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First published online 3 May 2006
doi: 10.1242/dev.02388
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1 University of Washington Department of Biochemistry, Box 357350, Seattle, WA
98195-7350, USA.
2 University of Washington Department of Biology, Box 351800, Seattle, WA 98195,
USA.
3 Center for Regenerative Medicine, Massachusetts General Hospital, 185
Cambridge Street, Boston, MA 02114, USA.
4 University of Washington Department of Biological Structure, Box 357420,
Seattle, WA 98195, USA.
* Author for correspondence (e-mail: kimelman{at}u.washington.edu)
Accepted 3 April 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Bmp signaling, Transgenic zebrafish, Ventral mesoderm, Cloaca, Proctodeum, Excretory system, Anus, Anorectal
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Hammerschmidt and
Mullins, 2002; Kishigami and
Mishina, 2005). Zebrafish mutants in the Bmp pathway, such as
swirl (bmp2b) and snailhouse (bmp7), have
severe deficits in ventral and posterior mesoderm, along with expansion of
dorsal and anterior tissues, such as the brain and notochord
(Mullins et al., 1996).
Conversely, analysis of mutants with increased Bmp signaling, combined with
overexpression experiments in Xenopus, have shown that Bmp signaling
is also sufficient for the formation of blood, vascular and kidney tissues,
which are derived from the ventral mesoderm in zebrafish
(Kimmel et al., 1990). For
example, overexpression of Bmp RNAs in Xenopus animal caps is
sufficient to induce the expression of red blood cell markers
(Zhang and Evans, 1996), and
chordino, a zebrafish mutant with excess Bmp signaling, forms extra
blood, kidney and vascular cells
(Hammerschmidt et al., 1996;
Leung et al., 2005). Thus, Bmp
signaling is important for the induction of ventral mesodermal derivatives
during development.
Despite the crucial requirement of Bmp signaling for ventral mesoderm
formation and differentiation, the temporal basis of this role is not well
understood. Studies to date have examined the effects of manipulating Bmp
signaling only at the earliest stages of development, and have not examined
later effects on the differentiation of ventral mesodermal derivatives.
Furthermore, early loss of Bmp signaling results in aberrant formation of the
ventral mesoderm, prohibiting the subsequent analysis of patterning within
this domain. Because Bmps are expressed at high levels in the ventral region
of the embryo until the end of somitogenesis
(Martinez-Barbera et al.,
1997), it is important to determine whether Bmp signaling plays
any role in the ventral mesodermal derivatives beyond their initial
formation.
To analyze the temporal roles of Bmp signaling in the patterning of ventral
mesodermal tissues, we used an inducible transgenic zebrafish line that we
recently generated (Pyati et al.,
2005). This line, containing the Hsp70 promoter driving a
dominant-negative Bmp receptor fused to GFP, allows stage-specific Bmp
inhibition upon heat shock of transgenic embryos. Initially, we used these
transgenic fish to describe the roles of Bmp in tail development. We showed
that during the early gastrula stages, Bmp signaling is crucial for the
development of the primary tail, but, by the mid-gastrula stage, Bmp signaling
is important only for ventral tail fin formation and for preventing the
development of secondary tail structures
(Pyati et al., 2005). Thus,
the roles of Bmp in the posterior mesoderm change over time.
In this study, we describe the patterning roles of Bmp in ventral mesodermal tissues, including blood, vascular and kidney precursors, after the earliest phase of gastrulation. Although Bmp signaling is needed at the early gastrula stage for these ventral derivatives to form, we find, surprisingly, that inhibition of Bmp signaling just two hours later at the mid-gastrula stage causes an expansion of the blood and vascular precursors into the extreme ventral embryonic domain. Lineage labeling of these cells shows that they normally contribute to ventral tail tissues, including the developing cloaca (the common opening of the gut and kidneys), and that the development of these structures is deficient in transgenic embryos. We find that patterning and function of the excretory system is dependent on early and sustained Bmp signaling, and we examine, for the first time, the morphogenesis of the presumptive cloaca. Finally, we show that the T-box transcription factor HrT is an important downstream mediator of Bmp signaling in excretory system development. These results reveal a novel role for Bmp signaling in posterior organogenesis beyond early ventral mesoderm specification, and they implicate misregulation of this major developmental signaling pathway as a possible cause of anorectal malformations during human embryogenesis.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), except that pre-warmed 40°C media was used for
30-minute heat shocks in a 40°C air incubator. This produced robust
transgene expression (marked by GFP) within 30 minutes.
In situ hybridization
In situ hybridization was performed as previously described
(Griffin et al., 1998).
Coloration reactions were stopped simultaneously for transgenic embryos and
wild-type siblings, and, for these experiments, all images shown are
representative of 100% of embryos examined with each probe
(n
30).
Lineage labeling
kaede mRNA (100 pg) (Ando et
al., 2002) was injected into one-cell-stage embryos from a
transgenic outcross and heat shocked at the mid-gastrula stage (as described
above). Embryos were then immobilized in 1% low melting point agarose on
slides at the 10-somite stage and UV light at 405 nm was focused on the
ventral tail bud and underlying cells for 5 minutes for photoconversion. At 24
hpf, wild-type and transgenic embryos were sorted based on loss of the ventral
tail fin (100% penetrant in transgenic embryos), and visualization of red
labeling was performed using a compound fluorescent microscope. Labeling was
performed in a total of 25 embryos.
Excretion assay
Fluorescent rhodamine dextran (Molecular Probes) was injected into the
anterior gut of wild-type and transgenic embryos at 4 dpf, when the gut was
fully formed in wild-type embryos. Dye excretion was visualized and
photographed using a Zeiss Axioplan 2 compound fluorescent microscope.
Injections were performed in at least five larvae per genotype, in two
separate experiments.
Vital staining and confocal microscopy
Embryos were incubated for 1 hour in BODIPY TR methyl ester dye
(Cooper et al., 2005), then
filmed using a Bio-Rad MRC-600 confocal microscope for 4 dpf experiments. For
time-lapse microscopy of cloacal opening, a Zeiss LSM Pascal confocal
microscope was used. The time-lapse movie was made by taking a
z-series through an msxb-gfp transgenic embryo once every 10
minutes, then using one optical section for each timepoint.
msxb-gfp transgenic fish
Exon 1, intron 1 and 5.5 kb upstream of the 5'UTR (
7.5 kb total)
of msxb were PCR-amplified and cloned upstream of EGFP-SV40polyA.
Embryos were injected with 80 ng/µl of linearized plasmid, grown to
adulthood, and the progeny were screened for EGFP fluorescence. Embryonic EGFP
expression faithfully recapitulates endogenous msxb expression, with
the exception of the early neural crest domain
(Akimenko et al., 1995).
Acridine orange staining
Examination of cell death using acridine orange was performed as previously
described (Clements and Kimelman,
2005). Forty embryos per genotype were stained at 24-somites to
image dying cells in the developing cloaca.
hrT morpholino injections
hrT translation blocking or mismatch morpholino (2 ng)
(Szeto et al., 2002) was
injected into one-cell-stage embryos, and cloaca formation was observed at
24-48 hpf.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
However, in this earlier study, we did not address the requirement for Bmp
signaling to regulate the differentiation of ventral mesoderm after its
initial formation. To determine whether Bmp plays any role in regulating the
development of ventral mesodermal tissues beyond the early phase of
gastrulation, we heat shocked hs::dnBmpr-GFP embryos and wild-type
siblings at the mid-gastrula stage (80% epiboly, when gastrulation was just
over halfway completed). In the same experiment, we heat shocked embryos at
dome stage (about 2 hours before gastrulation) to compare the effects of
inhibiting early ventral mesoderm development. We next performed whole-mount
in situ hybridization to examine the expression of gata1 [red blood
cell precursors (Detrich et al.,
1995)], flk1 [vascular precursors
(Sumoy et al., 1997);
kdr–Zebrafish Information Network], and pax2.1 [kidney
precursors (Pfeffer et al.,
1998)] at the 10-somite stage. These genes were expressed in
adjacent stripes of cells within the lateral posterior mesoderm of wild-type
embryos heat shocked at either stage (Fig.
1A,D,G). By contrast, heat shock of transgenic embryos before the
start of gastrulation (dome stage) resulted in a loss of all three ventral
mesodermal markers (Fig.
1B,E,H), consistent with an early gastrula role for Bmp signaling
in specifying the ventral mesoderm (Pyati
et al., 2005). Surprisingly, reduction of Bmp signaling at the
mid-gastrula stage led to an expansion of gata1- and
flk1-positive cells into the mesoderm ventral to the tail bud
(compare regions marked with arrows in Fig.
1C,F with regions in Fig.
1A,D). This expansion of blood and vascular progenitors was also
confirmed by the expanded expression of the early blood and vascular marker
fli1 (Brown et al.,
2000; Thompson et al.,
1998) into the same domain ventral to the tail bud (data not
shown). pax2.1, which was normally expressed at low levels around the
tail bud (Fig. 1G), was also
upregulated in this domain in transgenic embryos
(Fig. 1I), but to a much lesser
extent than the blood and vascular markers. Thus, kidney cell fates are not
dramatically altered at the expense of blood and vascular gene expression.
Because transgenic embryos heat shocked at the mid-gastrula stage normally
displayed C1 dorsalization (loss of the ventral tail fin) without obvious cell
movement defects during epiboly, we conclude that reduction of Bmp signaling
during mid-gastrulation caused the extreme ventral mesoderm to undergo a fate
change to blood and vascular progenitors.
Defective development of the cloaca and ventral yolk extension in embryos with reduced Bmp signaling
The expansion of blood and vascular cells into the region below the tail
bud when Bmp signaling was inhibited suggested that zebrafish have a domain
that is more ventral than blood and vascular cells, and that the fate of these
cells is dependent on Bmp signaling. As the fate of these ventral cells was
unknown, we performed lineage labeling in wild-type and transgenic siblings
heat shocked at mid-gastrula using Kaede, a photoconvertible fluorescent
protein (Fig. 2A)
(Ando et al., 2002). Kaede can
be photoconverted from green to red fluorescence with UV light (400 nm),
allowing the selection of well-injected embryos for lineage labeling. We
injected one-cell-stage embryos from a clutch of outcrossed transgenic fish
with kaede RNA, and then heat shocked the embryos at 80% epiboly. At
10-somites, which is the stage when we observed ectopic gata1 and
flk1 expression in these cells
(Fig. 1), we photoconverted the
Kaede with a pinhole focusing UV light on the tissue below the tail bud. As a
consequence of the size of the pinhole used, we also labeled the ventral
portion of the tail bud. As expected, we observed red fluorescence in somitic
tissue, which is derived from ventral tail bud cells in both transgenic and
wild-type siblings (Fig. 2C,D).
Strikingly, cells in the ventral tail (not shown) and those lining the ventral
yolk extension were also labeled red because of the labeling of cells below
the tail bud. Transgenic embryos had a severe reduction in these tissues
compared with wild-type siblings (compare bracketed regions in
Fig. 2C,D). We also observed
labeled cells in the presumptive cloaca at the posterior end of the yolk
extension, and transgenic embryos consistently had cyst-like swellings in this
tissue (boxed region in Fig.
2D). This swelling, as well as the reduction in ventral tail
tissues, could be clearly visualized using Nomarski brightfield optics
(compare boxed regions in Fig.
2E,F). Thus, we conclude that the extreme ventral cells at the
10-somite stage contribute to ventral tail tissue, presumptive cloaca, and a
population of cells lining the ventral part of the yolk extension.
Additionally, we find that mid-gastrula Bmp signaling is crucial for the
formation of these tissues.
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).
Intriguingly, morpholino knockdown of hrT causes expansion of
gata1-expressing cells into the ventralmost mesoderm, similar to the
effect of reducing Bmp signaling during the mid-gastrula stages
(Szeto et al., 2002). Because
this result suggested a possible link between the loss of Bmp signaling and
the expansion of gata1 expression ventrally, we examined hrT
expression in transgenic embryos heat shocked at mid-gastrula. We observed a
complete loss of hrT transcripts in transgenic embryos compared with
wild-type siblings at both the 10-somite stage in the ventral mesoderm below
the tail bud, (compare Fig. 3A-C with
E-G) and at the 18-somite stage in ventral mesodermal cells lining
the yolk extension (compare Fig. 3D with
3H). The expression of hrT in the heart field, however,
was not affected (Fig. 3A,E).
These results show that mid-gastrula Bmp signaling is necessary for
maintaining the fate of the extreme ventral mesoderm as marked by hrT
expression.
|
). RNA
encoding the T-box transcription factor Tbx2b marks the excretory system and
ventral border of the yolk extension in zebrafish
(Dheen et al., 1999). After a
mid-gastrula stage heat shock, we observed a clear reduction of tbx2b
expression in cells ventral to the yolk extension and at the extreme end of
the developing excretory system (the proctodeum) in transgenic embryos
(compare Fig. 3I with 3M). This
result shows that Bmp signaling is important for normal patterning of
excretory system tissues and reveals that the regulation of tbx2b
expression in the excretory system is similar to its Bmp-dependent regulation
in the developing heart (Yamada et al.,
2000). We next examined the expression of hoxd13, which
is expressed in the developing proctodeum
(van der Hoeven et al., 1996).
hoxd13 expression was greatly reduced in the proctodeal region of
transgenic embryos when compared with wild-type siblings, even though the
posterior expression of hoxd13 expanded in the tail bud (compare
Fig. 3J with 3N). These results
show that a reduction of Bmp signaling at the mid-gastrula stage perturbs the
development of proctodeal and ventral yolk extension domains.
Because the terminus of the pronephric ducts ultimately connects to the
urogenital system and opens to the outside of the embryo
(Drummond et al., 1998), we
examined whether there were defects in the formation or positioning of the
kidney terminus in transgenic embryos compared with wild type siblings. As
shown by the expression of gata3
(Neave et al., 1995), the
pronephric terminus was specified normally, but it was not properly positioned
at the periphery of the embryo in transgenics compared with controls at the
20-somite stage (compare Fig. 3K with
3O).
Next, we examined the expression of vox, a ventral gene that is
known to be a direct target of Bmp signaling
(Imai et al., 2001;
Kawahara et al., 2000;
Melby et al., 2000).
vox is expressed in ventral tail tissues, including the ventral tail
fin and developing proctodeal region, in wild-type embryos. In accordance with
reports showing that vox becomes dependent on Bmp signaling by mid to
late gastrulation (Melby et al.,
2000; Ramel and Lekven,
2004), we observed a total absence of vox expression in
the ventral domain of transgenic embryos when compared with wild-type siblings
at the 20-somite stage (Fig.
3L,P).
Finally, we assayed for loss of tissue specification in the cloacal region
by analyzing the expression of two cloaca markers at 24 hpf, prdm1
(Wilm and Solnica-Krezel,
2005) and evx1
(Thaeron et al., 2000). The
cloaca expression of both markers is absent in transgenic embryos when
compared with wild-type siblings (Fig.
3Q-T), supporting the hypothesis that loss of Bmp signaling beyond
the early gastrula stages causes a loss of cloacal cell fate. Overall, these
results clearly show that mid-gastrula Bmp signaling is necessary for the
expression of genes in the distal excretory system and the proper positioning
of the kidney terminus.
Bmp signaling is required through early somitogenesis for cloaca specification
We wished to analyze how long during embryogenesis Bmp signaling was
required for cloaca specification. After a 40°C heat shock, the
dominant-negative Bmp receptor is present for 10-15 hours (as indicated by GFP
fluorescence; data not shown), meaning that transgenic embryos heat shocked at
mid-gastrula would still have the dominant-negative receptor present
throughout much of somitogenesis. Thus, it was important to determine a window
of time that Bmp signaling was required for cloaca specification. To address
this question, we heat shocked transgenic and wild-type sibling embryos at 80%
epiboly, bud, 3-, 5-, 8-, 10-, 12- and 14-somites, then fixed 20 embryos of
each genotype per stage of heat shock for analysis of hoxd13
expression and left the remainder to score cloaca phenotypes at 24 hpf. As
shown in Table 1, cloacal
defects can be induced in transgenic embryos strongly between 80% epiboly and
8-somites, then to a lesser extent at 10-somites. However, heat-shocks after
this stage produced no effects on cloaca development. Absence of
hoxd13 expression closely parallels the morphological scoring,
although, by 10-somites, there is no obvious reduction in the expression of
this gene. This is likely to reflect a more subtle loss of proctodeal cells in
transgenic embryos heat shocked at 10-somites. Taken together, these results
show that sustained Bmp signaling is essential from 80% epiboly through the
early somite stages for proctodeal specification and cloaca development.
|
)]. We
injected fluorescent rhodamine dextran into the anterior gut of wild-type and
transgenic sibling embryos that had been heat shocked at the mid-gastrula
stage. Upon injection, we could visualize peristalsis of the gut as dye was
forced posteriorly in both wild type and transgenic embryos (data not shown).
However, upon peristalsis of the dye into the hindgut, we observed severe
defects in transgenic larvae. Contrary to wild-type siblings, in which dye was
excreted through the cloaca (Fig.
4B,C), transgenic animals could not eliminate dextran from their
gut cavity, and they developed an obvious swelling of their hindgut as a
consequence (Fig. 4D,E). These
results are consistent with our molecular analysis and show that cloacal
opening is dependent on proper Bmp signaling during development.
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Next, we took advantage of the optical transparency of the zebrafish embryo
to further dissect the mechanism of cloacal opening. For these experiments, we
used a transgenic zebrafish line that labels cells of the ventral epidermis
with GFP under the control of the msxb promoter. Endogenous
msxb is normally strongly expressed in cells of the ventral epidermis
during the stages of cloaca opening
(Akimenko et al., 1995), making
this transgenic line very useful for our studies. As shown by detailed
time-lapse filming of a wild-type msxb-gfp embryo starting at 24
somites (Fig. 6C-F, see also
Movie 1 in the supplementary material), cloacal opening involves massive
cellular rearrangements within the proctodeum and kidney terminus. A single
vacuolated cell within the proctodeum migrates ventrally and forms a pore
within the epidermis (labeled in green by msxb-gfp), possibly by
undergoing cell death. The kidney terminus connects to this pore and undergoes
a morphogenetic transformation to adopt a columnar appearance. The newly
formed presumptive cloaca thus consists of at least two cell types: kidney
cells in the dorsal aspect and epidermal cells at the ventral terminus.
Finally, we wished to examine the architecture of the mature excretory
system in living animals. Although the kidney connects to the presumptive
cloaca during late somitogenesis, the gut tube does not connect to the
presumptive cloaca until 4 dpf. Using BODIPY TR methyl ester
(Cooper et al., 2005), we
labeled all the tissues of 4-day old larvae for confocal microscopic analysis.
This labeling allowed us to assay the entire kidney and excretory system for
defects that occurred in transgenic larvae, and it provided a unique glimpse
into the morphology of the excretory system during development. Wild-type
larvae showed a clear opening of the gut into the cloaca, next to the kidney
opening (Fig. 6G). Consistent
with the lack of cloacal function that we observed earlier, transgenic larvae
failed to form a cloacal opening, and the kidneys and gut failed to open to
the outside of the body, resulting in large swellings in both structures
(compare Fig. 6H). This result
shows that development of both the urogenital and gut openings are dependent
on sustained Bmp signaling, and that the failure of the presumptive cloaca to
develop normally leads to major malformations of the excretory tissues.
|
),
mirroring the results observed here with a loss of Bmp signaling at the
mid-gastrula stage. To test whether HrT might be important for development of
the presumptive cloaca, we injected either a morpholino oligonucleotide (MO)
that blocks hrT translation or a four-base mismatch MO and examined
the structure of the presumptive cloaca at 24-48 hpf. As shown in
Fig. 7A, hrT morphants
had dysmorphic cloaca development (55%, n=128) compared with mismatch
control-injected embryos (Fig.
7B; 8%, n=118). However, unlike embryos with mid-gastrula
reductions in Bmp signaling, hrT morphants had normal ventral tail
fin formation (data not shown). Furthermore, the expression of tbx2b
(n=43), hoxd13 (n=41) and gata3
(n=69) was normal in morphant embryos
(Fig. 7C-H), suggesting that
HrT is involved in only a subset of the roles of Bmp within the developing
excretory system. We conclude that, in addition to its role in heart
development, HrT has an additional important role as a Bmp-regulated gene that
is required to allow the excretory system to form normally. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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),
indicating that precise Bmp signaling levels may be important for regulating
cell death in the developing cloaca. Specification of the kidney terminus is
normal in transgenic embryos, as revealed by gata3 expression,
supporting the hypothesis that defective proctodeal development is the primary
cause of excretory system defects when sustained Bmp signaling is compromised.
Overall, we suggest that the misspecification of extreme ventral cells results
in an inability of the kidney ducts and gut tube to migrate to the ventral
surface and interact with the proctodeum and epidermis to form the cloaca.
HrT: an important mediator of Bmp signaling in the ventral mesoderm
One of the key mediators of sustained Bmp signaling is the T-box
transcription factor HrT. hrT is expressed specifically in the
ventral mesodermal cells that normally do not express blood and vascular
genes, and reduction of Bmp signaling at the mid-gastrula stage caused a loss
of hrT expression in this region. Importantly, we had previously
observed that inhibition of HrT function with MOs caused the expression of
gata1 in the most ventral mesoderm
(Szeto et al., 2002), exactly
as is observed when Bmp signaling is inhibited at the mid-gastrula to early
somitogenesis stages. We now show that inhibition of HrT function causes a
defect in cloacal development, similar to that observed with inhibition of Bmp
signaling. However, the defects caused by inhibiting HrT function are much
less severe than those observed when Bmp signaling is inhibited, with no loss
of ventral tail fin or reduction of cells under the yolk extension, and no
loss of ventral gene expression. These results indicate that hrT is
one of several Bmp-regulated genes involved in regulating the formation of the
cloaca.
The Bmp gradient model
Our data suggest a modification to the Bmp gradient model, which posits
that the highest levels of Bmp signaling at the ventral limit of the mesoderm
are required for formation of the blood and vascular cells
(Dosch et al., 1997). Our
results reveal an extreme ventral mesodermal domain that is normally devoid of
blood and vascular cells, and which requires continuous Bmp signaling at early
gastrulation and beyond to maintain its fate. These findings raise two
possible models for the specification of the extreme ventral mesoderm, and
both involve sustained Bmp signaling. In the first model, the extreme ventral
mesoderm requires both the highest and most sustained Bmp signaling to be
specified, thus representing the highest point in the Bmp gradient. In the
second model, all of the ventral mesodermal derivatives require the same level
of Bmp signaling at the early gastrula stage, but only the extreme ventral
mesoderm requires sustained Bmp signaling. In this scenario, the extreme
ventral mesodermal cells would still need to encounter the highest effective
levels of Bmp signaling, as they would integrate moderate levels of signaling
over an extended time. In both scenarios, the extreme ventral mesoderm
requires the most Bmp signaling of any tissue, but the first model fits more
closely with the classical idea of a spatial gradient, as Bmp levels would be
highest where the extreme ventral mesoderm would form. Future experiments
using low, moderate and high levels of Bmp inhibition at the early gastrula
stage should distinguish between these two models. In either scenario, our
studies show that only the extreme ventral mesoderm is dependent on Bmp
signaling from the mid-gastrula stage. Moreover, we find that the role of Bmp
signaling changes dramatically between the early gastrula and mid-gastrula
stages; while Bmp signaling is required during the earliest phase of
gastrulation for blood, vascular and kidney cell specification, after this
stage, Bmps limit the domain of expression of blood and vascular markers.
A model for the roles of Bmp signaling in cloaca development
Based on the results discussed above, we propose the following model for
Bmp signaling in development of the excretory system
(Fig. 8). Beyond the early
gastrula stage, sustained Bmp signaling is required to specify the most
ventral region of the embryo that subsequently forms the presumptive cloacal
region, ventral tail fin and ventral lining of the yolk extension. One of the
crucial targets of the sustained Bmp signaling is hrT, which acts to
suppress early blood gene expression in the ventral mesodermal cells, and is
necessary for the morphogenesis of the presumptive cloaca. The ventral cells
at the end of the yolk extension undergo a complex remodeling that allows the
proctodeum to interact with the kidney terminus, possibly by emitting a signal
that allows the kidney tubules to migrate to the most extreme ventral region
of the embryo. Our data suggest that in response to a loss of Bmp signaling,
the misspecified ventral cells fail to spread underneath the tail as the tail
bud extends, thus leading to severe deficiencies in ventral tissues, including
the proctodeum. As a result, the extension of the kidney ducts is stalled, and
they fail to connect to the proctodeum and open to the outside of the embryo.
A similar morphogenesis is repeated three days later, when the gut tube needs
to connect to the outside of the embryo. As these events can be readily
visualized in living embryos by confocal microscopy, the zebrafish provides an
excellent system for examining the cellular dynamics that underlie these
important biological processes.
|
),
which range from surgically simple defects such as an imperforate anal
membrane to far more difficult problems such as failure of the anus and/or
rectum to form properly, aberrant connections between the rectum and urinary
tracts, and malformations within the cloaca
(Durston et al., 1989;
Nixon, 1988;
Pena et al., 2004). Notably,
cloacal malformations, particularly the more severe type, typically involve
other malformations within the body, suggesting that these defects are not the
failure of a single morphogenetic event but instead involve a more widespread
alteration during early development (Smith
and Saeki, 1988). The most common malformations associated with
cloacal defects involve alterations in the vertebral column, although
alterations in the kidneys and urinary tract, genitals and lower limbs are
also observed (Duhamel, 1961;
Smith and Saeki, 1988). Based
on the observation that these defects often involve the more posterior parts
of the body, Duhamel (Duhamel,
1961) proposed that the cloacal defects were part of a spectrum of
caudal defects in which the most extreme form manifests itself as sirenomelia,
in which the legs fuse into a single structure.
Intriguingly, a recent study in the mouse has shown that a loss of
Bmp7 combined with a half dose or complete loss of twisted
gastrulation (Tsg) results in sirenomelia
(Zakin et al., 2005). Bmp
signaling defects in caudal mesoderm have been implicated by clinical
researchers as one possible cause of analogous human malformations
(Jain and Weaver, 2004).
Zebrafish develop a ventral tail fin instead of a true set of hindlimbs, and
loss of the ventral tail fin has been regarded as a readout for defects in
ventral/caudal mesoderm (Tucker and Slack,
2004). Thus, we hypothesize that a reduction of Bmp signaling
after the early gastrula stages results in a global loss of the caudal
mesoderm, affecting the ventral tail fin and cloaca in zebrafish, and the
lower limbs and cloaca in humans. Based on the studies reported here, we
suggest that very subtle alterations in Bmp signaling, due to either genetic
or environmental perturbations, may be one cause of human cloacal
malformations.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/133/11/2275/DC1
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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