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doi: 10.1242/10.1242/dev.00400
1 Department of Developmental Biology, Stanford University School of Medicine,
Stanford, CA 94305, USA
2 Department of Biology, University of Rochester, Rochester, NY 14627, USA
3 Developmental Genetics Program, Skirball Institute of Biomolecular Medicine,
Department of Cell Biology, New York University School of Medicine, New York,
NY 10016, USA
* Present address: Department of Cellular Biology, University of Georgia, 724
Biological Sciences Building, Athens, GA 30602, USA
Author for correspondence (e-mail:
talbot{at}cmgm.stanford.edu)
Accepted 9 January 2003
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Nodal signals, Zebrafish, Spemann organizer, Gastrulation, Dorsoventral axis, Mesoderm
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Warga and
Nüsslein-Volhard, 1999). Cells slightly farther from the
margin involute at later stages and exclusively become mesodermal cell types.
In more animal regions, cells adopt ectodermal fates and do not involute. Each
germ layer is patterned along the dorsoventral axis, generating the diverse
array of cell types characteristic of the vertebrate body plan. The initial
animal-vegetal and dorsoventral asymmetries are established by maternal
transcription factors, which regulate zygotic genes controlling cell fate
specification and morphogenesis (Harland
and Gerhart, 1997; Heasman,
1997; Moon and Kimelman,
1998; Schier and Talbot,
1998; De Robertis et al.,
2000; Kimelman and Griffin,
2000; Schier,
2001). Although zygotic factors that can induce and pattern
mesoderm have been identified, significant questions remain unanswered about
how the mesoderm is patterned.
nodal-related genes encode zygotically acting TGFß family
proteins that are necessary for induction of mesoderm and endoderm
(Conlon et al., 1994;
Jones et al., 1995;
Joseph and Melton, 1997;
Schier and Shen, 2000;
Takahashi et al., 2000;
Schier and Talbot, 2001;
Whitman, 2001). Mouse
nodal mutant embryos lack a primitive streak and fail to form
mesodermal derivatives (Conlon et al.,
1994; Varlet et al.,
1997). In zebrafish, there are two known nodal-related
genes, squint (sqt; ndr1 — Zebrafish
Information Network) and cyclops (cyc; ndr2 —
Zebrafish Information Network) (Erter et
al., 1998; Feldman et al.,
1998; Rebagliati et al.,
1998a; Rebagliati et al.,
1998b; Sampath et al.,
1998). Whereas defects in sqt and cyc single
mutants are largely confined to dorsal axial structures
(Hatta et al., 1991;
Thisse et al., 1994;
Heisenberg and Nüsslein-Volhard,
1997; Feldman et al.,
1998; Warga and
Nüsslein-Volhard, 1999), almost all mesendodermal derivatives
are absent in sqt; cyc double mutants, including notochord, trunk
somites, pronephros, heart, blood and gut
(Feldman et al., 1998). Thus,
these nodal-related genes have both overlapping and essential
functions in mesendoderm development. Cell tracing experiments in sqt;
cyc double mutants and in maternal-zygotic one-eyed pinhead
(oep) (MZoep) mutants, which are completely unresponsive to
Nodal signals (Gritsman et al.,
1999), indicate that Nodal signals allocate marginal cells to
mesendodermal fates (Feldman et al.,
2000; Carmany-Rampey and
Schier, 2001).
Although it is now firmly established that Nodal signals induce
mesendoderm, a possible role for Nodal signals in specifying different
mesendodermal fates along the dorsoventral axis is still controversial. In
Xenopus, three classes of models have been proposed for the role of
activin-like ligands, a group that includes Activin and Nodal signals, in
mesoderm patterning (Harland and Gerhart,
1997; Heasman,
1997; McDowell and Gurdon,
1999; De Robertis et al.,
2000; Kimelman and Griffin,
2000). One group of models proposes that a gradient of
activin-like signals patterns the mesoderm along the dorsoventral axis. This
view is supported by explant experiments in which low doses of activin-like
signals induced ventrolateral mesodermal fates and high levels induced dorsal
fates (Smith et al., 1988; Ruiz i Altaba
and Melton, 1989; Green et
al., 1992; Agius et al.,
2000). Further support for graded action of nodal-related
genes comes from analysis of the distribution of phosphorylated Smad2, a
proposed transcriptional effector of Nodal signals that is initially elevated
in the dorsal marginal region in Xenopus blastulae
(Faure et al., 2000). In
addition, Nodal inhibitors block mesoderm formation in different dorsoventral
positions in a dosage-dependent manner when overexpressed in Xenopus
embryos, consistent with asymmetric action of endogenous Nodal signals
(Agius et al., 2000).
The second class of models proposes that activin-like signals act uniformly
along the dorsoventral axis to induce mesoderm in the marginal region, while
independent signals generate dorsoventral pattern
(Christian et al., 1992;
Kimelman et al., 1992;
Clements et al., 1999). This
view is supported by experiments with a synthetic activin/TGF-ß
responsive reporter, which found that the transcriptional output from these
signals is uniform along the dorsoventral axis at the beginning of
gastrulation (Watabe et al.,
1995). Furthermore, promoter analysis indicates that input from
both Wnt and activin-like signaling pathways is required for proper expression
of dorsal-specific genes, such as goosecoid and siamois
(Watabe et al., 1995;
Crease et al., 1998).
Recent work (Lee et al.,
2001) supports a third model emphasizing the dynamic action of
activin-like signals. The spatiotemporal distribution of phosphorylated Smad2
suggests that these signals are active predominantly in dorsal regions in the
late blastula, and that the activity shifts to ventral regions as gastrulation
progresses. In addition, activin-like ligands acting in dorsal regions elicit
responses at earlier stages than ventrally acting ligands
(Lee et al., 2001;
Schohl and Fagotto, 2002).
These results suggest that dorsoventral patterning involves distinct temporal
responses to activin-like signals in dorsal and ventral cells.
Drawing parallels from the work on activin-like signals in
Xenopus, analysis of Nodal pathway mutants has suggested at least two
possible models of Nodal function in zebrafish mesendoderm patterning. The
first model proposes graded action of Nodal signals along the dorsoventral
axis, with high levels inducing dorsal mesendoderm and low levels inducing
ventrolateral mesendoderm (Fig.
1A). In support of this view, dorsal axial structures are reduced
in mutants with impaired Nodal signaling, such as sqt, cyc and
schmalspur (sur) single mutants
(Hatta et al., 1991;
Heisenberg and Nüsslein-Volhard,
1997; Feldman et al.,
1998; Pogoda et al.,
2000; Sirotkin et al.,
2000a). The expression patterns of sqt and cyc
are consistent with an elevated dorsal requirement for nodal-related
gene function. Soon after the onset of zygotic transcription, sqt is
expressed specifically in the dorsal marginal region, where dorsal mesendoderm
originates, and cyc is strongly expressed in the axial mesendoderm
during gastrulation (Erter et al.,
1998; Feldman et al.,
1998; Rebagliati et al.,
1998a). Moreover, dorsal mesoderm is most sensitive to
overexpression of lefty1 (also known as activin), an inhibitor of activin-like
signals (Bisgrove et al., 1999;
Thisse and Thisse, 1999;
Meno et al., 1999;
Thisse et al., 2000). Finally,
overexpression of high levels of sqt or cyc induces
presumptive ectodermal cells to initiate dorsal mesendodermal gene expression,
while lower levels induce pan-mesodermal but not dorsal mesodermal markers
(Erter et al., 1998;
Sampath et al., 1998;
Chen and Schier, 2001). Models
in which Nodal activity gradients pattern the dorsoventral axis predict a
transformation of cell fates along the dorsoventral axis as Nodal dose is
lowered (one scenario is depicted in Fig.
1A, right), but this prediction has not yet been tested by
fate-mapping experiments.
|
; Shimizu et al.,
2000; Sirotkin et al.,
2000b). The sqt and cyc genes are expressed
uniformly along the dorsoventral margin at the late blastula stage
(Erter et al., 1998;
Feldman et al., 1998;
Rebagliati et al., 1998a;
Sampath et al., 1998),
consistent with an equivalent requirement in dorsal and ventrolateral regions.
Additional evidence derives from the analysis of zygotic oep
(Zoep) mutants (Schier et al.,
1997), in which the response to Nodal signals is impaired but not
eliminated (Gritsman et al.,
1999). Zoep mutants lack endodermal derivatives from both
the dorsal and ventral margin. Fate mapping of the dorsal marginal region in
Zoep mutants indicates that the marginal-most cells (prechordal plate
precursors) are transformed to a slightly more animal fate (notochord
precursors), suggesting that different levels of Nodal signaling distinguish
these cell fates (Gritsman et al.,
2000). Similarly, low levels of exogenous antivin block endoderm
but not mesoderm formation (Thisse and
Thisse, 1999; Thisse et al.,
2000). If Nodal signals are required uniformly around the margin,
reductions in Nodal levels should shift all marginal derivatives toward more
animal fates (Fig. 1B, right
panels) — a prediction that has not been tested by fate mapping cells in
ventrolateral regions. To test these models and to explore possible stage-specific requirements for nodal-related genes, we have assessed mesendodermal patterning and fate specification in sqt-/-; cyc+/+ and sqt-/-; cyc+/- embryos, in which the action of endogenous Nodal signals is reduced but not eliminated. Our results indicate that Nodal signals act in the marginal region to pattern the animal-vegetal axis and that ventrolateral mesendodermal fates can be induced by a lower level of nodal-related gene function than dorsal mesendoderm. Furthermore, we find that differential regulation of the cyclops gene in dorsal and ventrolateral cells contributes to the different requirements for nodal-related gene function in these cells. In addition, our analysis shows that dorsal mesendodermal precursors are competent to respond to Nodal signals over a surprisingly long period, ranging from late blastula through at least early gastrula stages.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Sampath et al., 1998). Embryos
were collected from natural crosses and staged as described
(Kimmel et al., 1995). Embryos
shown in Fig. 4D and
Fig. 2A-O were derived from
crosses of sqtcz35/+; cycm294/+
x sqtcz35/+; cycm294/+ parents.
After photography, DNA for genotype analysis was extracted from living embryos
as described by Gates et al. (Gates et
al., 1999), or from fixed and stained embryos as described by
Sirotkin et al. (Sirotkin et al.,
2000b). For analysis of gsc, embryos with defective
gsc expression at each stage were counted and a representative sample
was photographed; after photography, DNA was extracted for genotyping as
previously described. All other mutant embryos, except the sqt
mutants shown in Fig. 9G-J and
Fig. 3E,J were derived from
repeated crosses of a small group of sqtcz35/+;
cycm294/+ x sqtcz35/+ parents,
in which the majority of sqtcz35/sqtcz35;
cycm294/+ progeny consistently had a truncated notochord
and fused somites, as shown in Fig.
2P-U. For quantitation of foxa2/axial expression at 8
hours post-fertilization (h), stained embryos were photographed as wholemounts
in canada balsam: methyl salicylate (11:1), rehydrated in PBT (1x PBS
and 0.1% Tween-20), and equilibrated in 80% glycerol. The embryos were then
dissected along the ventral midline, photographed under low magnification, and
DNA was extracted to determine the genotype by PCR.
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;
Sampath et al., 1998). In
crosses with no cyc homozygotes, we used a primer pair that amplified
a fragment specific to the cycm294 allele to distinguish
cyc+/+ and cycm294/+ embryos: forward,
5'-GGTGGACATGCATGTGGATTT-3' and reverse,
5'-TCGGGCAGGCCCCCTCCCG-3'.
mRNA synthesis and embryo injection
The zß-catenin expression vector has been described previously
(Kelly et al., 1995).
Transcripts for injection were synthesized using the Message Machine kit
(Ambion). Wild-type embryos were injected with 100 pg ß-catenin
mRNA, while embryos from an intercross of sqtcz35/+
parents were injected with 500 pg ß-catenin mRNA. In this
experiment (Fig. 9G-J), mutant
genotypes were inferred from phenotypes rather than assessed by PCR. In the
control injection 24% (12/49) of the embryos had reduced gsc
expression typical of sqt mutants
(Fig. 9J), whereas the
remaining 76% (37/49) displayed normal gsc
(Fig. 9H). ß-catenin
overexpression induced ectopic or expanded gsc
(Fig. 9G) in over half of the
wild-type embryos (46/77). ß-catenin did not induce high levels of
gsc expression in sqt mutants, because 26% (16/62) of
injected embryos displayed reduced gsc typical of uninjected
sqt mutants (Fig.
9I).
Immunocytochemistry and in situ hybridization
For analysis of the distribution of ß-catenin protein, wild-type
embryos were fixed at 15 minute intervals during the first 4 hours of
embryogenesis, processed for immunocytochemistry with ß-catenin
antibodies and sectioned. Polyclonal anti-ß-catenin antibodies
(Schneider et al., 1996) were
used at a 1:1000 dilution. Immunostaining was carried out as described
(Schier et al., 1997). For
sectioning, embryos were embedded in Eponate-12 resin and sectioned at 3
µm. Twenty embryos were analyzed at each time-point, and two or three of
these were sectioned to confirm the presence or absence of nuclear
ß-catenin. Although the majority of embryos after the 1000-cell stage
exhibited ß-catenin protein in dorsal nuclei (in both the YSL and
blastomeres), only three embryos at the 128-cell stage, three embryos at the
256-cell stage and three embryos at the 512 stage exhibited ß-catenin
protein in dorsal nuclei. At all stages, staining of membrane localized
ß-catenin protein served as a positive control for the antibody
reaction.
Synthesis of probes and in situ hybridization were conducted as described
(Sirotkin et al., 2000b).
After in situ hybridization, the number of mutant embryos from each cross was
counted and representative examples of each phenotype were photographed and
genotyped by PCR, except when the genotype could be inferred from morphology
as in the analysis of gsc at 10 h in sqt-/-;
cyc+/- and sqt-/-;
cyc-/- embryos and sox17 expression in
sqt-/-; cyc+/- mutants. The number of
mutants was usually close to the expected values, and the same mutant
phenotypes were observed in repeated crosses of the same parents.
Lineage-tracing and fete map analysis
Embryos of sqtcz35/+; cycm294/+
x sqtcz35/+ parents were injected
(Kimmel et al., 1990) with the
lineage tracer dye tetramethylrhodamine-isocyanate dextran (Molecular Probes,
Eugene, OR; 10x103 Mr, diluted to 5%
(wt/vol) in 0.2 M KCl) in single blastomeres of the surface enveloping layer
(EVL) at the 1000-cell stage. Injected embryos were mounted in 0.125% agarose
in embryo medium and oriented so that the fluorescent cells faced toward the
microscope objective; the position of the clone along the animal-vegetal axis
was then determined. The clonal position relative to the dorsoventral axis was
determined at 6 h, when the dorsal side is first morphologically apparent in
wild-type embryos, or at 8 h, when the dorsal side is morphologically apparent
in sqt-/-; cyc+/- embryos. Some
embryos were filmed during gastrulation with time-lapse photography as
previously described (Warga and Kimmel,
1990). After filming, embryos were removed from agarose and the
fates of the resulting clones were determined at 24 h and 48 h, after which
all embryos were genotyped by PCR.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Kupffer's vesicle, a derivative of the dorsal forerunner cells
(Cooper and D'Amico, 1996;
Melby et al., 1996), was
reduced or absent at 12 h (Fig.
2T). Forerunner cells were also reduced or absent in
sqt-/-; cyc+/+ mutants (see
Fig. 5J-L, arrows), as revealed
by expression of sox17 (Alexander
and Stainier, 1999). Thus, most or all sqt-/-;
cyc+/+ single mutants have defects in dorsal marginal
structures at the onset of gastrulation. At 24 h, a variable fraction of
sqt mutants display mild cyclopia and reduction of ventral
diencephalon (Table 1;
Fig. 2I) and some
sqt-/-; cyc+/+ single mutants are
morphologically indistinguishable from wild type
(Table 1; data not shown). The
amelioration of the sqt mutant phenotype at later stages depends on
the function of cyc, because sqt; cyc double mutants lack
all mesendodermal derivatives in the head and trunk at 24 h
(Fig. 2A-C)
(Feldman et al., 1998).
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)
(Fig. 6A-F). In addition,
sqt-/-; cyc+/- embryos have more
pronounced cyclopia than sqt-/-;
cyc+/+ embryos (Fig.
2F,I), suggesting a greater reduction in prechordal plate
mesoderm. The severity of these defects varies with the genetic background,
but within a group of siblings the typical sqt-/-;
cyc+/- embryo has a stronger phenotype than the typical
sqt-/-; cyc+/+ sibling. These results
indicate that the formation of dorsal axial structures in sqt mutants
is strongly dependent on cyc gene dosage, such that the phenotype of
sqt-/-; cyc+/+ embryos is compounded
by the loss of a single copy of cyc (i.e. in
sqt-/-; cyc+/- embryos). Conversely,
we did not note any differences between the phenotypes of
sqt+/+; cyc-/- and
sqt+/-; cyc-/- embryos
(Fig. 2J-L;
Table 1; data not shown).
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;
Pogoda et al., 2000;
Sirotkin et al., 2000a).
Therefore we investigated the role of crossregulation in the dosage-sensitive
interaction between sqt and cyc by examining cyc
expression in sqt mutants and sqt expression in cyc
mutants. Expression of sqt appeared normal in a time course of
cyc mutant embryos (compare Fig.
3F-I with 3A-D), but was reduced in sqt mutants
(Fig. 3E,J). cyc
expression was altered in sqt-/-;
cyc+/+ mutants (compare
Fig. 3L-O with 3P-S). Although
cyc expression initiated in sqt mutants
(Fig. 3P), it was reduced or
absent at the dorsal margin at 5 h (Fig.
3Q) and was evident at the dorsal midline at reduced levels during
gastrulation (Fig. 3R,S).
Expression of cyc was reduced significantly in
sqt-/-; cyc+/- mutants
(Fig. 3T-V) in comparison with
sqt-/-; cyc+/+ embryos
(Fig. 3Q-S). At the onset of
gastrulation in sqt-/-; cyc+/-
mutants, cyc transcripts were present in only two weak dorsolateral
patches (Fig. 3U); the dorsal
gap between these patches closed as gastrulation progressed, such that a
narrow strip of cyc expression was visible at the dorsal midline at 8
h (Fig. 3V). These results show
that dorsal cyc expression is activated by sqt at 5 h, the
first stage where a reduction of cyc expression is evident in
sqt mutants. The reduction of cyc expression in
sqt-/-; cyc+/- embryos when compared
with sqt-/-; cyc+/+ mutants suggests
that cyc expression also activates its own transcription. It is not
likely that a reduction of mutant mRNA stability is the cause of reduced
cyc expression in sqt-/-;
cyc+/- mutants, because the level and pattern of
cyc mRNA expression was not altered in sqt+/+;
cyc+/- embryos when compared with wild type (data not
shown).
Mesendoderm induction requires different levels of
nodal-related gene function in dorsal and ventrolateral regions
To examine the effect of reduced Nodal signaling on dorsoventral patterning
of the mesendoderm, we analyzed the time course of expression of an array of
marker genes in sqt-/-; cyc+/+ and
sqt-/-; cyc+/- mutants. For these
experiments, we analyzed clutches of embryos in which most
sqt-/-; cyc+/- mutants had strong
cyclopia, truncated notochords, and medially fused somites. Embryos from
appropriate crosses were harvested at blastula and gastrula stages, analyzed
by whole-mount in situ hybridization, assigned to phenotypic classes based on
expression patterns, and then genotyped by PCR assays to determine which
genotypes constituted each phenotypic class. As expected from morphological
analysis, markers for dorsal mesendoderm such as gsc and
axial/foxa2 were reduced more severely in sqt-/-;
cyc+/- embryos than in sqt-/-;
cyc+/+ single mutants. Expression of gsc, which
marks the prechordal plate during gastrulation
(Stachel et al., 1993),
initiated in the dorsal marginal region in sqt-/-;
cyc+/+, sqt-/-;
cyc+/- and sqt-/-;
cyc-/- embryos (Fig.
4B1,C1,D1), but it rapidly faded in embryos of all three mutant
genotypes (Fig. 4B2,C2,D2). By
5 h (40% epiboly), gsc transcripts were prominent at the dorsal
margin of wild-type embryos (Fig.
4A2), but were detectable in only a few cells in
sqt-/-; cyc+/+ mutants
(Fig. 4B2). We were unable to
detect gsc expression in sqt-/-;
cyc+/- and sqt-/-;
cyc-/- embryos at this stage
(Fig. 4C2,D2). gsc
expression recovered during gastrulation in sqt-/-;
cyc+/+ mutants (Fig.
4B3-B6), but not in sqt-/-;
cyc+/- and sqt-/-;
cyc-/- mutant siblings
(Fig. 4C3-C5,D3-D5). After
gastrulation, weak ectodermal gsc expression initiated in both
sqt-/-; cyc+/- and
sqt-/-; cyc-/- mutants
(Fig. 4C6,D6). Like
gsc, expression of axial/foxa2 in axial mesoderm
(Strähle et al., 1993) is
reduced to a much greater extent in sqt-/-;
cyc+/- embryos than sqt-/-;
cyc+/+ siblings (Fig.
5A-F). And like gsc, axial/foxa2 expression at
the midline later recovers in sqt-/-;
cyc+/+ embryos (data not shown). Thus, early dorsal
mesendodermal gene expression is reduced in sqt-/-;
cyc+/+ single mutants, and the further loss of a single
copy of the cyclops gene compounds this defect. The recovery of
dorsal mesoderm gene expression in some sqt mutants suggests that
dorsal marginal cells can adopt dorsal mesendoderm fates in response to
cyc signaling during gastrulation, more than 2 hours after dorsal
mesendoderm induction occurs in wild type.
To examine how the endoderm is affected in sqt-/-;
cyc+/+ and sqt-/-;
cyc+/- mutants embryos, we analyzed expression of
axial/foxa2 and sox17
(Fig. 5A-L)
(Schier et al., 1997;
Alexander and Stainier, 1999).
In order to quantify endodermal progenitors, we made fillets of mid-gastrula
stage wild-type and mutant embryos stained for axial/foxa2 expression
and counted the total number of labeled endodermal cells
(Fig. 5M). At mid-gastrulation,
wild-type embryos (n=4) had an average of 270 (±54)
axial/foxa2-expressing endodermal cells, sqt-/-;
cyc+/+ embryos (n=5) had an average of 164
(±35), and sqt-/-; cyc+/-
embryos (n=5) had only 65 (±12). A similar distribution of
endodermal cells was observed in sqt-/-;
cyc+/+ and sqt-/-;
cyc+/- mutants at 10 h and 12 h, as detected by
sox17 expression (data not shown). The reduction in endodermal
precursors was not uniform along the dorsoventral axis
(Fig. 5N). In wild-type
embryos, previous work showed that endoderm is asymmetrically distributed at
mid-gastrulation (Warga and
Nüsslein-Volhard, 1999), and we found an average of 13 rows
of axial/foxa2-expressing endodermal cells dorsally, compared with
only four rows ventrally (Fig.
5N). This asymmetry was less pronounced in
sqt-/-; cyc+/+ embryos, which had an
average of only seven tiers of axial-expressing cells dorsally and 2.5 tiers
ventrally. Dorsal endoderm was completely eliminated in
sqt-/-; cyc+/- embryos, although one
to three tiers of axial/foxa2-expressing cells remained ventrally and
laterally. Thus, like mesoderm, endoderm in dorsal locations in sqt
mutants is more sensitive to reductions in cyc gene dosage than
endoderm in lateral and ventral positions.
We next investigated whether the loss of dorsal mesendodermal markers is
accompanied by a corresponding alteration in ventrolateral gene expression
(Fig. 6). The ventrolateral
mesoderm markers spadetial (spt; tbx16 — Zebrafish
Information Network), tbx6 and vox
(Hug et al., 1997;
Griffin et al., 1998;
Kawahara et al., 2000;
Melby et al., 2000) are each
expressed around the margin but are largely excluded from dorsal marginal
regions (Fig. 6G,J,M,P).
Ventrolateral expression of these genes was not significantly altered in
sqt-/-; cyc+/+ or
sqt-/-; cyc+/- mutant embryos
(Fig. 6H,K,N,Q), except that
each of these genes was excluded from a larger dorsal sector in
sqt-/-; cyc+/- embryos
(Fig. 6I,L,O,R). The different
effects on dorsal and ventrolateral mesendodermal gene expression in
sqt-/-; cyc+/+ and
sqt-/-; cyc+/- mutants suggests that
the formation of mesoderm and endoderm requires higher levels of cyc
function at the dorsal margin than at the ventrolateral margin.
Dorsal marginal cells adopt neural fates in
sqt-/-; cyc+/+ and
sqt-/-; cyc+/- mutants
As dorsal marginal cells fail to express markers for both dorsal and
ventrolateral mesendoderm in sqt-/-;
cyc+/- mutants, we wished to determine the fates of dorsal
marginal cells in these embryos. Expression of the pan-mesodermal marker
ntl/brachyury (Schulte-Merker et
al., 1992), was reduced on the dorsal side of
sqt-/-; cyc+/+ embryos
(Fig. 6T) and absent from a
dorsal sector in sqt-/-; cyc+/-
embryos (Fig. 6U), raising the
possibility that dorsal marginal cells adopt a neurectodermal fate in
sqt-/-; cyc+/- mutants. Accordingly,
the neural plate marker cyp26
(White et al., 1996;
Thisse and Thisse, 1999;
Kudoh et al., 2001) was
positioned closer to the margin in sqt-/-;
cyc+/+ and sqt-/-;
cyc+/- mutants than in wild type
(Fig. 6W,X, arrowheads).
The gene expression studies prompted us to determine directly the fates of
marginal cells in sqt-/-; cyc+/+ and
sqt-/-; cyc+/- mutants. We labeled
single blastomeres at the 1000-cell stage by injection of a lineage tracer dye
and ascertained the fates of their progeny between 1 and 3 d, when most cells
have differentiated. Dorsal cells closest to the margin in wild-type embryos
exclusively form mesodermal and endodermal derivatives, including hatching
gland, foregut and notochord (Kimmel et
al., 1990; Melby et al.,
1996; Warga and
Nüsslein-Volhard, 1999). By contrast, some dorsal marginal
cells in sqt-/-; cyc+/+ and
sqt-/-; cyc+/- mutants adopt neural
fates, including spinal cord, hindbrain and midbrain
(Fig. 7B,C upper panels), which
derive from more animal regions in wild-type embryos
(Fig. 7A). In two cases, mutant
dorsal marginal clones exclusively generated neural fates
(sqt-/-; cyc+/+ n=1;
sqt-/-; cyc+/- n=1), which is
never observed in wild type. In addition, dorsal marginal cells formed foregut
endoderm and hatching gland rarely if at all in sqt-/-;
cyc+/+ and sqt-/-;
cyc+/- mutants, although these cells can form notochord.
As previously reported (Feldman et al.,
2000; Carmany-Rampey and
Schier, 2001), all dorsal marginal cells adopt neural fates in the
absence of Nodal signaling (Fig.
7D).
|
Abnormal morphogenesis of dorsal marginal cells in
sqt-/-; cyc+/- mutants
In addition to changes in cell fate, we observed that dorsal marginal cells
undergo an aberrant morphogenetic behavior in sqt-/-;
cyc+/- embryos, consistent with their altered cell fates
(Fig. 7 and
Fig. 8B). At the onset of
gastrulation, dorsal marginal cells enter the mesendodermal germ layer and
rapidly move toward the animal pole, forming the prechordal plate
(n=16 clones, Fig. 8A)
(Warga and Nüsslein-Volhard,
1999). Instead of undergoing normal involution movements and
moving toward the animal pole, dorsal marginal cells in
sqt-/-; cyc+/- embryos dispersed along
the margin. Many dorsal cells in mutants rolled inwards, temporarily altered
their course and then continued migrating toward the vegetal pole
(Fig. 8B n=3 clones in
sqt-/-; cyc+/- embryos). Despite their
anomalous behavior, some of these dorsal cells were able to contribute to
trunk notochord, much as they did in wild type
(Fig. 7,
Fig. 8A'',B'',
arrows). In contrast to dorsal marginal cells, the morphogenetic program of
cells on the ventrolateral margin appeared normal in
sqt-/-; cyc+/- embryos
(Fig. 8D, n=33
wild-type and 3 sqt-/-; cyc+/-
embryos).
|
;
Feldman et al., 1998;
Rebagliati et al., 1998a;
Sampath et al., 1998). To
understand how the asymmetry of sqt and cyc expression is
generated, we asked if cyc is controlled by the maternal dorsal
determinant ß-catenin, which has been implicated as a regulator of
sqt and Xenopus xnr-1
(Hyde and Old, 2000;
Shimizu et al., 2000).
ß-catenin mRNA overexpression induced expanded or ectopic
cyc expression at 6 h in about half (32/60) of injected wild-type
embryos (Fig. 9A), similar to
the fraction of injected embryos (12/18) with ectopic or expanded gsc
expression at 5 h (Fig. 9G).
Although ß-catenin did not affect cyc expression prior
to 6 h (Fig. 9C-F), it induced
ectopic or expanded sqt expression at 3.3 h (79/112)
(Fig. 9K), as previously
reported (Shimizu et al.,
2000). ß-catenin had no effect on sqt
expression at 5 h (Fig. 9M).
Thus, activation of sqt and cyc by ß-catenin follow
distinct time courses, suggesting that sqt may be a direct target,
while cyc could be an indirect target of ß-catenin, mediated by
genes such as sqt or boz that are induced at earlier stages
(Fekany et al., 1999).
Because squint expression is rapidly upregulated by
ß-catenin, we asked if it is required for
ß-catenin function. We injected ß-catenin mRNA
into embryos from sqt+/- parents and analyzed gsc
expression at 5 h (40% epiboly). At this stage, gsc is reduced to
only a few cells in control injected and uninjected sqt mutants
(Fig. 9J). Although
ß-catenin mRNA can induce mutant secondary axes in the absence
of sqt, as indicated by extra patches of cells expressing low levels
of gsc (Fig. 9I,
arrow), it cannot induce the high levels of gsc typical in wild-type
embryos (Fig. 9G)
(Kelly et al., 1995;
Pelegri and Maischein, 1998).
Thus, ß-catenin requires squint function to induce high levels
of dorsal mesodermal gene expression. Nevertheless, ß-catenin, acting
directly or through other factors, is sufficient to induce low levels of
ectopic gsc expression in sqt mutants.
Consistent with the possibility that ß-catenin activates early
sqt expression, we found that ß-catenin protein is localized to
the nuclei of presumed dorsal blastomeres prior to 3 h
(Fig. 10A-C), when
sqt transcripts are first detected
(Erter et al., 1998;
Feldman et al., 1998). These
results extend a prior analysis of ß-catenin distribution, which reported
that ß-catenin accumulates in dorsal nuclei at 3.3 h
(Schneider et al., 1996).
Moreover, we detected nuclear ß-catenin in embryos as early as the
128-cell stage (2.25 h) (Fig.
10A), prior to formation of the yolk syncytial layer (YSL), an
extra-embryonic structure proposed to have a role in establishing dorsoventral
asymmetry in the overlying blastoderm (reviewed by
Schier and Talbot, 1998).
Nuclear ß-catenin was detected in only a small fraction (15%,
n=3/20, at the 128-cell stage) of the embryos prior to the 1000-cell
stage, perhaps because of intermittent accumulation of nuclear ß-catenin
during rapid cleavage divisions. Although there are no other markers of
dorsoventral polarity in zebrafish embryos younger than 3 h, the dorsal
accumulation of nuclear ß-catenin after 3 h has been confirmed by
colocalization with dorsal specific genes
(Koos and Ho, 1998).
Therefore, we presume that ß-catenin accumulates in dorsal nuclei prior
to 3 h, as it does after 3 h. Our results show that dorsoventral asymmetry is
established in the blastoderm before the YSL forms.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Thisse et al., 2000;
Chen and Schier, 2001). The
analysis of sqt-/-; cyc+/+ or
sqt-/-; cyc+/- mutants extends
previous fate mapping studies of Nodal pathway mutants
(Feldman et al., 2000;
Gritsman et al., 2000;
Carmany-Rampey and Schier,
2001) by demonstrating a dosage-sensitive role for Nodal genes in
patterning the animal-vegetal axis in dorsal and ventrolateral marginal
regions.
Our genetic analyses indicate that mesendodermal progenitors at different
positions along the dorsoventral axis have distinct requirements for
nodal-related genes. In sqt-/-;
cyc+/- mutants, dorsal axial structures and dorsal
expression of early mesendodermal genes such as gsc, axial, sox17,
ntl and cyc, are strongly reduced or eliminated, whereas
ventrolateral fates are relatively mildly affected (Figs
2,
5 and
6). This shows that a
nodal gene dosage sufficient to induce mesendoderm at the
ventrolateral margin is insufficient to induce dorsal mesendoderm. In
addition, we noted that dorsal, but not ventrolateral, mesendoderm development
is quite sensitive to nodal-related gene dosage. Dorsal mesendoderm
is reduced in sqt-/-; cyc+/+ mutants,
reduced more strongly in sqt-/-;
cyc+/- mutants, and, as shown in previous studies
(Feldman et al., 1998;
Gritsman et al., 1999),
completely lacking in the absence of Nodal signaling. These differences along
the dorsoventral axis are not anticipated by simple models in which Nodal
signals act uniformly to induce dorsal and ventrolateral mesendoderm
(Fig. 1B).
Differential regulation of sqt and cyc in dorsal
and ventrolateral mesendoderm
Dorsal sqt expression is activated by ß-catenin
(Fig. 9) (Shimizu et al., 2000;
Kelly et al., 2000). Previous
work (Schneider et al., 1996)
has shown that ß-catenin localized to dorsal nuclei in the YSL at the
2000-cell stage (3.3 h). We found that ß-catenin was detectable in the
nuclei of dorsal blastomeres as early as the 128-cell stage (2.25 h)
(Fig. 10), prior to formation
of the YSL. These results show that dorsoventral asymmetry is established in
the blastoderm before the YSL forms. In addition, RNase treatment experiments
indicate that RNAs in the YSL are not essential for specification of dorsal
identity in overlying blastomeres (Chen and
Kimelman, 2000). Taken together, these results establish that
ß-catenin in dorsal blastomeres specifies dorsal expression of target
genes including sqt and boz
(Shimizu et al., 2000;
Kelly et al., 2000) and show
that the YSL is not required for the initial dorsal identity within the
blastoderm. By contrast, the YSL is required for expression of sqt
and cyc in ventrolateral marginal blastomeres
(Chen and Kimelman, 2000).
We found that differential regulation of sqt and cyc
expression in dorsal and ventrolateral marginal cells of the late blastula
accounts, at least in part, for the differential requirement of
nodal-related genes along the dorsoventral axis. In the late
blastula, cyc is expressed in a marginal ring that includes cells at
all positions along the dorsoventral axis, but our results show that different
mechanisms control cyc expression in dorsal and ventrolateral cells.
Several observations indicate that sqt activates dorsal cyc
expression in response to the maternal dorsal determinant ß-catenin.
Overexpression of ß-catenin induces sqt expression soon
after the midblastula transition (Fig.
9K) (Shimizu et al.,
2000), and nuclear ß-catenin protein is present at the
correct time and place to directly activate sqt expression
(Fig. 10). In addition,
ß-catenin requires sqt function for high-level activation of
dorsal gene expression (Fig.
9G-J). Dorsal expression of cyc in the late blastula is
reduced in sqt mutants (Fig.
3Q), and overexpression of ß-catenin does not
activate cyc expression until the early gastrula stage
(Fig. 9A,C,E). This suggests
that ß-catenin directly activates sqt, which in turn induces
dorsal cyc expression in the late blastula. At later stages, dorsal
cyc expression is dependent on an autoregulatory loop, as evidenced
by the strong reduction of dorsal cyc mRNA in
sqt-/-; cyc+/- when compared with
sqt-/-; cyc+/+ mutants
(Fig. 3) and by previous
studies of cyc expression in Nodal pathway mutants
(Meno et al., 1999;
Pogoda et al., 2000;
Sirotkin et al., 2000a).
By contrast, ventrolateral expression of cyc is induced
independently of Nodal signals but requires Nodal activity to achieve normal
levels. cyc expression is induced normally in ventrolateral marginal
cells in sqt-/-; cyc+/+ embryos,
indicating that this expression does not depend on sqt function
(Fig. 3). In embryos lacking
all nodal gene function, cyc expression is initiated in
ventrolateral marginal cells, but does not reach normal levels, whereas dorsal
cyc expression is not detectable
(Meno et al., 1999). These
observations indicate that ventrolateral cyc expression is induced by
as yet unknown factors and maintained at normal levels by an autoregulatory
loop. Thus cyc expression is controlled differently in dorsal and
ventrolateral marginal cells, with dorsal cyc expression entirely
dependent on sqt and cyc gene function.
The marked dependence of dorsal, but not ventrolateral, cyc expression on Nodal signaling results in a non-uniform reduction of Nodal signals in sqt-/-; cyc+/+ and sqt-/-; cyc+/- mutants. Some dorsal marginal cells in sqt-/-; cyc+/+ and sqt-/-; cyc+/- mutants adopt neural fates because they are exposed to little or no Nodal signals before gastrulation. By contrast, ventrolateral marginal cells in sqt-/-; cyc+/+ and sqt-/-; cyc+/- mutants are exposed to sufficient levels of cyc at the late blastula stage to induce ventrolateral mesendoderm. Thus, the role of autoregulation in the reinforcement of dorsal cyc expression can explain the severe reduction of dorsal but not ventrolateral fates in sqt-/-; cyc+/- mutants.
Experiments in zebrafish, frogs and mice indicate that autoregulation is a
conserved feature of the transcriptional control of nodal-related
genes (this work) (Meno et al.,
1999; Hyde and Old,
2000; Osada et al.,
2000; Norris et al.,
2002). Our results extend this observation by demonstrating that,
in zebrafish, cells differ in their sensitivities to the autoregulatory
feedback loop depending on their position. cyc expression was
completely eliminated from dorsal marginal cells in late blastula stage
sqt-/-; cyc+/+ embryos, but was present at
reduced levels in ventrolateral marginal cells
(Fig. 3P-S). This aspect of the
autoregulatory control of nodal-related gene expression may also be
conserved in other systems, introducing a point of caution in interpreting
experiments in which Nodal signaling activity is reduced by genetic methods or
by overexpression of Nodal antagonists. It is possible that some cell types
are preferentially affected in these experiments.
Dorsoventral patterning of mesendoderm is independent of Nodal
signals
Our cell-tracing and gene expression experiments are inconsistent with
models proposing that the graded action of Nodal signals patterns the
dorsoventral axis (Fig. 1A).
Such models predict that dorsal marginal clones should contain fates normally
arising at ventrolateral positions, such as muscle, when Nodal signaling is
reduced. Rather than muscle or other ventrolateral derivatives, dorsal
mar
