Department of Anatomy and Cell Biology, Roy J. and Lucille A. Carver
College of Medicine, University of Iowa, Iowa City, IA 52242, USA
* Present address: Neuroscience Graduate Group, University of California, Davis,
CA 95616, USA
Author for correspondence (e-mail:
michael-rebagliati{at}uiowa.edu)
Accepted 12 February 2003
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SUMMARY |
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Key words: Zebrafish, southpaw, Nodal-related, Left-right, Asymmetry
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INTRODUCTION |
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The lack of congruence between the cyclops and oep/sur
phenotypes raises the issue as to whether the role of asymmetric nodal
signaling in LR patterning is conserved in zebrafish vis à vis other
vertebrates. One possibility is that nodal signaling is not needed in the left
lateral plate in zebrafish, and that the oep and sur
mutations disrupt nodal-dependent signaling from or within the dorsal
organizer (reviewed by Burdine and Schier,
2000). Alternatively, the discordance between the cyclops
and oep/sur phenotypes could be reconciled if another nodal-related
gene besides cyclops were expressed asymmetrically in the lateral
plate. The action of this protein, rather than Cyclops, would then provide the
requisite level of nodal signaling on the left side to establish visceral
organ LR asymmetry in the zebrafish. Although mutations in the other zebrafish
nodal-related gene, squint (sqt; ndr1
Zebrafish Information Network) do disrupt LR asymmetry, sqt itself
shows no LR asymmetry of expression and it is never expressed within the
lateral plate (Feldman et al.,
1998
; Karlen and Rebagliati,
2001
; Rebagliati et al.,
1998a
). Thus, if Nodal signaling were required within the left
lateral plate, this function would have to reflect the action of a new
nodal-related gene.
To evaluate these possibilities, we have undertaken a degenerate RT-PCR screen for additional zebrafish nodal-related genes. We have identified a new zebrafish nodal-related gene, which we have named the southpaw locus (spaw). As implied, spaw shows strong left-sided expression within the trunk, as well as bilateral expression within segmental plate (paraxial) mesoderm. Asymmetric expression commences at the 10-12 somite stage, making spaw the earliest known molecular marker of LR asymmetry for the zebrafish. We show through loss-of-function experiments with morpholinos that spaw is crucially required for the establishment of visceral LR asymmetry, both at the organ level and at the molecular level. Despite the fact that spaw is not expressed within the head, we find that spaw is also required for the establishment of the earliest aspects of diencephalic LR asymmetry. The anterior limit of asymmetric spaw expression lies just rostral to the developing heart. Consequently, our results raise the intriguing possibility that diencephalic LR asymmetry may arise through an Spaw-dependent relay that emanates from the anterior part of the left lateral plate.
In summary, our studies provide strong support for the hypothesis that the role of Nodal signaling in vertebrate LR asymmetry is conserved in the zebrafish. Moreover, these experiments show that the pathways establishing visceral organ and diencephalic LR asymmetry require the same asymmetric cue, the Nodal protein Spaw. Finally, these observations suggest a specific model for how neural and visceral LR asymmetry are coordinated during vertebrate embryogenesis.
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MATERIALS AND METHODS |
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Construction of spaw plasmids
We made an Spaw expression construct, pCS2+ActHASpaw, in which the
mature region of Spaw is fused to the complete prepro region of Xenopus
Activin ßA. An HA epitope tag is also present between the proteolytic
cleavage site and the N terminus of the mature region. pCS2+ActHASpaw
was constructed by amplifying the mature region of spaw with primers
(HASPAWF1) and (HASPAWR1). After XhoI/XbaI digestion, the
spaw fragment was ligated directionally into
XhoI/XbaI cut pCS2+preproAct. The
XhoI/XbaI pCS2+preproAct backbone was prepared by
digestion of pCS2+AXnr1 (Piccolo
et al., 1999) to remove the xnr-1 mature region and gel
isolation of the resulting vector/preproactivin/HAtag fragment.
pSpaw(MATR) was derived from pSpaw-1.4 by digesting
with NcoI and AfeI to delete the mature region and then
blunting and religating the vector+preproSpaw backbone.
pSpaw(MATR) was used to generate an spaw
prepro-region-specific probe.
DNA oligonucleotides
Morpholino oligonucleotides
Lower case letters: substitutions to reduce MO secondary structure.
The specificity and efficacy of Cyclops-MO1 has been validated previously
(Karlen and Rebagliati, 2001).
Spaw-MO1 is complementary to a region spanning the putative translation start
site (Fig. 1A). For comparison,
corresponding sequences of the cyclops, sqt and spaw mRNAs
are 5'-CATCATGCACGCGCTCGGAGTCGCG-3' (initiation codon in
bold), 5'-TGACATGTTTTCCTGCGGGCTCCTG-3' and
5'-CGCAATGCAGCCGGTCATAGCGTGC-3', respectively.
|
The mismatches between Spaw-MO1/2 and the cyclops and sqt mRNAs/pre-mRNAs are sufficient to provide specificity. The specificity of Spaw-MO1 and Spaw-MO2 was confirmed by the absence of cyclops and sqt phenotypes, the absence of dorsal midline defects, and by rescue (see text).
Embryo injections
For production of native Spaw prepro protein, pSpaw-1.4 was
linearized with NcoI and transcribed with SP6 polymerase. For
production of ActHASpaw, pCS2+ActHASpaw was linearized with
NotI and transcribed with SP6. RNA and DNA injections into two- to
16-cell embryos were carried out as described
(Rebagliati et al., 1998a).
Morpholinos were diluted into 1xDanieau's and injected at the one- to
eight-cell stage into the yolk, just under the blastoderm. Typically, 5-10 ng
of a morpholino was injected per embryo in a volume of 2-3 nl.
Whole-mount in situ hybridization analysis and
immunohistochemistry
Embryos were fixed in 4% paraformaldehyde. Single probe in situ was carried
out using a digoxigenin-labeled antisense RNA probe
(Tsang et al., 2000) or with a
dinitrophenol-labeled antisense RNA probe as described
(Long and Rebagliati, 2002
).
Two-color (BM Purple and INT RED)/two-probe in situ hybridization was carried
out using Dig- and Fl-labeled probes
(Liang et al., 2000
) or, for
less abundant transcripts, using Dig- and DNP-labeled probes
(Long and Rebagliati, 2002
).
pSpaw-1.4 was used to make the spaw probe. We used a
full-length pitx2c cDNA to make a pitx2 probe that
hybridizes to all the pitx2 mRNA isoforms
(Essner et al., 2000
).
Immunohistochemistry with the myosin MF20 antibody was carried out described
(Rebagliati et al., 1998a
) but
using cobalt-enhanced DAB.
Mutant analysis
The following alleles were used: sqtcz35,
bozm168, cycb16,
ntlb160 and flhn1.
bozm168 homozygotes were identified by the presence of
severe notochord defects (corresponding to classes III-V of the boz
phenotypic series) (Fekany et al.,
1999).
Scoring organ LR asymmetry
Cardiac jogging was assayed visually or by whole-mount in situ
hybridization with an nkx2.5 probe. Heart looping was assayed
visually or by staining with the myosin heavy chain antibody MF20. Pancreatic
LR asymmetry was determined at 48-72 hours post-fertilization using a
zebrafish preproinsulin probe
(Milewski et al., 1998).
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RESULTS |
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One can draw an additional distinction by looking at the spacing of the
seven conserved cysteines within the mature ligand domain of the Spaw
polypeptide. Nodal-related proteins fall into two subclasses based on two
distinct patterns of cysteine residues
(Jones et al., 1995). One
subclass includes the mouse nodal and zebrafish Cyclops proteins; the other
includes the Xenopus Nodal-related 1 (Xnr1) and zebrafish Sqt
proteins. The Spaw protein contains the split-cysteine arrangement that is
characteristic of the Xnr1 subtype (Fig.
1A). Overall, Spaw shares more amino acid identity with Xenopus
Nodal-related 1 than with zebrafish Cyclops
(Fig. 1B); this is also
consistent with its classification within the Xnr1 subgroup.
Developmental expression profile of spaw
We determined the developmental profile of spaw expression using
whole-mount RNA in situ hybridization. No spaw expression is detected
prior to somitogenesis. The earliest detectable spaw expression
consists of two bilateral domains flanking the tailbud; these domains first
appear at the four- to six-somite stage and persist into later stages of
somitogenesis (Fig. 2A, inset
in Fig. 2M). By 24 hours
post-fertilization, expression in these areas is strongly downregulated but
trace amounts of spaw expression are still visible (not shown). To
determine within which anlagen these bilateral domains lie, we performed
double probe in situ hybridization with a spaw probe and a
spadetail (spt) or no tail (ntl) probe.
spaw transcripts overlap with spt expression within adaxial
cells and presomitic mesoderm but are excluded from the ntl-positive
notochord (Fig. 2B-D). There
may be overlap between spt, ntl and spaw in the area where
presomitic mesoderm merges into the tailbud. These results indicate that, at
least during early somitogenesis, the bilateral spaw domains lie
within the regions containing paraxial mesoderm precursors
(Griffin et al., 1998;
Schulte-Merker et al.,
1994
).
|
As the protein One-eyed Pinhead (Oep) is required for signaling by
Nodal-related proteins, oep and spaw expression would be
expected to overlap or to be adjacent
(Gritsman et al., 1999). In
situ hybridization with oep and spaw probes show that, from
the 10-to 17-somite stages, oep and spaw expression coincide
temporally and either overlap in the lateral plate or are in close proximity
(Fig. 2F,G). However, unlike
oep, spaw is not expressed in the head
(Fig. 2H,I).
Two-color in situ hybridization was carried out to compare the expression
domains of spaw with those of lefty2 and pitx2: two
asymmetrically expressed genes that are known to be Nodal dependent in other
organisms. To improve sensitivity, we have modified the standard protocol by
replacing the fluorescein label with a different hapten: dinitrophenol
(Long and Rebagliati, 2002).
The observed in situ patterns are consistent with the interpretation that
left-sided spaw precedes (Fig.
3A) and then transiently overlaps the left-sided lefty2
and pitx2 expression domains in the trunk
(Fig. 3B-E for lefty2;
pitx2 data not shown). The anteroposterior (AP) boundary of the
left-sided spaw domain terminates just rostral to that of
lefty2 (Fig. 3D);
lefty2 expression marks the heart field at these stages. Our results
echo previous studies comparing cyclops, lefty1/2 and pitx2;
these studies also found overlapping and exclusive domains for these four
genes within the heart field (Bisgrove et
al., 2000
). pitx2, cyclops and lefty1 also
exhibit overlapping left-sided asymmetry within the epiphyseal region of the
diencephalon. However, spaw is not expressed in the diencephalon
(Fig. 2H; Fig. 3F; data not shown).
|
In terms of LR asymmetry, the main effect of the ntl and
floating head (flh) mutations is to cause bilateral
expression of normally asymmetric markers (lefty2, pitx2) within the
diencephalon and lateral plate [however, note that the primary effect of
flh on lefty2 in the LPM was reported by one group to be
loss of lefty2 expression (Chin et
al., 2000)]. Consistent with these reports, we observed mainly
bilateral expression of spaw in the lateral plate of
ntlb160 or flhn1 homozygotes
(Table 1,
Fig. 4A,B). The
bozm168 and cycb16/hhex alleles were
reported to abolish LPM expression of lefty1, lefty2 and
pitx2 (Chin et al.,
2000
). By contrast, we observed that the primary effect of
bozm168 and cycb16/hhex was
to cause bilateral expression of spaw within the LPM
(Table 1). We do not know the
reason for this difference, but the known variability of the boz
phenotype could be a contributing factor
(Fekany et al., 1999
).
sqtcz35 causes bilateral expression of pitx2,
lefty2 and cyclops in the diencephalon, but effects on visceral
asymmetry have not been reported (Liang et
al., 2000
). We find that the predominant effect of this mutation
is bilateral expression of spaw within the lateral plate
(Table 1,
Fig. 4D). Right-sided
(reversed) expression of spaw was observed only at a low frequency,
most often in bozm168 and cycb16/hhex
homozygotes (Table 1,
Fig. 4E). Overall, our results
indicate that correct left-sided regulation of spaw also depends on
the normal development of the dorsal midline.
|
|
Functional analysis of the spaw locus: overexpression
studies
In order to determine the inductive properties of Spaw, we constructed a
chimeric expression vector (pCS2+ActHASpaw) in which an HA-epitope
tagged version of the Spaw mature domain is fused to the prepro region of
Xenopus Activin ßA. Endogenous spaw transcripts can be
distinguished from the chimeric transcripts by using a probe specific for the
spaw prepro-region sequences. Injection experiments with
ActHASpaw mRNA or pCS2+ActHASpaw DNA show that the mature
Spaw protein has similar inductive properties to Sqt. Like Sqt, Spaw can
induce ectopic goosecoid and cyclops expression in shield
stage embryos, i.e. Spaw has dorsalizing activity
(Fig. 4G,H). We also tested a
synthetic spaw mRNA transcript encoding the native Spaw precursor
protein. This showed a qualitatively similar, albeit much weaker dorsalizing
activity (data not shown). The lower activity may reflect the absence of some
native 5' and 3' UTR sequences but other explanations have not
been ruled out. Overexpression of Spaw also disrupts LR asymmetry, as shown by
randomization of cardiac LR asymmetry and induction of bilateral expression of
endogenous spaw transcripts (data not shown). However, dorsalizing
mutations are known to disrupt LR asymmetry
(Chen et al., 1997). Thus, the
effects of Spaw overexpression on LR asymmetry probably reflect the fact that
early zygotic mis-expression of Spaw dorsalizes the embryo.
Functional analysis of the spaw locus: morpholino-mediated
inhibition of Spaw activity
To block Spaw activity, we designed two spaw antisense morpholino
oligonucleotides (Fig. 1A).
Spaw-MO1 is complementary to sequences near the putative start codon of the
spaw open reading frame. Spaw-MO2 is complementary to the splice
acceptor site for the last exon. The last two exons encode the mature ligand
domain of the Spaw polypeptide. The use of two morpholinos targeted to
different spaw sequences provides a stringent control for
specificity. In addition, we also compared the effects of the spaw
morpholinos to the effects of a cyclops morpholino (Cyclops-MO1)
and/or a standard negative control morpholino (NC-MO) that is not
complementary to spaw sequences.
Given that disruptions of the dorsal midline can perturb LR asymmetry, it was important to verify that Spaw-MOs do not disrupt the normal development of dorsal structures. By both molecular and morphological criteria, dorsal midline development proceeded normally in Spaw-MO1-injected embryos (Fig. 5A-D; see Fig. 7B). There was no cyclopia (Fig. 5D) or floor plate defect (see Fig. 7B), which suggests that prechordal mesoderm and ventral CNS development are normal. These observations also indicate that the spaw morpholino did not inhibit the other two nodal-related genes, cyclops and sqt, as such ventral CNS defects are a hallmark of the cyclops and sqt phenotypes. The somites and the notochord were also grossly normal, comparing Spaw-MO1 and uninjected embryos (Fig. 5A-D). Likewise, Spaw-MO2-injected embryos did not exhibit any cyclopia or axial mesodermal defects, again showing that neither cyclops/sqt function nor midline development was affected significantly (data not shown). To verify these conclusions using molecular criteria, we examined the expression of five dorsal midline markers (goosecoid, sqt, ntl, netrin and lefty1) at various times during gastrulation (60-70% epiboly) or somitogenesis (1-3 somites, 6-7 somites, 17-18 somites). In all cases, gene expression was similar between uninjected and Spaw-MO1-injected embryos (Fig. 5E, Fig. 6, Fig. 7B,C; data not shown). There were no obvious changes in Spaw-MO2-injected embryos, either; this was checked using two midline markers that are sensitive to reductions in Sqt or Cyclops activity [goosecoid (shield stage) and netrin (16-20 somites); data not shown]. As these experiments suggested that LR phenotypes would be interpretable in Spaw-deficient embryos, we proceeded to analyze the effect of these morpholinos on LR asymmetry.
|
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In Spaw-MO-injected embryos, the LR asymmetry of cardiac jogging was severely disrupted. There was a large increase in the frequency of embryos with either right (reversed) or no jogs, with the latter being the more prevalent effect (Table 2A; Fig. 5G,H). These effects were seen with Spaw-MO1 and Spaw-MO2 but were not seen with doses of a Cyclops-MO that are sufficient to phenocopy the cyclops mutation or with comparable doses of an unrelated morpholino (Table 2A).
|
Left-right asymmetry of the pancreas requires spaw
It was of interest to know whether spaw function is also required
for the asymmetry of other visceral organs. In zebrafish, the embryonic
pancreas is normally positioned to the right of the dorsal midline and this is
first evident at 36 hours after fertilization. Defective LR patterning
causes the pancreas to be mispositioned to the left-side or to remain in a
midline position (Yan et al.,
1999
). The displacement of the endocrine pancreas can be
visualized using an in situ probe for the preproinsulin gene
(Fig. 5K,L)
(Milewski et al., 1998
).
Inhibition of spaw led to an increase in the frequency of
misplacement of the pancreas, either to a reversed (left-sided) position or,
more frequently, to a midline placement
(Table 2C). These results are
in agreement with earlier observations that a partial loss of zygotic
oep activity disrupts pancreatic LR asymmetry
(Yan et al., 1999
).
Regulation of asymmetrically expressed genes by spaw: trunk
mesoderm
As spaw is required for visceral organ LR asymmetry, one would
expect asymmetric gene expression within the left lateral plate mesoderm to be
regulated by spaw. To test this prediction, we examined the
expression of four such left-sided genes, cyclops, lefty1, lefty2 and
pitx2, as well as expression of spaw itself within the
lateral plate. The first four genes initiate asymmetric expression after the
onset of left-sided spaw expression and therefore are candidates for
downstream, Spaw-dependent genes. Asymmetric spaw expression was
blocked by Spaw-MO1, but the bilateral spaw domains near the tailbud
were unaffected (Fig. 7A-C). The effect on spaw expression itself is not unexpected, as studies in
other animals have demonstrated the existence of an autoregulatory loop for a
subset of the expression domains of several nodal-related genes
(reviewed by Hamada et al.,
2002; Wright,
2001
). Asymmetric mesodermal expression of cyclops, lefty1,
lefty2 and pitx2 was lost in 80-100% of Spaw-MO1-injected
embryos (Table 3;
Fig. 7E-G). lefty1
expression in the posterior notochord was also consistently lost or reduced
(Fig. 7H) (Spaw dependence of
lefty1 in anterior and middle parts of the notochord could not be
determined because of the variability of wild-type lefty1 expression
in these regions). A similar dependence of midline (floorplate)
lefty1 expression on late Nodal signaling has been reported
previously for some mouse nodal alleles
(Lowe et al., 2001
;
Norris et al., 2002
). None of
these effects was seen when a control MO was injected
(Table 3;
Fig. 7A,E-H). Spaw-MO2 had
similar effects on lefty1 and spaw expression as seen with
the translation-inhibiting morpholino Spaw-MO1
(Table 3). As mentioned before,
expression of midline markers was not affected at either early or late stages
(Fig. 5E,
Fig. 6,
Fig. 7B-D). Likewise, the
spaw morpholinos did not perturb bilateral domains of gene
expression, as evidenced by normal bilateral expression of oep within
the lateral plate mesoderm (Fig.
7D), bilateral expression of oep and flh in the
dorsal diencephalon (Fig. 7L-O)
and the bilateral expression of pitx2 in the putative Rohon-Beard
cells, the ventral CNS and the hatching gland
(Fig. 7E,P-S; data not
shown).
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DISCUSSION |
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Analyses of the mouse nodal and Xenopus xnr1 promoters
have revealed that stable nodal gene expression often requires a
positive autoregulatory loop. This loop is mediated by cisacting elements
containing Foxh1/Fast-binding sites
(Norris and Robertson, 1999;
Osada et al., 2000
;
Saijoh et al., 2000
). The
phenotype of zebrafish foxh1/sur mutations implies a similar
mechanism for fish, as sqt and cyclops expression is
initiated but not maintained in sur/
homozygotes (Pogoda et al.,
2000
; Sirotkin et al.,
2000b
). Consistent with the existence of a positive feedback loop,
we find that stable expression of spaw transcripts within the left
lateral plate requires Spaw activity.
Regulation of Nodal signaling in Xenopus and mouse embryos also
involves negative feedback, whereby nodal ligands induce the synthesis of
their own antagonists, the Lefty1 and Lefty2 proteins
(Cheng et al., 2000;
Lowe et al., 2001
).
lefty1 and lefty2 are also downregulated in zebrafish
embryos with reduced levels of Oep, a co-factor for Nodal signaling
(Yan et al., 1999
). Our
experiments provide additional evidence for a negative feedback loop in the
zebrafish. Morpholino-mediated knockdown of spaw essentially
abolishes lefty2 and lefty1 expression in the LPM and
downregulates lefty1 expression in the posterior part of the
notochord. In summary, many of the salient features of the
nodal-lefty-pitx2 `cassette' are conserved in the zebrafish in the
context of spaw expression, regulation and function.
Implications for the lateral transfer of LR asymmetry cues from the
organizer
The pattern of spaw expression in normal and mutant embryos
provides insight into the mechanism by which LR asymmetry cues are relayed
from the early organizer (shield) to the lateral plate. Asymmetric
spaw expression initiates near the tailbud before spreading
anteriorly; this occurs well after the completion of gastrulation. This
observation suggests that the transfer of laterality cues from the early
organizer to the lateral plate is mediated by a cell population within or near
the tailbud, whose lineage can be traced back to the early organizer. One
group of cells that fulfills this criterion is the dorsal forerunner cells.
These cells originate in the part of the dorsal organizer that does not
involute during gastrulation. The descendants of the forerunner cells
ultimately come to reside near the tailbud in a structure known as Kuppfer's
vesicle (KV). The cells lining KV are monociliated
(Essner et al., 2002), similar
to cells within the mouse node that have been implicated in LR patterning of
the mouse LPM (reviewed by Hamada et al.,
2002
) (Hamada et al.,
2002
; Nonaka et al.,
2002
; Supp et al.,
1997
). The fact that mutations in the boz, sqt, ntl and
flh genes all cause bilateral expression of spaw is at least
consistent with a role for the forerunners cells in regulating LR asymmetry.
sqt, ntl and flh expression overlap within the forerunner
cells, and boz mutations lead to a downregulation of sqt
within the forerunner domain (Feldman et
al., 1998
; Melby et al.,
1997
; Rebagliati et al.,
1998a
; Schulte-Merker et al.,
1994
; Shimizu et al.,
2000
; Sirotkin et al.,
2000a
). Likewise, others have suggested a role for the forerunner
cells/KV in LR patterning based both on forerunner expression of the zebrafish
ortholog of left-right dynein, an axonemal dynein required for visceral LR
asymmetry in the mouse (Supp et al.,
1997
) and on the aforementioned KV expression of monocilia
(Essner et al., 2002
); both
their embryological and genetic data indicate that dorsal forerunner cells and
KV are essential for normal LR development (H. J. Yost, personal
communication). However, the forerunners are not the only candidates for the
transfer of laterality cues from the dorsal organizer to the tailbud. The YSL
nuclei that underlie the early organizer will also shift in a manner that
parallels the movement of the forerunner cells. In principle, these YSL nuclei
could also relay additional LR cues from the early organizer to the
tailbud.
Coordination of visceral and diencephalic LR asymmetry
The Swiss embryologist Von Woellarth made some of the earliest observations
of linkage between neural and visceral LR asymmetry. He noted an association
between reversal of organ situs and reversal of diencephalic LR asymmetry in
amphibians in the wild (Von Woellwarth,
1950). Consistent with such a linkage, others have shown that
Nodal signaling is required for both visceral and diencephalic LR patterning
in the zebrafish. oep mutants cannot transduce Nodal signals and fail
to express left-side-specific asymmetries within both the head and trunk
(Concha et al., 2000
;
Gamse et al., 2002
;
Liang et al., 2000
). Our
experiments identify the Spaw protein as a key Nodal signal that is needed for
both diencephalic and visceral organ LR asymmetry in the zebrafish.
How are visceral organ and diencephalic LR asymmetry coordinated during
embryogenesis? Our data are consistent with the hypothesis that Spaw signaling
from the anterior left lateral plate mesoderm induces diencephalic LR
asymmetry, either directly through long-range diffusion or through a signaling
relay (Fig. 8). The ultimate
effect would then be either activation of a left-sided program or,
equivalently, the antagonism of a repressor
(Concha et al., 2000), in
either case leading to left-sided diencephalic expression of cyclops,
pitx2 and lefty1. Although this model provides a simple
explanation of how visceral and diencephalic LR asymmetry can be coordinated,
other models cannot be ruled out. For example, because the asymmetric
spaw pattern is highly dynamic, we cannot yet exclude the possibility
of a more anterior, transient domain of spaw expression that cues
diencephalic LR asymmetry.
|
Our studies in the zebrafish and those in the chicken suggest that Nodal signaling from the anterior lateral plate may have a critical role in organizing LR asymmetry in the head. One goal of future experiments will be to test directly the hypothesis that spaw expression in the anterior lateral plate triggers diencephalic asymmetry.
Note added in proof
Gamse et al. have shown that the left-right placement of the parapineal
organ, first evident at 28 hours, subsequently organizes later aspects of
diencephalic LR asymmetry in the zebrafish
(Gamse et al., 2003). The
left-right displacement of the parapineal from the midline may depend on the
earlier asymmetric expression of cyclops, pitx2 and lefty1
within the epiphyseal region.
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ACKNOWLEDGMENTS |
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