral margin acts as the major secreting center of BMPs during
gastrulation (Agathon et al.,
2003; Schier and Talbot,
2005; Yamamoto and
Oelgeschlager, 2004) and is in close proximity to dorsal cells at
the end of gastrulation. However, because BMP signaling initiates
positive-feedback mechanisms in target cells
(Yamamoto and Oelgeschlager,
2004), an alternate source of BMP ligands might initiate from
PSMAD1/5/8-positive chordamesoderm. To demonstrate that BMP signals originate
from the ventral margin, we removed 100 cells from the ventral side of
the embryo at 80% epiboly and examined the effects on chordamesoderm. Embryos
lacking most of their ventral margin resembled Bmp4 morphants; the sizes of
ventral fin and ventral somitic mesoderm were reduced (see Fig. S4A in the
supplementary material). Both the domain of chordamesoderm as well as cell
proliferation in this tissue was reduced, resembling that of Bmp4 morphants
and tBR embryos heat-shocked at 80% epiboly (see Fig. S4B-E'' in
the supplementary material). This suggests that chordamesoderm possesses a
requirement for ventrally derived BMP ligands during late gastrulation.
BMP activity levels influence the temporal state of the notochord
In addition to notochord size, we wished to determine whether alteration of
BMP activity affects the timing of notochord differentiation. In both
tBR embryos heat-shocked at 80% epiboly and Bmp4 morphants, the
expression of markers indicative of chordamesoderm was shifted such that they
resembled expression patterns seen in more mature notochord. At the 9-somite
stage, expression of ihhb, which can be detected throughout the
entire notochord in control embryos, was maintained only in the caudal
notochord of embryos lacking Bmp4 (Fig.
5A-C). Likewise, shha expression was lost from the
notochord but maintained in floor plate and the diencephalon in 24 hpf
tBR embryos, Bmp4 morphants and embryos lacking ventral margin
(Fig. 5E-G,I-K; see Fig. S3G I
the supplementary material). These results were confirmed by loss of
ptc1 expression from the trunk and tail, where the expression pattern
of 24 hpf embryos lacking Bmp4 resembled 36 hpf controls
(Fig. 5M-O,DD; see Fig. S4H in
the supplementary material). col2a1a expression was absent from the
floor plate of 36 hpf tBR embryos heat-shocked at 80% epiboly and
Bmp4 morphants, and more closely resembled the expression observed in 48 hpf
controls (Fig. 5Q-S,GG).
Conversely, in Fstl1/2 morphants, chordamesodermal gene expression was
prolonged when compared with controls, resembling that of embryos at younger
developmental stages. Expression of ihhb, which is restricted to the
caudal notochord in 18-somite stage embryos, was maintained in the rostral
notochord of Fstl1/2 morphant embryos (Fig.
5U,V). We also observed shha expression in vacuolated
notochord cells and ptc1 expression in axial and paraxial tissues of
the trunk of 36 hpf Fstl1/2 morphants, where they are not expressed in
controls (Fig.
5X,Y,AA,BB,DD,EE). col2a1a expression was maintained in
the notochord of 48 hpf Fstl1/2 morphants
(Fig. 5GG,HH), resembling
expression of 24 hpf controls (Yan et al.,
1995). The gene expression patterns observed in Fstl1/2 morphants
are consistent with a delay in transitioning into differentiated notochord, as
is observed in embryos lacking Col15a1
(Pagnon-Minot et al., 2008)
and Laminin1/4/5 (Parsons et al.,
2002; Pollard et al.,
2006).
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The proliferative state of chordamesoderm establishes the timing of notochord maturation
Alterations in BMP activity levels influence both the proliferative
properties of chordamesoderm, as well as the timing of its differentiation. To
determine whether the proliferative capacity of chordamesoderm influences the
timing of its maturation, we used small molecule inhibitors of the cell cycle
(Stern et al., 2005) to block
cell proliferation during the window in which BMP activity influences the
proliferative capacity of chordamesoderm. We found that treating embryos from
80% epiboly to bud stage with a combination of hydroxyurea and aphidicolin
effectively blocked cell proliferation during late gastrulation (see Fig.
S5A,B in the supplementary material) (also data not shown). Removing the
inhibitors at bud stage allowed proliferation to resume 2 hours after washing,
although BrdU incorporating cells were not observed in chordamesoderm (see
Fig. S5C-C'' in the supplementary material). The chordamesoderm defects
observed in embryos treated with cell cycle inhibitors during late
gastrulation resembled the defects observed in embryos lacking Bmp4.
Expression of the chordamesodermal markers ihhb, shha and
col2a1a resembled expression in Bmp4 morphants
(Fig. 5D,H,L,P,T), and
notochord cells at the level of the cloaca became vacuolated before those of
controls (Fig. 6K,L).
In contrast to embryos with reduced BMP activity, chordamesoderm proliferation is increased and transition into mature notochord is delayed in Fstl1/2 morphants. By treating Fstl1/2 morphants with hydroxyurea and aphidicolin from 80% epiboly to bud stage, we were able to rescue the prolonged expression of chordamesoderm markers. Expression of ihhb and shha was restricted to caudal notochord in these embryos, resembling comparably staged controls (Fig. 5W,Z,CC). Similarly, col2a1a expression was lost from the notochord and floor plate of 48 hpf Fstl1/2 morphants treated with cell cycle inhibitors, also resembling controls (Fig. 5II). This suggests that the proliferative state of chordamesoderm, which is established during late gastrulation, determines the timing of the chordamesoderm transition into mature notochord.
A direct role for BMP signaling in notochord development
The presence of PSMAD1/5/8-positive cells
(Fig. 4) suggests that
chordamesoderm is directly influenced by BMP activity. To demonstrate that
this tissue autonomously responds to BMP signaling, we removed 30 cells
from the ventral margin of shield stage embryos and transplanted them into the
dorsal organizer of an equivalently staged host
(Fig. 6O). Upon placing
wild-type cells into a wild-type host, these cells reliably populated the
notochord, consistent with previous fate mapping and transplantation studies
(Gritsman et al., 2000;
Saude et al., 2000).
Transplantation of ventral wild-type cells into tBR embryos
heat-shocked after transplantation resulted in donor cell morphology
resembling that of control embryos (94/132)
(Fig. 6P,Q). Surprisingly, we
found that these cells populated the entire notochord at the exclusion of
endogenous tBR cells. Following the transplantation of ventral
tBR cells into wild-type embryos, the tBR cells consistently
failed to populate the notochord (0/151)
(Fig. 6R,S). They did, however,
populate adjacent paraxial mesoderm, contributing to myofibers and somitic
cells. Thus, BMP activity biases cells in proximity of the dorsal organizer to
become chordamesoderm.
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Because we observed that axial cells remained PSMAD1/5/8-positive when Bmp4 was knocked down, we wished to examine the effects of other BMPs on notochord development (see Fig. S6 in the supplementary material). Implantation of beads soaked with recombinant human Bmp2/7 did not noticeably alter the domain of chordamesoderm or notochord morphology when compared with controls.
The timing of notochord maturation influences myotome patterning
Because delays in notochord maturation disrupt myotome patterning
(Pagnon-Minot et al., 2008),
we examined slow and medial fast muscle specification in Fstl1/2 morphant
embryos. During early somitogenesis stages, Hedgehog (Hh) activity maintains
myod1 expression in slow and medial fast muscle precursors
(Barresi et al., 2000;
Linker et al., 2003;
Wolff et al., 2003). In
embryos lacking Fstl1/2, myod1 expression was upregulated in the
presumptive slow and medial fast territories of the somite (see Fig. S7A,B in
the supplementary material). Immunostaining with antibodies against Myod1
confirmed a significant increase in Myod1-positive cells when compared with
controls (see Fig. S7 in the supplementary material) (74.6 versus 120.4,
n=5; P<0.001). Fstl1/2 morphant embryos also displayed an
increase in horizontal myoseptum size, as evidenced by immunostaining with F59
antibodies that recognize mainly slow muscle (see Fig. S7G,H in the
supplementary material) (Devoto et al.,
1996). Likewise, the number of Engrailed-positive cells, which are
induced in response to Hh activity (Wolff
et al., 2003), was increased in Fstl1/2 morphants (see Fig.
S7J,K,M,N in the supplementary material).
|
|
DISCUSSION |
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|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Axial requirements for BMP activity change as gastrulation proceeds
Models addressing BMP activity in embryogenesis have established the
ventralizing role of BMP signaling in specifying DV fate
(Kimelman, 2006;
Schier and Talbot, 2005).
Surprisingly, our study demonstrates a dynamic requirement of axial tissue for
BMP activity during gastrulation. Following establishment of the DV axis,
axial cells require tightly regulated levels of BMP activity for proper
patterning of chordamesoderm and its precursor pool. Changes in axial
requirements for BMP activity are consistent with two recent studies in
zebrafish examining BMP and Nodal activity during zebrafish gastrulation.
Tucker and colleagues (Tucker et al.,
2008) demonstrated that BMP patterning of the DV axis occurs
progressively in an anterior to posterior fashion as gastrulation proceeds.
Hagos and Dougan (Hagos and Dougan,
2007) demonstrated that cells responsive to Nodal signaling
acquire their fate based upon the cumulative dose of Nodal signals the cell
receives. This is a function of both the competence of the receiving cells as
well as the strength of the signal. These studies suggest that cells possess
different requirements for activity gradients as gastrulation proceeds. This
is in agreement with the characteristic migration of two margins into
proximity with the other during gastrulation: the ventral margin secreting BMP
ligands and the dorsal organizer secreting BMP antagonists
(Agathon et al., 2003). As the
circumference of the margins decrease during epiboly, cells populating ventral
and dorsal margins receive increasing amounts of factors secreted from the
other margin. The question is, at what point does distance between the two
margins influence the BMP activity gradient on the dorsal side of the
gastrula?
|
It is possible, although unlikely, that axial cells require BMP activity
prior to late gastrulation. Labeling embryos with PSMAD1/5/8 antibodies at
hourly intervals has revealed that PSMAD1/5/8-positive cells are not observed
in the dorsal gastrula until late gastrulation
(Tucker et al., 2008).
Therefore, the rise of PSMAD1/5/8 levels after 80% epiboly in Fstl1/2
morphants is consistent with a role in mediating BMP activity in axial
tissue.
Selective requirement of BMP activity in the chordamesoderm
Nodal activity is crucial for both the induction of mesoderm and the
specification of axial cell fate
(Kimelman, 2006;
Schier and Talbot, 2005;
Stemple, 2005). The location
of cells within the dorsal organizer dictates the level of Nodal activity that
axial mesoderm receives. High levels of Nodal signaling specify deep
gsc-expressing prechordal plate, whereas lower levels specify more
superficial flh-expressing chordamesoderm
(Gritsman et al., 2000;
Saude et al., 2000). As
Follistatin binds Activin ligands
(Harrington et al., 2006;
Nakamura et al., 1991), one
possibility for the notochord phenotype observed in Fstl1/2 morphants is an
alteration in Nodal activity. If this activity were significantly altered, one
would expect a transfating between types of axial mesoderm, as has been
observed in mutants lacking zygotic contribution of the Nodal co-receptor Oep
(Gritsman et al., 2000).
However, we observe no changes in the prechordal domain of Fstl1/2 morphants,
as assayed by the expression of the Nodal-responsive gene gsc and
PSMAD2 levels. Furthermore, the phenotypes observed in chordamesoderm and
notochord of embryos with reduced BMP activity are complementary to those
observed in Fstl1/2 morphants. Thus, the main role of Fstl1/2 is to modulate
BMP activity.
Two models can explain the inability of prechordal cells to respond to BMP signaling: the location of these cells during gastrulation, or their competence to respond to Bmp4. As gastrulation proceeds, prechordal plate cells rapidly increase their distance from the ventral margin, distancing themselves from BMP-secreting cells. If prechordal cells were competent to respond to Bmp4, gsc expression would be altered in embryos receiving Bmp4-soaked beads. Therefore, a more plausible model is that prechordal plate cells are not competent to respond to Bmp4 during mid- to late-gastrulation, which would make them distinct from chordamesoderm precursors in the dorsal organizer.
A balance between proliferation of the CNH and differentiation of chordamesoderm
Our results indicate that during late gastrulation through early
somitogenesis, BMP signaling regulates the size of the chordamesodermal domain
by establishing its proliferative state. In the absence of Bmp4,
chordamesodermal cells prematurely transition into differentiated notochord.
Conversely, chordamesodermal proliferation is increased in Fstl1/2 morphants
and maturation of the notochord is delayed. BMP signaling may play a dual
independent role to control the proliferative status of the CNH and
chordamesoderm and to regulate the timely differentiation of notochord
precursors. By inhibiting cell proliferation over the same window at which BMP
activity influences the proliferative state of chordamesoderm, we were able to
mimic the notochord phenotype observed in tBR embryos and Bmp4
morphants. Additionally, blocking cell proliferation inhibits the prolonged
maintenance of chordamesodermal character observed in Fstl1/2 morphants. As
heat-shocking tBR embryos after the 3-somite stage has no apparent
effect on indicators of notochord differentiation, it is likely that BMP
activity does not directly delay chordamesoderm maturation. Rather, the
evidence presented suggests a single role for BMP signaling in controlling
proliferation and favors a causal link between CNH proliferation and notochord
maturation. We propose a model where the size of the axial precursor pool,
established by Bmp4 and tightly controlled by two Fstl proteins, ultimately
determines the maturation schedule of the notochord. Although we demonstrate
the requirement of Fstl1 and Fstl2 in this process, both chordin
(chd) and noggin1/2 (nog1/2) are also
chordamesodermally expressed, with nog1/2 expression persisting in
the CNH through notochord maturation
(Furthauer et al., 1999;
Miller-Bertoglio et al.,
1997). Although their requirement in DV specification prevents
simple morpholino-mediated inactivation
(Dal-Pra et al., 2006;
Hammerschmidt et al., 1996a;
Hammerschmidt et al., 1996b;
Schulte-Merker et al., 1997),
we cannot rule out that either chd or nog1/2 might act
synergistically with fstl1/2 in chordamesoderm development as they do
in early DV patterning. Similar models for a role of Bmp4 signaling in
balancing proliferation and differentiation have been proposed in other
contexts. The influence Bmp4 exerts over hematopoietic stem cell (HSC)
proliferation (Zhang and Li,
2005) draws parallels with the effect of losing Bmp4 on the CNH.
Bmp4 promotes the proliferative state of HSCs while delaying differentiation
(Zhang et al., 2003). In the
absence of Bmp4, HSCs exit the cell cycle and differentiate prematurely
(Bhatia et al., 1999).
In addition to notochord contributions, cells from the CNH give rise to
floor plate and tailbud mesoderm (Cambray
and Wilson, 2002; Charrier et
al., 1999; Davis and
Kirschner, 2000). Our transplantation experiments suggest a
particular requirement for BMP responsiveness in chordamesoderm:
BMP-responsive cells preferentially populate the notochord, whereas cells
unresponsive to BMP signaling contribute to paraxial and tailbud mesoderm. In
addition to its role in proliferation and differentiation, differential levels
of BMP activity appear to be crucial in relegating CNH cells to a particular
fate.
Experiments by Agathon and colleagues
(Agathon et al., 2003) have
shown that the BMP-rich ventral margin in zebrafish posses tail organizer
activity that contributes to non-axial derivatives in the tail, whereas the
CNH, which is derived from the dorsal organizer, contributes to axial
components. Their data, along with other studies
(Beck et al., 2001;
Gont et al., 1993;
Pyati et al., 2005;
Tucker and Slack, 1995)
suggest that axial and non-axial tissues of the tailbud can develop
independently under certain experimental conditions. However, as these two
organizing centers move into close proximity at the end of gastrulation, they
probably need to interact with each other to promote the coordinated growth,
patterning and differentiation of the tailbud. Our studies have revealed a
requirement for BMP signaling from the ventral margin on the dorsal organizer,
providing a mechanism by which such coordination can be achieved and
demonstrating previously unappreciated roles of BMP activity in the patterning
and maturation of axial structures.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/23/3891/DC1
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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