Huntsman Cancer Institute, Center for Children, Department of Oncological Sciences, University of Utah, Salt Lake City, UT 84112, USA
Author for correspondence (e-mail:
joseph.yost{at}hci.utah.edu)
Accepted 23 December 2004
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SUMMARY |
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Key words: Left-right patterning, Cilia, Kupffer's vesicle, Dorsal forerunner cells, Left-right dynein, Organogenesis
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Introduction |
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A central question in developmental biology is what mechanism initiates
asymmetric gene expression and subsequent asymmetric organogenesis? In mice,
an elegant model has been proposed in which monocilia protruding from cells in
the late gastrula node direct an asymmetric flow of extracellular fluid that
results in the establishment of asymmetric gene expression
(Nonaka et al., 1998). This
`nodal flow' model is supported by the observations that LR defects are caused
by mutations in microtubule motor proteins that affect either ciliogenesis,
such as Kif3a (Marszalek et al.,
1999
; Takeda et al.,
1999
), Kif3b (Nonaka et al.,
1998
) and Polaris (Murcia et
al., 2000
), or cilia motility, such as Left-right dynein (Lrd),
which is encoded by the iv gene
(Supp et al., 1999
). The
strongest arguments for a role for nodal flow in the establishment of LR
asymmetries come from cultured mouse embryos in which externally applied
rightward fluid flow can reverse LR development in wild-type embryos and
externally applied leftward flow can rescue LR development in mutants that
would normally have inverted LR orientation
(Nonaka et al., 2002
).
The nodal flow model was initially proposed to move a peptide-signaling
factor, perhaps a member of the FGF, TGFß or Hedgehog signaling families.
More recently, nodal flow has been proposed to function by asymmetrically
activating non-motile mechanosensory cilia on the periphery of the node and
initiating an asymmetric Ca2+ flux
(McGrath et al., 2003)
(reviewed by Tabin and Vogan,
2003
; Yost, 2003
).
Nodal flow is the earliest known event in murine LR development. However,
genes implicated in ciliated node cell function are also expressed in non-node
cells, and mutations of these genes give rise to a variety of laterality
phenotypes and more pleiotropic phenotypes, making it impossible to exclude
other mechanisms for LR development during early embryogenesis (for reviews,
see Tabin and Vogan, 2003
;
Wagner and Yost, 2000
;
Yost, 2003
). Furthermore, it
is not known whether nodal flow is a murine-specific mechanism. Cilia
formation and expression of lrd homologues have recently been
observed in structures analogous to the node in chick, frog (Xenopus
laevis) and zebrafish embryos (Essner
et al., 2002
), but the existence of motile cilia and nodal flow
has not been demonstrated in non-murine embryos. Further confounding the
issue, there are molecular asymmetries that precede the appearance of
lrd expression and monocilia in Xenopus
(Kramer et al., 2002
;
Kramer and Yost, 2002
;
Levin et al., 2002
) and
perhaps in chick (Levin et al.,
1995
; Stern et al.,
1995
). This raises the issue of whether ciliated cells in other
vertebrate embryos generate fluid flow and have a conserved function for
ciliogenesis genes in LR development.
In zebrafish, ciliated cells arise in the tailbud at the end of
gastrulation (Essner et al.,
2002) in a transient spherical organ called Kupffer's vesicle
(KV). KV, first described in 1868
(Kupffer, 1868
), is a
conserved structure among teleost fishes. Electron microscopy studies in the
bait fish Fundulus heteroclitus have shown that a single cilium (i.e.
monocilium) protrudes from each cell lining KV into the lumen
(Brummett and Dumont, 1978
). In
zebrafish, KV is formed from a group of approximately two-dozen cells, known
as dorsal forerunner cells (DFCs), that migrate at the leading edge of the
embryonic shield (the zebrafish equivalent of the mouse node) during
gastrulation. In contrast to other cells in this region, DFCs do not involute
during gastrulation, but remain at the leading edge of epibolic movements. At
the end of gastrulation, DFCs migrate deep into the embryo and organize to
form KV (Cooper and D'Amico,
1996
; D'Amico and Cooper,
1997
; Melby et al.,
1996
). During subsequent somite stages, KV is found ventral to the
forming notochord in the tailbud and adjacent to the yolk cell. Although KV
was first described well over 100 years ago, it remains unknown whether DFCs
and KV are mesodermal or endodermal in origin, and it is unclear what role
they play during development, leading to the categorization of KV as an
embryonic `organ of ambiguity' (Warga and
Stainier, 2002
).
Recently, using a novel technique to knockdown gene expression specifically
in DFCs, we reported the first evidence that DFCs and/or KV function in LR
patterning (Amack and Yost,
2004). Here, we show that cilia that arise inside KV are motile
and generate a consistent counterclockwise fluid flow. A combination of laser
ablations, embryological manipulations, analyses of mutants and antisense
morpholinos against zebrafish left-right dynein-related1
(lrdr1) injected either into all embryonic cells or specifically
targeted to DFCs, demonstrates that ciliated KV cells control LR development
of the brain, heart and gut. The presence of motile cilia in zebrafish embryos
supports the idea that fluid flow is a conserved LR mechanism used in all
vertebrates. In addition, we propose a multi-step genetic pathway in which
both the T-box transcription factor no tail (ntl) and
components of the Nodal signaling pathway are necessary, either directly or
indirectly, for the expression of lrdr1 in DFCs and for the
morphogenesis of KV. Based on these analyses, we propose that KV is a
transient embryonic `organ of asymmetry' that regulates the earliest known
step in LR axis specification in zebrafish. These results comprise the first
evidence that ciliated cells function during LR development in a non-murine
embryo.
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Materials and methods |
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Laser ablations
DFCs were ablated from wild-type embryos between shield and 80% epiboly
stages using a VSL-337ND-S Tunable Nitrogen Laser (Laser Science) set to 440
nm. The laser was interfaced to a Leica DMRA compound microscope through a
fiber optic cable (Photonic Instruments). Heart laterality was scored in
DFC-ablated embryos (and all other embryos in this study) by scoring heart
looping as normal, reversed or midline in anesthetized embryos at 2 days
post-fertilization.
KV disruptions
Embryonic dissections were carried out on agarose pads using 29 gauge
needles on dechorionated embryos. Control embryos were also dechorionated at
the same time and kept under similar conditions. All dissections were carried
out stereotactically in that both the left and right sides were disrupted.
Embryos that showed any disruption of the yolk cell were discarded. Embryos
with normal ntl staining in the notochord were selected for LR marker
analysis.
Antisense depletion of Lrdr1
To isolate the 5' end of zebrafish lrdr1, also referred to
as dynein axonemal heavy chain 11 (Unigene # Dr20984), we performed
5' RACE (rapid amplification of cDNA ends). lrdr1 cDNA sequence
was compared with a genomic DNA contig in the Sanger Institute genome database
(www.ensembl.org)
that contained the 5' end of the zebrafish lrdr1 gene in order
to assign exon and intron boundries for the first seven exons. An updated
version of the zebrafish genome from Ensembl includes an annotated fragment of
lrdr1 containing the first 10 exons (Gene ID: ENSDARG00000004221).
Antisense morpholino oligonucleotides (MO) directed against the lrdr1
start codon (lrdr1-AUG MO:
5'-GCGGTTCCTGCTCCTCCATCGCGCC-3'), the lrdr1 exon 2/intron
2 splice site (lrdr1E2/I2 MO:
5'-ACTCCAAGCCCTCACCTCTTATCTA-3'), the start codon of
lefty1 (lft1 MO:
5'-GGCGCGGACTGAAGTCATCTTTTCA-3') and a standard negative control
MO (5'-CCTCTTACCTCAGTTACAATTTATA-3') were obtained from
Gene-Tools. To knock down gene expression in all cells, MO were injected
between the one- and four-cell stages as previously described
(Nasevicius and Ekker, 2000).
To deliver MO specifically to DFCs, fluorescein-labeled MO were injected into
the yolk cell between the 512-cell and 1000-cell stages, and embryos with
fluorescent DFCs were selected for analysis, as described
(Amack and Yost, 2004
). RT-PCR
was used to determine the effect of lrdr1E2/I2 MO on lrdr1
mRNA splicing. Total RNA was extracted from embryos using TRIzol Reagent
(Invitrogen) and cDNA was synthesized by MMLV-RT primed by random decamers
(Ambion Retroscript kit). lrdr1 cDNA was amplified by 35 cycles of
PCR using a forward primer (5'-ACATTCACGCCCTTCAAAAC-3') in exon 2
and a reverse primer (5'-ACGTCCTGGATCATTTTTGC-3') in exon 4.
Amplicons were confirmed by DNA sequencing.
In situ hybridization
Antisense RNA probes were transcribed in the presence of digoxigenin-11-UTP
or fluorescein-12-UTP (BMB) from linearized DNA templates using the Maxiscript
kit (Ambion). Free nucleotides were removed using Bio-Gel P-6 Micro Bio Spin
columns (BioRad). cDNA templates used included ntl
(Schulte-Merker et al., 1994),
pitx2 (Essner et al.,
2000
), shh (Krauss et
al., 1993
), lft1 and lft2
(Bisgrove et al., 1999
).
Different probes and combinations of probes were used for lrdr1: the
previously reported lrdr1 RT-PCR product
(Essner et al., 2002
), a
genomic HindIII to HindIII fragment subcloned from a PAC
corresponding to a region 5' to the RT-PCR product, and a 5' RACE
PCR product. All lrdr1 probes produced similar results individually.
In situ hybridizations were performed as previously described
(Essner et al., 2000
), and in
some cases were automated using a Biolane HTI in situ machine (Huller and
Huttner AG). Embryos were cleared in 70% glycerol in PBST and photographed
with a Leica MZ12 stereoscope using a Dage-MTI DC330 CCD camera.
Immunohistochemistry
Embryos were fixed overnight in 4% paraformaldehyde at 4°C and
dehydrated stepwise into methanol for storage at -20°C. After stepwise
re-hydration, embryos were blocked for 1 hour in 5% goat serum (Sigma), 2%
bovine serum albumin (BSA, Sigma) and 1% DMSO in PBST. Embryos were incubated
with an anti-acetylated tubulin antibody (1:400, Sigma) and anti-Ntl antibody
(1:100, a gift from D. J. Grunwald)
(Schulte-Merker et al., 1992)
in blocking solution. After washing embryos in 2% BSA and 1% DMSO in PBST,
embryos were incubated with FITC-labeled goat anti-mouse IgG2b antibodies
(1:500, Southern Biotech) and Alexa 568-labeled goat anti-rabbit antibodies
(1:500, Molecular Probes). After washing, embryos were cleared in Slow Fade
(Molecular Probes) and the tail region was removed and mounted. Embryos were
imaged using an Olympus Fluoview scanning laser confocal microscope with a
60x objective.
Videomicroscopy of cilia
Live embryos were mounted in 2% methylcellulose in a depression slide and
cilia were imaged using a Zeiss Axioskop microscope with a 63x DIC
objective. Movies were captured with a Photometrics Coolsnap HQ digital camera
and Metamorph imaging software.
Fluorescent bead injections
Embryos were removed from their chorions and mounted in 1% low melt agarose
in system water on coverslips. Fluorescent beads (0.5-2 µm) (Polysciences)
were pressure injected into KV at the six-somite stage. Embryos were imaged on
a Zeiss Axioskop 2 microscope with a 10x Plan Apo objective. Movies were
taken with a Nikon Coolpix 995 digital camera and manipulated using QuickTime
Pro and Photoshop 7.
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Results |
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The progeny of DFCs in KV assemble cilia similar to those observed at the
mouse node (Essner et al.,
2002). To examine the developmental timing of cilia formation in
DFC/KV, we used anti-acetylated Tubulin antibodies to label cilia at different
stages during gastrulation and somitogenesis. To aid in the identification of
DFCs and KV in whole embryos, anti-Ntl antibodies were used to label nuclei in
the notochord, mesoderm and DFCs during gastrulation and in the notochord,
tailbud and KV during somitogenesis. Cilia were not observed in the DFC domain
during gastrulation (data not shown) but were detected at 4 SS in Ntl-positive
cells under the notochord, in the region of KV
(Fig. 1H). These cilia
initially appeared as short projections into the lumen of the forming KV.
Cilia in the well-formed KV were elongated at 10 SS
(Fig. 1I) and remained
elongated at 15 SS (data not shown). At 18 SS, the lumen of the KV was smaller
or absent, although some of the dispersed KV cells remained ciliated
(Fig. 1J). Confocal analyses of
embryos at 6-8 SS (n=5) revealed a 1:1 ratio between the number of
cilia visualized with the anti-acetylated tubulin antibody and the number of
nuclei staining positive for Ntl in KV, suggesting KV cilia are monocilia.
To determine whether KV cilia are motile, we used videomicroscopy to
examine KV in living embryos. At 6-10 SS, cilia were motile in the lumen of KV
(see Movie 1 in the supplementary material). To test whether these cilia are
capable of generating fluid flow inside KV, fluorescent beads were injected
into KV at the 6 SS. These beads revealed a consistent directional
counterclockwise flow inside KV (see Movie 2 in the supplementary material).
Flow persisted until 10-12 SS, when asymmetric expression of the Nodal-related
gene southpaw begins in left lateral plate mesoderm cells at the
anterioposterior level of KV (Long et al.,
2003). These observations suggested that cilia in KV might
function in a manner analogous to cilia in the mouse node during LR
development.
Ablation of DFCs alters LR development without other effects on embryogenesis
Murine genes implicated in nodal flow are also expressed in non-ciliated
cells, and mutations in these genes, which result in a diverse array of
laterality phenotypes and pleiotropic defects in other tissues, do not provide
direct evidence that ciliated cells have a role in LR patterning. To directly
test whether ciliated cells are required for LR patterning, DFCs were ablated
from wild-type zebrafish embryos during gastrulation using a nitrogen laser.
To verify that the ablation technique was efficient, embryos were fixed
following ablation at 60% epiboly, and stained for ntl expression
(Fig. 2B) or lrdr1
expression (data not shown). With this laser-ablation technique, each cell is
individually ablated at a specified focal plane, so it is unlikely that
underlying yolk nuclei or adjacent mesoderm cells are affected. Ablated
embryos lacked ntl (n=8/8) and lrdr1
(n=7/8) gene expression in the DFCs, but had wild-type expression of
ntl in adjacent mesoderm cells
(Fig. 2B). Control embryos
displayed wild-type ntl expression in both the mesoderm and DFCs
(n=5/5, Fig. 2A).
These controls indicate that laser ablation provides an efficient approach to
eliminate DFCs during gastrulation without altering gene expression in
neighboring cells. To examine the effects of DFC ablation on KV formation,
DFC-ablated and unablated control embryos were immunostained at 6 SS with
anti-Ntl and anti-acetylated Tubulin antibodies
(Fig. 2C,D). Although two out
of six DFC-ablated embryos had a ciliated KV that appeared similar to control
embryos (Fig. 2C), two out of
six of the DFC-ablated embryos formed a dismorphic KV that was misshapen (data
not shown) and two out of six of the embryos did not form KV
(Fig. 2D). These results
indicate that ablation can result in a range of effects on KV morphogenesis,
which probably reflects the efficacy of DFC ablation.
|
In contrast to normal anterioposterior and dorsoventral development, ablation of DFCs at 60-80% epiboly had significant effects on brain and heart LR development as assessed by the molecular markers lefty1 (lft1) and lefty2 (lft2). lft1 and lft2 are normally expressed in the left dorsal diencephalon and left heart field, respectively (Fig. 2G,I). In the diencephalon, DFC-ablated embryos showed equal numbers of embryos with right-sided (35%, Fig. 2H) or absent lft1 expression (35%) and a few with bilateral or left-sided expression (Fig. 2K). Unablated controls showed wild-type expression of lft1 (89%, Fig. 2G,K). In the heart field, the majority (53%) of DFC-ablated embryos had inverted expression of lft2 (Fig. 2J,L). Taken together, our analyses revealed that 82% of DFC-ablated embryos had laterality defects (Fig. 2M), whereas only 11% of unablated control embryos had a laterality marker defect (n=71, data not shown). When heart loop orientation was scored, DFC ablation at 60-70% epiboly resulted in 24% reversed hearts (n=46) and DFC ablation at 70-80% epiboly resulted in 29% reversed hearts (n=14), with control, non-ablated siblings having 2-3% heart reversals (n=274).
Kupffer's vesicle is an embryonic organ of asymmetry
The results from laser ablations of DFCs indicate that DFCs or KV cells are
essential for normal LR patterning, but do not define the developmental stages
at which this patterning occurs. To directly test the role of KV in the
generation of laterality, KV was disrupted by microsurgery at distinct stages
during somitogenesis. The efficacy of KV disruption was analyzed by
immunostaining with anti-Ntl and Tubulin antibodies. In control embryos, cilia
and KV cells were organized in a normal spherical pattern
(Fig. 3C), whereas in KV
disrupted embryos this morphology was severely perturbed
(Fig. 3D,E, n=13/17).
In embryos in which KV was disrupted between the 3-7 SS, notochord (assessed
by ntl expression) and tail development was normal, but expression of
the left-side markers pitx2 in the dorsal diencephalon and lateral
plate and lft2 in heart primordia was altered
(Fig. 3G,H,J). By contrast,
when the KV disruptions were carried out at 13-15 SS, asymmetric expression of
pitx2 and lft2 was normal
(Fig. 3K). Consistent with the
asymmetric gene expression data, heart laterality was reversed in 35% of
embryos in which KV was disrupted at 3-7 SS (n=23), while KV
disruptions at 10-15 SS resulted in a low rate of heart laterality
disturbances (4%, n=45). Although these results do not exclude the
possibility that DFCs play an earlier and separate role in LR development
(i.e. during gastrulation) before giving rise to KV, they indicate that KV is
a necessary organ for LR patterning at 3-7 SS but is dispensable after 10
SS.
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Controls for DFCMO targeting experiments
Two controls were designed to test whether LR defects caused by injecting
lrdr1 MO at mid-blastula stages were specific to lrdr1
knockdown in DFCs and their progeny. First, we used MO against lft1,
which is expressed in adjacent shield cells but not in DFCs during
gastrulation (Fig. 1B-D).
Consistent with a previous report (Feldman
et al., 2002), 1.5 ng lft1 MO co-injected with 2 ng of
fluorescein-tagged negative control MO at the one- to four-cell stages
affected the midline and resulted in bilateral expression of lft1 in
the brain (n=27/28) and lft2 in the heart field
(n=28/28). Injecting lft1 MO + control MO into the yolk at
mid-blastula stages resulted in predominantly normal lft1 and
lft2 asymmetric expression (Table
1). This indicates that MO injected into the yolk at mid-blastula
stages do not affect non-DFC cells (e.g. neighboring midline cells) and do not
have non-specific effects on LR development. Second, to test whether
lrdr1 MO must enter DFCs to affect LR development, we co-injected 1
ng of lrdr1-AUG MO and 1.6-2.5 ng of lrdr1E2/I2 MO into the
yolk at late blastula stages (dome stage to 30% epiboly), when connections
between the yolk and DFCs are thought to be closed
(Cooper and D'Amico, 1996
). In
these embryos, MO diffused throughout the yolk but did not accumulate in DFCs,
did not affect splicing of lrdr1 RNA, which is expressed only in DFCs
at 90% epiboly (Fig. 4J, lane
7), and did not disrupt asymmetric expression of lft1 or
lft2 (Table 1).
Although this control does not exclude the possibility that yolk syncitial
layer (YSL) nuclei underlying DFCs
(D'Amico and Cooper, 2001
;
Kimmel and Law, 1985
) might
have a role in LR development, it indicates that if lrdr1 is
expressed in yolk cells, it does not have a detectable role in LR development.
Together, these results indicate that lrdr1 expression in DFCs, which
proceed to make ciliated cells in the KV, is essential for normal LR
development of the viscera and brain.
lrdr1 is required for fluid flow in KV
Loss of Lrd in mutant mice renders nodal cilia immotile
(Supp et al., 1999). To
determine if MO knockdown of zebrafish lrdr1 affected cilia motility
and fluid flow in KV, fluorescent beads were injected into KV of
lrdr1-AUG MO injected embryos. KV appeared normal in these embryos,
but flow of fluorescent beads was reduced or absent
(Table 2 and see Movie 3 in the
supplementary material). Embryos injected with negative control MO showed the
characteristic counterclockwise flow seen in wild-type embryos
(Table 2). Importantly,
lrdr1-AUG MO injected embryos from these experiments that were not
injected with fluorescent beads showed a high rate (24%, n=58) of
heart laterality reversal, whereas all embryos injected with negative control
MO (n=61) had normal heart laterality. This indicates that, as in
mouse, lrdr1 is required for cilia motility and provides evidence
that fluid flow inside KV is required for zebrafish LR determination.
|
Zebrafish laterality mutants have been classified based on the disruption
of the normally left-sided markers lft1, lft2 and pitx2
(Bisgrove et al., 2000;
Bisgrove et al., 2003
). In the
present study, we examined the following laterality mutants: class I mutants
ntl and floating head (flh) have defects in
notochord formation and bilateral expression of asymmetry markers; class II
mutants spadetail (spt) and casanova (cas)
have randomized and discordant expression of laterality markers; class III
mutant cyclops (cyc) lacks asymmetric expression of
lft1 in the diencephalon but has normal expression of other asymmetry
markers in the heart and gut. Class IV mutants one-eyed pinhead
(oep) and schmalspur (sur) fail to asymmetrically
express lft1, lft2 or pitx2 in the diencephalon, heart and
gut, probably owing to a direct disruption of the Nodal signaling pathway in
responding cells (Gritsman et al.,
1999
; Pogoda et al.,
2000
).
DFCs were observed by DIC microscopy from 60% to 80% epiboly, and usually
comprised 15 to 27 cells in wild-type embryos. DFCs have strong endocytic
activity from the dome stage to 70% epiboly that can be assessed by staining
with the fluorescent vital dye SYTO-11
(Cooper and D'Amico, 1996).
Each of the laterality mutants, with the exception of cas, had DFCs
that were endocytically active and had normal ntl expression
(Table 3). In our study,
cas mutants had DFCs, but they were fewer (average 10.5
cells/embryo), flatter, more disperse and did not take up SYTO-11. This
somewhat concurs with a previous report
(Alexander et al., 1999
) that
cas mutants lack DFCs, and indicates that cas, a Sox-related
transcription factor, is required for DFC formation and endocytic activity.
With the exception of cas, the formation of DFCs, their organization
and their endocytic activity during gastrulation do not appear to be
correlated with later laterality defects in the mutants examined.
|
|
Last, we screened laterality mutants for KV organogenesis and ciliogenesis within KV. ntl mutants (identified by a lack of Ntl immunostaining) formed patches of cells with anti-Tubulin labeled cilia, but these embryos failed to develop a normal KV (Fig. 5G; Table 3). In spt mutants, lrdr1 expression was normal and tubulin immunostaining detected cilia, but KV was disorganized (Fig. 5H and Table 3). cas and oep mutants showed an absence of both cilia and KV (Fig. 5I,J; Table 3). Notably, ntl, spt, oep and cas mutants all lead to the same endpoint of defective KV morphogenesis, but block distinct steps leading to a functional KV and affect downstream LR gene expression in dramatically different manners.
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Discussion |
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Here, using zebrafish embryos, we provide the first evidence that ciliated cells function during LR development in a non-murine embryo. Laser ablation of DFCs, which perturbs KV morphogenesis, and surgical disruption of KV directly show that DFC/KV cells play an essential role in zebrafish LR patterning. We also show that cilia in KV are motile and generate a directional fluid flow that is severely impaired by MO knockdown of lrdr1, which consequently alters LR development. Finally, analysis of LR mutations in zebrafish has revealed novel roles for both the ntl and Nodal pathways in the regulation, either directly or indirectly, of lrdr1 expression for the regulation of LR development.
Using a recently developed technique that targets MO to DFC/KV cells
(Amack and Yost, 2004), we have
found that lrdr1 expression in DFC/KV cells is required for normal LR
development. MO injected into the yolk cell at mid-blastula stages often enter
DFCs, and not other cells, probably via cytoplasmic bridges first
characterized by Cooper and D'Amico
(1996
) by loading fluorescent
dextran into the yolk between the 1000-cell to oblong stages. Dextran
accumulated in at least some DFCs in
60% of these embryos and was also
often found in some non-DFC marginal cells, indicating that both DFCs and
other margin cells are potentially in cytoplasmic confluence with the yolk. We
have observed similar results injecting fluorescent-tagged MO into the yolk
cell at mid-blastula stages, including a high degree of embryo-to-embryo
variability. Fluorescence microscopy was used to select embryos in which MO
have loaded into DFCs but not other embryonic cells. It is possible that small
amounts of MO, below detection by fluorescent microscopy, accumulate in cells
other than DFCs. To test this, we targeted DFCs with MO against another gene
that is required for normal LR development, lft1, which is not
expressed DFCs, but in cells neighboring DFCs
(Fig. 1B-D). Eighty-two percent
of DFClft1MO embryos showed normal asymmetric
lft2 expression (Table
1), indicating MO injected into the yolk at mid-blastula stages do
not affect non-DFC cells. In a separate control for DFCMO
experiments, embryos injected with lrdr1 MO into the underlying yolk
syncitial layer (YSL) cells at stages when bridges between the YSL and DFCs
have closed (dome stage to 30% epiboly) have normal LR development. Although
this does not exclude a possible role for the YSL in LR development, it
indicates that lrdr1 (this study) and ntl
(Amack and Yost, 2004
) do not
function in YSL during LR patterning. Taken together, the results from three
approaches - DFC ablation, KV surgical disruption and lrdr1 knockdown
in DFC/KV cells - demonstrate that the ciliated KV is an embryonic organ of
asymmetry that is required for the earliest known step in the generation of LR
asymmetry in zebrafish.
Multiple pathways for Kupffer's Vesicle development and establishment of LR asymmetry
Our analysis of KV formation and function in zebrafish mutants provides a
template for reconsidering distinct phenotypes seen in mouse and human
laterality syndromes (Bisgrove et al.,
2003). Based on our results, we propose five distinct steps in KV
that precede the earliest known asymmetric gene expression patterns in
zebrafish (Fig. 6). In the
first step, DFCs are induced during the blastula period. As previously
reported (Alexander et al.,
1999
) and shown here, cas mutant embryos, which are
deficient for a novel Sox-related gene and lack all endoderm, also lack normal
DFCs. The few DFCs present in cas mutants are misshapen and defective
in: endocytosis; lrdr1, ntl and sqt expression;
ciliogenesis; and formation of KV. As cas was originally isolated as
a cDNA responsive to Nodal signaling
(Dickmeis et al., 2001
) and
two Nodal-related ligands, sqt and cyc, are expressed in
close proximity to DFCs (Rebagliati et
al., 1998
), the Nodal pathway is strongly implicated in the
induction of the DFCs. However, as cyctf219 mutants form
DFCs that are SYTO-11 positive and express lrdr1, it is likely that
other members of the Nodal family, perhaps maternally stored, are important
for the formation of DFCs.
The second step in the KV pathway is the activation of lrdr1
expression in DFCs at the end of gastrulation. DFCs in mutants that lack
oep, a Nodal signaling co-receptor, or mzsur, a
foxa1 transcription factor in the Nodal response pathway, fail to
express lrdr1. In addition to Nodal signaling, ntl mutant
embryos fail to express lrdr1 in the DFCs, suggesting that Ntl
transcription factor is upstream of lrdr1. As ntl mutants
have a normal number of DFCs that stain with SYTO-11, express ntl
mutant RNA and sqt RNA, and form cilia
(Table 3), it is unlikely that
the absence of lrdr1 expression in ntl mutants is due to
non-specific changes in DFC determination. In some cases, ntl can be
induced by the Activin/Nodal pathway
(Bisgrove et al., 1999;
Toyama et al., 1995
); one
could propose that Nodal signaling is upstream of ntl, which is then
upstream of lrdr1. However, Ntl protein is expressed in oep
mutants that lack lrdr1 expression, although there might be a subtle
and undetectable decrease in Ntl in oep mutants. In addition, DFCs in
ntl and oep mutants differ in sqt expression and
cilia formation. Together, this suggests that the ntl-dependent
induction of lrdr1 is a pathway parallel to the Nodal pathway.
Obviously, mutant analysis indicates that lrdr1 expression is
dependent on ntl and components of the Nodal pathway, but does not
distinguish whether this dependence is direct or indirect. Clearly, both of
these genetic pathways have other functions in KV development beyond
regulation of lrdr1 expression
(Fig. 6), as lrdr1
morphants do not display defects in KV organogenesis and cilia formation seen
in ntl and oep mutants. Conversely, although knockdown of
lrdr1 disrupts directional fluid flow in the KV, one cannot exclude
other roles for lrdr1 within DFC/KV cells for LR development.
In the third step, DFCs organize to form KV at the completion of
gastrulation. This process involves ingression into the forming tailbud and
formation of an epithelial sheet at the base of the notochord. KV formation is
dependent on Nodal signaling components (exemplified by oep) and Ntl,
as well as on additional genes that are not required for lrdr1
expression, including the T-box transcription factor Spt. Using the
DFCMO technique, KV organogenesis was found to be cell-autonomously
dependent on ntl (Amack and Yost,
2004). It is not yet known whether defects in KV development
described here (Table 3) are
cell autonomous for spt and components of the Nodal pathway.
In the fourth step, cilia form on KV cells and project into the lumen.
Surprisingly, cilia formation can be uncoupled from the normally coincident
formation of KV in step 3, as spt and ntl mutants do not
form an organized KV but form cilia (Fig.
5G,H; Table 3). In
agreement with observations in Dnahc11/Dnahc11 mice
(Supp et al., 1999), cilia
formation in zebrafish does not appear to be dependent on lrdr1, as
cilia are found in ntl mutants that lack normal lrdr1
expression (Fig. 5G) and in
lrdr1 morphant embryos (Fig.
4H). In the fifth step, cilia motility and fluid flow in KV is
dependent on lrdr1, which determines normal asymmetric gene
expression patterns in lateral tissues.
This mutant analysis points out that perturbation of one or combinations of
the five steps in KV function outlined above results in altered LR development
in the brain, heart and gut. Of the characteristics we examined, defects in KV
organogenesis alone (e.g. spt), KV organogenesis plus lrdr1
expression (e.g. ntl), KV ciliogenesis, organogenesis and
lrd1 expression (e.g. oep), or all of the above plus DFC
formation (e.g. cas) results in LR abnormalities. Interestingly, each
of the mutants has distinct expression patterns of downstream laterality
markers (Bisgrove et al.,
2000). Although it might be tempting to speculate that defects in
the distinct KV steps outlined above result in distinct laterality phenotypes
in the rest of the embryo, it is important to note that the genes involved are
expressed in other cells besides DFC/KV cells, and such a conclusion can not
be made until the gene of interest is knocked down specifically in DFC/KV
cells. For example, DFCMO knockdown of ntl has a
laterality phenotype that is distinct from bilateral expression patterns seen
in ntl mutants and whole-embryo ntl morphants, indicating
that ntl has distinct roles in DFC/KV and in midline cells for LR
development (Amack and Yost,
2004
). Our surgical disruptions of KV indicate that KV is no
longer needed for LR development by 10 SS, but do not exclude the possibility
that different LR signals come from the DFCs or the KV at earlier stages in
DFC/KV development.
How does KV fluid flow control lateral asymmetric gene expression?
Several models have been proposed to explain how cilia movement and fluid
flow influences LR development, and how this information is transmitted to
lateral plate mesoderm to initiate asymmetric expression of Nodal, Lefty and
Pitx2 (for review (Mercola,
2003; Raya and Belmonte,
2004
)). Flow has been proposed to influence the localization of a
signaling molecule (Okada et al.,
1999
) or asymmetric activation of mechanosensory cilia and
intracellular Ca2+ (McGrath et
al., 2003
) in mice. In chick, nodal cilia are present
(Essner et al., 2002
).
Although motility and nodal flow have not been reported, extracellular
Ca2+ has been proposed to activate lateral asymmetric nodal
expression via a Notch-dependent pathway
(Raya et al., 2004
). Here, we
show in zebrafish that lrdr1-dependent asymmetric fluid flow in KV
probably results in asymmetric gene expression that sweeps from the tail to
the head of the developing embryo. However, the mechanisms by which
counterclockwise flow in KV is translated to molecular asymmetries near KV are
not known.
In zebrafish, charon, an inhibitor of nodal signaling, is
expressed in the tailbud at 2-3 SS during the formation of KV and then
symmetrically adjacent to KV until the 14 SS
(Hashimoto et al., 2004).
Southpaw (spaw), a member of the nodal family, is bilaterally
expressed immediately adjacent to charon
(Hashimoto et al., 2004
;
Long et al., 2003
). Morpholino
knockdown of charon results in bilateral expression of spaw
and other asymmetric markers in lateral plate mesoderm (cyc, lefty
and pitx2). Conversely, knockdown of spaw results in absence
of downstream asymmetric markers. It is not known how the symmetric expression
patterns of charon and spaw control subsequent asymmetric
expression of spaw and other downstream genes. However, the bilateral
spaw and charon expression near KV during the period that we
have shown KV is important in LR development suggests that information derived
from the lrdr1-dependent asymmetric fluid flow might influence the
balance or interactions of spaw and its antagonist charon in
adjacent cells.
There is elegance in the frugality of using the same signaling pathway at
multiple stages in LR development. The T-box transcription factor Ntl
independently drives lrdr1 expression and KV morphogenesis
(Fig. 6) and midline formation
in mesoderm cells, both of which are necessary for distinct aspects of normal
asymmetric gene expression in lateral plate
(Amack and Yost, 2004).
Similarly, the Nodal signaling pathway is required for the induction of
lrdr1 gene expression in DFCs in order to drive motile cilia in
Kupffer's vesicle, and ciliated KV is necessary for driving normal LR
asymmetric expression of components of the Nodal signaling pathway in lateral
mesoderm. This novel aspect of the Nodal pathway, upstream of lrdr1
function, suggests that analyses that have implicated the Nodal pathway in LR
development in mice, chick, frog and zebrafish should be re-investigated for
proximal defects in the formation and function of ciliated cells.
Conservation of a ciliary-based mechanism for LR development
Several lines of evidence presented here indicate that KV cilia in
zebrafish are required in LR development and are analogous to cilia located on
the ventral mouse node. However, it is not known whether the generation of a
flow in KV is the first symmetry breaking event in zebrafish. Certainly, the
appearance of motile cilia in the KV, and the developmental period during
which DFC/KV manipulations can alter LR development, precedes the appearance
of asymmetric gene expression of spaw, cyc, lft1 and lft2 in
lateral tissues. As such, it is the earliest known event in zebrafish LR
development. However, recent evidence from frog embryos suggests that
asymmetries are generated long before the appearance of ciliated cells
(Kramer et al., 2002;
Kramer and Yost, 2002
;
Levin et al., 2002
). It is not
yet known whether these mechanisms are amphibian specific, and future work
will be focused on investigating potential connections between early molecular
asymmetries and nodal flow. In chick, lrdr expression and cilia are
found at stage HH4 (Essner et al.,
2002
), but more detailed expression analysis, cilia motility and
fluid flow have not been reported, making it difficult to know whether early
molecular asymmetries (Levin et al.,
1995
; Stern et al.,
1995
) precede a role for cilia. The results presented here
demonstrate the importance of cilia in Kupffer's vesicle, a transient
embryonic organ of asymmetry, for the establishment of left-right asymmetry in
the gut, heart and brain in zebrafish. These data provide the first direct
functional evidence of a ciliary-based mechanism operating in a non-murine
vertebrate embryo during LR patterning.
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Supplementary material |
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: Discovery Genomics, Minneapolis, MN 55413, USA
Present address: University of Wisconsin, Madison, WI 53706, USA
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