Department of Biochemistry, University of Washington, Seattle, WA 98195-7350, USA
* Author for correspondence (e-mail: kimelman{at}u.washington.edu)
Accepted 28 February 2005
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SUMMARY |
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Key words: Bmp signaling, Transgenic zebrafish, Ventral mesoderm, Tail
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Introduction |
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One crucial signaling pathway for vertebrate trunk and tail mesoderm
formation is the Bone morphogenetic protein (Bmp) pathway (reviewed by
Dale and Jones, 1999). Bmps are
members of the TGFß superfamily of secreted proteins, and they bind
TypeI/TypeII receptor complexes to initiate a signaling cascade that activates
transcription of downstream targets such as the homeobox genes vox
and vent (Onichtchouk et al.,
1996
; Melby et al.,
2000
; von Bubnoff and Cho,
2001
; Ramel and Lekven,
2004
). During gastrulation in Xenopus and zebrafish, Bmp
signaling is highest on the ventral side of the embryo, where it patterns the
mesoderm to adopt fates including blood, vasculature, pronephros and tail
muscle. Loss of Bmp signaling in both animals leads to expansion of dorsal
structures, such as trunk muscle, at the expense of ventral structures
(Re'em-Kalma et al., 1995
;
Mullins et al., 1996
;
Kishimoto et al., 1997
).
Zebrafish mutants support the model that a gradient of Bmp signaling patterns
various tissue types during gastrulation in the developing embryo (reviewed by
Dale and Wardle, 1999
). For
example, the swirl mutant, which lacks bmp2b, has radialized
trunk somites, reduced blood, vasculature and pronephros, and no tail tissue
(Kishimoto et al., 1997
).
Other mutants in the Bmp pathway, including snailhouse hypomorphs
(Dick et al., 2000
),
piggytail (Kramer et al.,
2002
), lost-a-fin
(Mintzer et al., 2001
) and
minifin (Connors et al.,
1999
), have progressively less severe phenotypes than
swirl mutants. For instance, in zygotic lost-a-fin embryos,
which are mutant for the Bmp receptor alk8, the patterning of the
ventral and posterior mesoderm is normal, but the ventral tail fin is absent
(Mintzer et al., 2001
). Such
data suggest that the highest levels of Bmp signaling are required for
formation of the ventral tail fin, while lower levels of Bmp signaling are
required for other ventral and posterior tissues.
The role of Bmps in tail development has been further elucidated by
experiments involving overexpression of bmp RNA in the early
zebrafish embryo. When injected in combination with RNA encoding Wnt8 and the
Nodal ligand Cyclops (Cyc), Bmp induces the formation of ectopic tails
(Agathon et al., 2003).
Moreover, whereas tails can form at lower efficiency when Bmp is expressed
with either Wnt8 or Cyc alone, Bmp is indispensable for tail formation. A
second set of experiments suggests that Bmp signaling is important for
reserving a population of tailbud cells for somitogenesis in the tail. When
dorsal determinants, which activate Bmp antagonists such as chordin
(Sasai et al., 1994
) and
noggin (Zimmerman et al.,
1996
), are removed before gastrulation, embryos do not develop
trunk somites and instead only develop tail somites
(Ober and Schulte-Merker,
1999
). If, however, dorsal determinants are removed in a
swirl background, which lacks zygotic Bmp signaling, the embryos
recover trunk somite formation and lose the tail somites. These experiments
indicate that Bmp signaling negatively regulates trunk somitogenesis while
promoting tail somitogenesis, potentially by setting aside a population of
cells to form tail somites rather than trunk somites.
Such studies show a requirement for Bmp signaling in formation of the
zebrafish tail organizer. However, they do not determine whether Bmps exert
their influence on tailbud cells only during gastrulation, or if Bmps continue
to be required in the post-gastrula tailbud. Bmps are expressed in the tailbud
and surrounding tissues throughout somitogenesis
(Martinez-Barbera et al.,
1997; Dick et al.,
2000
), making it seem likely that they are playing important roles
during trunk and tail formation. As mutants are defective in Bmp signaling
from the onset of zygotic transcription, and overexpression, dominant-negative
and morpholino experiments have been performed only before the gastrula
stages, it is unclear what the role of Bmp is in the ventral and posterior
mesoderm after the end of gastrulation. To address this question, we generated
a zebrafish transgenic line for conditionally reducing Bmp signaling. We found
that Bmp signaling plays an important role in primary tail formation during
early gastrulation, and that it prevents the formation of ectopic tails from
the mid-gastrula stages through early somitogenesis. High levels of Bmp
signaling appear to be important for the development of most ventral
mesodermal derivatives during early gastrulation, whereas the ventral fin
requires Bmp signaling from early gastrulation through the early somitogenesis
stages.
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Materials and methods |
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Generation of a stable transgenic line
Uncut DNA (1 nl) was injected into 1-cell stage WIK/AB embryos.
IsceI meganuclease was co-injected with the DNA to maximize the
number of integration events. The injection mix was: 100 pg/nl DNA, 0.5
x IsceI Buffer, 10% IsceI enzyme. The F1
generation was screened for heatshock-inducible GFP fluorescence, and a stable
line was generated.
Heatshock conditions
Embryos from an F1 out-cross were typically heatshocked for 1
hour at desired stages by placing them for 1 hour in a 37°C air incubator,
and then screened for GFP fluorescence after 1.5 hours at 28.5°C. Embryos
were either fixed at 2 hours post-heatshock for in-situ hybridization analysis
or left in a 28.5°C air incubator for later visualization. For PCR machine
heatshocks, single embryos were separated into PCR tubes with 20 µl of
embryo medium. The PCR block was ramped to 37°C for 1 hour, then held at
28.5°C.
Confocal microscopy
Truncated Bmp receptor-GFP/tbx6-gfp embryos were heatshocked from
shield to bud stage in a 37°C air incubator. For late tail growth, embryos
with ectopic tails and GFP fluorescence in the tail were identified at the
22-24-somite stage. Embryos were mounted in embryo medium containing 1%
low-melt agarose. For early tail growth, 20 embryos were mounted at the
16-17-somite stage in 1% low-melt agarose with their tails exposed to the
medium. Images were acquired using a Zeiss LSM Pascal confocal microscope and
a 40 x lens. Slices (1.6 µm) were taken once per hour.
Transplants
For wild-type transplants (Moens and
Fritz, 1999), 1-cell stage WIK/AB embryos were injected with
fluorescein-dextran (Mr 10,000). At the dome stage, cells
were transplanted from labeled donor embryos to the ventral side of
shield-stage host embryos from a transgenic in-cross. Host embryos were
immediately heatshocked in an air incubator at 37°C through bud stage to
maximize ectopic tail formation. Embryos with ectopic tails at 24 hours were
scored for the presence or absence of fluorescein-labeled cells within the
ectopic tails. Control transplants from transgenic donors were performed in
the same manner, but donor embryos were collected from in-crosses of
transgenic fish. PCR analysis of genomic DNA was used to determine whether
donor embryos were wild type or transgenic.
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Results |
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To express the truncated Bmp receptor-GFP fusion at different times of
development, we placed this construct under the control of the hsp70
promoter (Halloran, 2000) and generated a zebrafish transgenic line. To
determine if the induction of the transgene would recapitulate phenotypes of
known Bmp pathway mutants, we heatshocked embryos before the onset of
gastrulation and observed their phenotypes during the development of the
posterior body. Transgenic embryos were sorted by GFP fluorescence 1 hour
after the completion of the heatshock and scored at both the 14-somite stage
and 30 hours post-fertilization (hpf) for the degree of dorsalization using
the scale developed by Mullins et al.
(Mullins et al., 1996). In
this scale, C5 represents an embryo completely lacking Bmp signaling
(swirl phenotype), whereas C1 is a mildly dorsalized embryo.
Heatshocking embryos at 3 hpf produced severely dorsalized phenotypes in
transgenic embryos (Fig. 2B,C).
By contrast, sibling non-transgenic embryos that received the same heatshock
treatment had no discernible defects (Fig.
2A), demonstrating that the effects we saw were not due to the
heat treatment. The majority of transgenic embryos were in the C4
dorsalization class, with severe expansion of somites and curling of the trunk
and tail (Fig. 2B;
Table 1). Embryos in this class
resembled hypomorphic snailhouse (bmp7) mutants
(Dick et al., 2000
). Some
embryos were severely dorsalized at the end of gastrulation (10 hpf, data not
shown), but they did not survive beyond the end of somitogenesis. These
embryos were scored as a C5 phenotype, resembling swirl (bmp2b)
mutant embryos (Kishimoto et al.,
1997
). A third class of embryos had C3 phenotypes, with expansion
of posterior somites and curling at the end of the tail
(Fig. 2C). Embryos in the C3
class resemble piggytail (smad5) mutants
(Kramer et al., 2002
).
Interestingly, heatshocking embryos at 3 hpf in a PCR machine rather than an
air incubator produced a more severe dorsalization, with all the embryos
having a C5 phenotype (Table
2). These data demonstrate that induction of the transgene before
gastrulation phenocopies zebrafish mutants with severe reductions in Bmp
signaling.
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Effects of reducing Bmp signaling on ventral and posterior mesoderm at different stages of development
Having developed a system for inducibly interfering with Bmp signaling, we
wished to examine the effects of reducing Bmp signaling at different stages of
development. Heatshocking embryos at shield stage caused phenotypes that were
far less severe than those resulting from 3 hpf heatshocks. At 30 hpf, all
transgenic embryos had the C1 dorsalized phenotype, with normal tails lacking
ventral tail fins (Fig. 2E;
Table 1). The ventral tail fin
is thought to be the tissue that is most sensitive to losses of Bmp signaling,
revealed by minifin (tolloid) and lost-a-fin (alk8) mutants
(Connors et al., 1999;
Mintzer et al., 2001
). In
addition to the C1 phenotype, typically one-fifth of transgenic embryos formed
ectopic tail-like structures at their posterior ends
(Fig. 2F;
Table 1), although in one
clutch as many as 45% of the embryos had these structures, which we henceforth
refer to as ectopic tails. Heatshocking at the end of gastrulation (bud
stage), and during somitogenesis (3-somite and 6-somite stages) also caused
loss of the ventral tail fin (Table
1), indicating that ventral tail fin tissue is dependent upon
continued Bmp signaling after gastrulation and through the early somitogenesis
stages. Ectopic tail phenotypes were also induced by heatshocking at bud
through 6-somite stages, but with lower penetrance than heatshocking at shield
stage (Table 1). Heatshocking
after the 6-somite stage had no effect on posterior development.
We reasoned that heatshocking individual embryos in a PCR machine might decrease the lag time of transgene expression, since this method caused a C5 phenotype in 100% of embryos heatshocked at 3 hpf. Indeed, this method resulted in GFP fluorescence within half an hour of heatshock. As shown in Table 2, heatshocking at shield (early gastrulation) using the PCR machine caused a more severe effect than similar heatshocks in an air incubator, with 41% of embryos having a C3 phenotype. By 70% epiboly (mid-gastrulation), however, heatshocking in the PCR machine caused only a C1 phenotype in transgenic embryos. Percentages of ectopic tails caused by heatshocking at various stages matched closely with similar heatshocks in the air incubator (compare Tables 1 and 2). Thus, Bmp signaling is important for ventral tail fin formation and suppression of ectopic tails from the mid-gastrula stage through early somitogenesis.
eve1 is a zebrafish homeobox gene expressed in the ventral
mesoderm during gastrulation and in the tailbud during somitogenesis, thus
serving as a good marker of ventroposterior fates within the embryo
(Joly et al., 1993;
Mullins et al., 1996
). As
eve1 expression is reduced in bmp mutants
(Joly et al., 1993
;
Mullins et al., 1996
), we
wished to examine whether eve1 expression is dependent on Bmp
signaling only during gastrulation, or whether it continues to be affected by
reduction of Bmp signaling after gastrulation. We heatshocked embryos at
shield stage, bud stage and 3-somite stage, then assayed for eve1
expression by whole-mount in-situ hybridization. As predicted from mutant
analyses, heatshocking at shield stage reduced eve1 expression at the
ventral margin in transgenic embryos compared with wild-type siblings (compare
Fig. 3B with 3A). Interestingly, heatshocking after the end of gastrulation (bud and 3-somite
stages) still caused reduced eve1 levels in the tailbuds of
transgenic embryos (compare Fig. 3C,E with
3D,F). The size of the tailbud was not decreased, as the homeobox
gene vent1 (Kawahara et al.,
2000
; Melby et al.,
2000
) was expressed at normal levels in the tailbuds of transgenic
embryos after heatshock (data not shown). These data show that eve1
expression is dependent upon Bmp signaling during both gastrulation and early
somitogenesis.
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Transplantation of cells with reduced Bmp signaling
We next asked whether cells with normal Bmp signaling capabilities could
populate ectopic tails. We reasoned that trunk and/or tail cells are
mis-specified to form ectopic tails when Bmp signaling levels are reduced at
mid-gastrulation or early somitogenesis. To test this hypothesis, we performed
transplants of fluorescein-labeled wild-type donor cells into ventral regions
of transgenic hosts and heatshocked the hosts at shield stage
(Fig. 7A). We then scored for
the presence of donor cells in the ectopic tails at 24 hpf. As controls, we
performed the same experiment with transgenic donor cells to be sure that
cells with reduced Bmp signaling would move into ectopic tails. As shown in
Fig. 7, wild-type transplanted
cells did not populate ectopic tails (Fig.
7B; 0%, n=12), while transgenic cells did
(Fig. 7C; 57%, n=14).
The transgenic cells could also populate the primary tail
(Fig. 7C). By contrast, the
transgenic cells transplanted into wild-type embryos never produced an ectopic
tail (data not shown), potentially because we could not get a large enough
group of transgenic cells in one location to form an ectopic tail. These data
indicate that cells capable of receiving Bmp signals are actively recruited
into primary trunk or tail tissue, while cells that have reduced Bmp signaling
capacity can go into either the primary trunk and tail or the ectopic
tail.
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Discussion |
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Combinatorial signaling in trunk and tail somite formation
Patterning of trunk and tail mesoderm involves the input of signals as
diverse as Wnts (Lekven et al.,
2001), Fgfs (Griffin et al.,
1995
) and Nodals (Thisse et
al., 2000
) in addition to Bmps. The importance of each of these
pathways in forming the posterior body has been revealed by mutant studies,
but their temporal-specific roles are less clear. A pharmacological Fgf
inhibitor revealed the necessity of Fgf signaling for somite formation during
posterior body outgrowth (Griffin and
Kimelman, 2003
), and we have observed that the Wnt pathway is
needed continuously during the somitogenesis stages for trunk and tail
development (E. Herbig, U.J.P. and D.K., unpublished). As all four signaling
molecules are expressed in the ventral region during gastrulation and in the
posterior body during somitogenesis, it is likely that they are not only
playing individual roles in forming the trunk and tail, but combinatorial
roles as well. Indeed, combinatorial signaling between Bmps and Wnts has been
recently shown to be important for maximal expression of the zebrafish tailbud
gene tbx6 (Szeto and Kimelman,
2004
), and the zebrafish organizer-repressing genes vox
and vent (Ramel and Lekven,
2004
). Our data suggest that Bmps are individually important for
formation of the tail organizer during early gastrulation, but they are not
crucial for trunk or tail formation after this stage. One reason that the role
of Bmp in tail development may shift so dramatically is that signaling
pathways such as those for Wnt and Fgf can compensate for the loss of Bmp
signaling beyond the early gastrula stages. The ventroposterior gene
eve1, for example, is regulated by Bmp and Fgf signals
(Joly et al., 1993
;
Griffin et al., 1995
). While
Bmp is needed for initiation and maintenance of maximal eve1
expression, Fgf may be sufficient to maintain enough eve1 expression
after early gastrulation for proper tail organizer function. Another
possibility is that Bmp signaling initially establishes the ventroposterior
domain of mesodermal cells during early gastrulation, but it has no role at
later times other than to regulate the formation of the ventral fin. While
this is a relatively small domain within the embryo, it is important for
normal development and would provide a reason for the embryo to maintain Bmp
signals in the tailbud during somitogenesis. With the ability to conditionally
reduce Bmp activity in zebrafish embryos it will now be possible to examine
the interactions with other signaling pathways to determine if Bmp acts
combinatorially with other pathways after gastrulation, or whether Bmp has a
much more circumscribed role during this time.
Bmp signaling and ventral mesoderm development
In the gradient model for Bmp signaling, ventral mesodermal derivatives
require the highest levels of Bmp activity during gastrulation. Bmps in the
most ventral part of the margin specify a population of cells to form
multipotent hemangioblast cells (Walmsley
et al., 2002) as well as cardiac precursors
(Kishimoto et al., 1997
) and
pronephros cells (Mullins et al.,
1996
). These cells are subsequently patterned to give rise to
different subpopulations of the cardiovascular, blood and pronephric systems.
Data in Xenopus using an inducible smad6 Bmp inhibitor
construct suggest that Bmp signaling is important for maintenance of blood
precursors even after gastrulation
(Schmerer and Evans, 2003
).
However, our data indicate that in zebrafish, neither blood, vascular nor
pronephric precursors are affected by reduction of Bmp activity after the
gastrula stages. Like the tail mesodermal cells, it seems likely that Bmp
induces a group of cells in the margin to adopt the most ventral fates, while
other signals maintain and pattern these cells. Such a model is supported by
mutant analyses, in which zygotic mutants for somitabun and
lost-a-fin have normal ventral mesoderm, but maternal zygotic mutants
for these genes have deficient ventral mesoderm
(Mintzer et al., 2001
;
Kramer et al., 2002
).
Therefore, the early acting maternal supplies of smad5 and
alk8 are sufficient for ventral mesoderm formation in the absence of
functional zygotic protein.
We cannot exclude a later role for Bmps in regulating downstream patterning
or morphogenesis of the ventral mesodermal derivatives. Indeed, the analysis
of radar morphants suggests that reducing the post-gastrula activity
of this Bmp family member compromises vascular integrity in the trunk and tail
(Hall et al., 2002). While we
did not observe such a phenotype in later heatshocks (data not shown), it is
possible that the dominant-negative Bmp receptor does not inhibit Radar
signaling or that we did not heatshock at the appropriate stage. However, we
can conclude that Bmps are required for early patterning of ventral mesodermal
derivatives during gastrulation, but not afterward.
Roles of Bmp signaling after early gastrula stages
Our data show that Bmp signaling plays very different roles in posterior
development after the early gastrula stages
(Fig. 8). Rather than
maintaining tail formation during somitogenesis, Bmp is important for
preventing the formation of ectopic tails and for promoting the formation of
the ventral fin. Heatshocks between shield stage and early somitogenesis
stages cause phenotypes closely resembling those of severe minifin
mutants (Connors et al., 1999).
In minifin mutants, the extracellular metalloprotease Tolloid is
mutated, leading to reductions in Bmp signaling levels when zygotic Tolloid
has been depleted. As discussed earlier, these mutants do not develop ventral
tail fins. Our transgenic analysis allows us to postulate that the zygotic
minifin mutant phenotype is due to reduced Bmp signaling after the
early gastrula stages, when specification of the primary tail has already
occurred. This model is in agreement with data from Connors and Mullins (S. A.
Connors and M. C. Mullins, personal communication), in which transgenic rescue
of minifin mutant embryos is most efficiently achieved from the late
gastrula stages onward. Similar to our results, they find that rescue of the
minifin phenotype can only be effectively achieved through the early
somitogenesis stages. Using two different approaches, these results
demonstrate a role for Bmp signaling during the late gastrula and early
somitogenesis stages in ventral tail fin formation.
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As bmps are strongly expressed in the ventral portion of the primary tail, it is likely that this signaling normally prevents the organization of secondary tail structures. In line with this, our transplant data indicate that only posterior cells lacking Bmp signaling can contribute to ectopic tail tissues. The formation of ectopic tails is, therefore, a cell-autonomous effect. Wild-type cells in a background of cells with low Bmp signaling behaved like wild-type cells in a wild-type background, whereas only cells with a lowered capacity to respond to Bmp signaling became part of the ectopic tail. Thus, Bmp signaling may be important for limiting the number of tail precursors during the mid-gastrula through early somitogenesis stages.
Two different morphogenetic mechanisms could explain the emergence and
growth of secondary tails. In the first, loss of Bmp signaling through the
early somitogenesis stages could lead to a split in the tail later in
development. In this view, the ventral portion of the primary tailbud splits
away from the remainder of the tailbud, pulling away tissue such as the
hypochord and notochord from the primary tail during outgrowth. A second
mechanism is that the most ventral tail mesoderm could be converted to a new
tail organizer when Bmp signaling is disrupted. As this organizer grows out,
it forms primarily somites, and it also recruits the hypochord and notochord
away from the primary axis. These two models are not mutually exclusive, and
we favor a combination of both. Our experiments examining the emergence of the
ectopic tail at the 17-18-somite stage suggest that the ventral portion of the
primary tailbud dissociates from the main tailbud to form an ectopic tail.
This is not, however, a simple split of the tail into equal parts, as is
commonly seen in Xenopus embryos with disrupted convergence extension
movements (reviewed by Ueno and Greene,
2003). Rather, a very specific domain of the embryo gives rise to
mesodermal tail tissues independent of the primary tail. We suggest that the
ectopic tail forms from a population of tail progenitor cells that detach from
the primary tailbud at the onset of tail formation and are left behind in the
ventral region of the embryo. Furthermore, we propose that these are the cells
that would otherwise form ventral tail mesoderm, but their fate changes due to
an early disruption in Bmp signaling. Future studies will be needed to
determine why in some cases these cells are left behind to form an ectopic
tail, whereas in most cases the cells integrate into the primary tailbud.
New avenues of research
Bmp signaling is used at many times in development to affect a wide variety
of cell types. Although its role has been extensively studied during early
times in the lower vertebrates, the conditional dominant-negative fish
described here will allow investigators to study Bmp signaling throughout
development and in adult fish. Because the dominant-negative Bmp receptor acts
cell-autonomously, it is well suited for transplant studies. Moreover, as the
receptor is GFP-tagged, in crosses of heterozygous fish it is easy to identify
the embryos expressing the transgene. Finally, because the dominant-negative
Bmp receptor turns over fairly rapidly, it is possible to do timecourse
experiments to determine when Bmp signaling is needed for particular
developmental events. We expect that these fish will be a useful resource for
analysis of Bmp signaling in zebrafish.
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ACKNOWLEDGMENTS |
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