Department of Biological Chemistry and Molecular Pharmacology, Harvard
Medical School, 240 Longwood Avenue, Boston, MA 02115, USA
* Present address: Department of Anatomy, University of California, San
Francisco, CA 94143, USA
Present address: The Evergreen State College, Lab I Room 3009, Olympia, WA
98505, USA
Author for correspondence (e-mail:
donaldm{at}evergreen.edu)
Accepted 21 August 2002
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SUMMARY |
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Key words: Dorsoventral polarity, easter, spätzle, dorsal, Serine protease, Drosophila
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INTRODUCTION |
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Spatial information originating in the follicular epithelium is later
transmitted to the embryo through the perivitelline space, which lies between
the vitelline layer of the eggshell and the embryo plasma membrane (reviewed
by Morisato and Anderson,
1995). The establishment of embryonic polarity is dependent on the
ventrally restricted activation of the uniformly distributed receptor Toll
(Hashimoto et al., 1988
;
Hashimoto et al., 1991
;
Stein et al., 1991
). The
ligand for Toll is apparently encoded by the spätzle gene, which
produces a protein containing a C-terminal cystine knot motif found in many
vertebrate growth factors (Morisato and
Anderson, 1994
). The Spätzle protein is secreted into the
perivitelline space as an inactive precursor, and is cleaved into the active
ligand (Morisato and Anderson,
1994
; Schneider et al.,
1994
) through the activity of a serine protease cascade that
includes the products of the genes nudel
(Hong and Hashimoto, 1995
),
gastrulation defective (Konrad et
al., 1998
), snake
(DeLotto and Spierer, 1986
)
and easter (ea) (Chasan
and Anderson, 1989
).
Activation of Toll initiates an intracellular signaling pathway that
results in the nuclear translocation of the transcription factor encoded by
dorsal, a member of the NF-B/rel family (reviewed by
Drier and Steward, 1997
).
Dorsal is initially present throughout the embryonic cytoplasm, where it is
retained by the inhibitory I
B protein encoded by cactus.
Signaling on the ventral side leads to the proteolysis of Cactus, thereby
releasing Dorsal (Belvin et al.,
1995
; Bergmann et al.,
1996
; Reach et al.,
1996
). Along the dorsoventral axis, high levels of Dorsal protein
are present in ventral nuclei, progressively lower levels in lateral nuclei
and no detectable protein in dorsal nuclei
(Roth et al., 1989
;
Rushlow et al., 1989
;
Steward, 1989
). The shape of
the Dorsal gradient is characterized by the size of the ventral domain
(measured by the number of nuclei expressing peak Dorsal) and a distinct slope
(assessed by the number of nuclei that lie between highest and lowest nuclear
Dorsal). Changing the shape of the Dorsal gradient causes patterning defects
that lead to embryonic lethality.
The Dorsal gradient subdivides the axis into distinct domains by setting
the expression limits of key zygotic regulatory genes, which are responsible
for initiating the differentiation of various tissues. High levels of nuclear
Dorsal lead to the transcription of twist in mesodermal precursor
cells (Thisse et al., 1988;
Jiang et al., 1991
;
Pan et al., 1991
). The Twist
protein is itself expressed in a graded fashion in the most ventral 16-18
cells, and this domain can be subdivided into smaller threshold responses
(reviewed by Rusch and Levine,
1996
). Intermediate levels of nuclear Dorsal activate the
transcription of short gastrulation (sog) in two lateral
stripes flanking the ventral Twist domain, each about 14-16 cells wide
(François et al.,
1994
). The rhomboid (rho) gene is transcribed in
a ventral subset of 8-10 cells in each sog domain
(Bier et al., 1990
;
Ip et al., 1992
). The
zerknüllt (zen) gene is transcribed in the dorsal
40% of the embryo circumference, in the region where Dorsal is absent
from nuclei (Rushlow et al.,
1987
). In the experiments described below, we characterize changes
in the Dorsal gradient by examining the expression domains of these zygotic
genes.
We are keenly interested in understanding how the shape of the Dorsal
gradient is regulated. Two classes of mutations produce particularly
interesting effects on the wild-type shape. In the first class, embryos
produced by grk- and Egfr- females
show two peaks of nuclear Dorsal separated by a shallow ventral minimum
(Schüpbach, 1987;
Roth and Schüpbach,
1994
). These ventralized embryos proceed to gastrulate with two
ventral furrows instead of the single wild-type ventral furrow. Recent studies
showed that this phenotype could be mimicked by overexpression of
Spätzle, suggesting that partial axis duplication arises from events in
the perivitelline fluid of the embryo
(Morisato, 2001
). Despite the
dramatic reshaping of the ventral domain in these mutant embryos, the slope of
the Dorsal gradient remains wild type
(Roth and Schüpbach,
1994
; Morisato,
2001
).
In the second class, dominant alleles of easter
(eaD) cause a more symmetric distribution of nuclear
Dorsal (Steward, 1989). As a
consequence, females carrying eaD mutations produce
ventralized embryos, in which ventrolateral structures are expanded at the
expense of dorsal structures, or lateralized embryos, in which dorsoventral
polarity is largely lost (Chasan and
Anderson, 1989
; Jin and
Anderson, 1990
).
The easter gene encodes the final member of the protease cascade
required to activate Spätzle. Easter is initially synthesized as an
inactive zymogen containing an N-terminal pro-domain and a C-terminal
catalytic domain. Proteolytic cleavage at the activation site between these
two domains by Snake presumably generates active Easter in vivo
(Chasan et al., 1992;
Dissing et al., 2001
;
LeMosy et al., 2001
). Yet, in
wild-type embryo extracts, active Easter is found in a high
Mr complex called Ea-X, which is hypothesized to contain a
protease inhibitor of the serpin family
(Misra et al., 1998
). Easter
is proposed to be active only on the ventral side of the embryo. The
eaD mutations, which map to conserved regions within the
catalytic domain (Jin and Anderson,
1990
), somehow cause a loss of this spatial regulation.
We present our analysis of a group of representative eaD alleles. We have characterized changes in the shape of the Dorsal gradient caused by these mutations, by examining the expression domains of the zygotic genes zen, sog, rho and twist. Within the allelic series, dorsoventral asymmetry was progressively lost and the slope of the Dorsal gradient flattened. When production of activated Easter was examined in eaD embryo extracts, we detected an Easter form corresponding to the free catalytic domain, which was never observed in wild type. The EaD catalytic domain exhibited protease activity, as measured by its ability to generate processed Spätzle in the embryo. In the case of the strongest lateralizing eaD allele, protease activity was detected several hours after the blastoderm stage in perivitelline fluid transfer experiments. Finally, mutant EaD proteins expressed in cultured Drosophila S2 cells were shown to cleave precursor Spätzle. These data suggest that the eaD mutations interfere with Easter inactivation by the inhibitor X, and support a model in which regulation by X is required for shaping the Dorsal gradient.
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MATERIALS AND METHODS |
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Analysis of embryos
Expression of zen, sog and rho RNA in embryos was
detected by antisense RNA probes. Hybridization and detection was carried out
as described (Tautz and Pfeifle,
1989). Embryos were embedded in Spurr resin (Polysciences) and
sectioned every 10 µm. The size of each domain was calculated by counting
the number of stained cells and dividing by the total number of cells in the
embryo circumference at 50% egg length. In order to ensure that only young
blastoderm embryos were analyzed, the quantitation presented in
Table 1 was restricted to
embryo cross-sections that contained 80-100 cells.
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Expression of Twist protein was detected with rabbit anti-Twist antibodies,
kindly provided by Siegfried Roth (Universität zu Köln). Primary
antibodies were visualized with biotin-conjugated anti-rabbit antibodies and
streptavidin-horseradish peroxidase (HRP) using Vectastain ABC (Vector
Laboratories) as described (Patel,
1994). Embryos were embedded in Spurr resin and sectioned every 10
µm.
Cuticles were manually dissected out of their vitelline membrane cases and
prepared as described (Wieschaus and
Nüsslein-Volhard, 1986).
Characterization of embryo extracts
Embryonic extracts were prepared as previously described
(Morisato and Anderson, 1994).
Protein samples were separated on a polyacrylamide gel and blotted to PVDF.
Rabbit antibodies generated against bacterially produced Spätzle protein
were affinity purified against the N- or C-terminal domain of Spätzle
protein using previously described methods
(Morisato and Anderson, 1994
).
Rabbit antibodies generated against bacterially produced TrpE-Easter fusion
protein (Chasan et al., 1992
)
were affinity purified against T7 protein 10-Easter fusion protein bound to
Affigel 10/15 (BioRad).
Expression of secreted Spätzle and EaN in S2 cells
The spätzle 1.9 kb cDNA
(Morisato and Anderson, 1994)
and ea
N cDNA (Chasan et
al., 1992
) were expressed under the control of the metallothionein
promoter in the pRMHa-3 vector (Bunch et
al., 1988
). The single base substitutions in
ea83l and ea5.13
(Jin and Anderson, 1990
) were
introduced by the QuikChange (Stratagene) site-directed mutagenesis
protocol.
Cultured Drosophila S2 cells were transiently transfected with a
total of 4.0 µg DNA (final amount adjusted with vector DNA) using
Cellfectin (Invitrogen). After transfection (5 hours), cells were incubated in
serum-free medium overnight, and protein production was induced with 0.7 mM
cupric sulfate. Conditioned medium was collected 20 hours later and analyzed
for the presence of secreted Spätzle and EaN.
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RESULTS |
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Among the ventralizing alleles, embryos produced by
ea125.3/+ females exhibited the weakest effects, with only
slight expansion of ventral denticle bands
(Fig. 1C)
(Chasan and Anderson, 1989).
Embryos laid by ea83l/+ and ea5022/+
females showed stronger phenotypes, characterized by the expansion of ventral
denticle bands, reduction or absence of dorsolaterally derived structures,
such as the filzkörper, and near absence of dorsal hairs
(Fig. 1E,G)
(Chasan and Anderson, 1989
).
Embryos laid by ea5022/+ females exhibited more
disorganized denticles and more severe head deformities than embryos laid by
ea83l/+ females. At gastrulation, embryos produced by
females carrying all three alleles invaginated an apparently normal ventral
furrow, but initiation of the lateral cephalic furrow was shifted to a more
dorsal position and fewer dorsal folds were formed.
Embryos produced by females carrying the lateralizing alleles
ea20n and ea5.13 show a marked
reduction in dorsoventral asymmetry. Unlike the other eaD
alleles, ea20n/+ females laid a mixture of ventralized and
lateralized embryos, as determined by analyzing both differentiated cuticles
and gastrulation movements. The ventralized embryos
(Fig. 1I) showed a stronger
phenotype than did embryos laid by ea83l/+ and
ea5022/+ females. By comparison, all of the embryos laid
by ea5.13/+ females showed a reduction in both dorsal
pattern elements and ventrally derived mesoderm, developing a cuticle with
rings of ventral and lateral denticle bands
(Fig. 1K)
(Chasan and Anderson, 1989). At
gastrulation, embryos from ea5.13/+ females failed to
invaginate a ventral furrow and exhibited a widened head fold.
We examined the phenotypes of embryos produced by eaD females in the absence of wild-type maternal easter activity, in order to study the genetic dominance exerted by these eaD mutations. For the ventralizing alleles, embryos produced by eaD/+ and eaD/ea- females were virtually indistinguishable (Fig. 1C-H). By contrast, the embryos produced by ea20n/ea- and ea5.13/ea- females were markedly more elongated and had fewer ventral denticle bands than embryos laid by ea20n/+ and ea5.13/+ females (Fig. 1I-L). Notably, all the embryos laid by ea20n/ea- females developed a lateralized head fold during gastrulation, when compared with the mixed population laid by ea20n/+ females. In summary, the presence of a wild-type dose of easter is able to confer detectable dorsoventral asymmetry to embryos produced by the lateralizing eaD alleles.
Taken together, the analysis of gastrulation behavior and differentiated cuticles suggests that the five eaD mutations can be ordered in the following allelic series, with the strongest allele exhibiting the greatest loss of dorsoventral polarity: wild type>ea125.3>ea83l>ea5022>ea20n>ea5.13.
Changes in the expression of zygotic markers caused by
eaD mutations
Our primary interest in the eaD mutations was to
understand their effects on the Dorsal gradient. Although we could stain
embryos directly for the expression of Dorsal protein, we felt that changes in
the shape of the gradient would be too subtle to discern and difficult to
quantitate. Therefore, we characterized the expression domains of the zygotic
genes zen, sog, rho and twist, each corresponding to a
specific concentration range of nuclear Dorsal (Figs
2,3,4,
Table 1). In order to simplify
comparisons between embryos, we expressed domain size as a percentage of the
embryo circumference (see Materials and Methods). The major points from these
data are summarized below.
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zen
The zen gene is transcribed in the dorsal 38% of the wild-type
embryo circumference, in those cells that lack nuclear Dorsal
(Table 1) (Rushlow et al., 1987). In
dorsalized embryos laid by ea- females, zen
expression expands across the entire dorsoventral axis. By contrast, the
zen domain is completely absent in embryos produced by
eaD/+ and eaD/ea-
females for both the ventralizing and lateralizing eaD
alleles (Table 1). Thus, even
in embryos produced by the weakest ventralizing allele
ea125.3, Dorsal protein is present in dorsal nuclei.
sog
The sog gene is transcribed in two ventrolateral stripes that
total 31% of the wild-type embryo circumference, with each stripe abutting the
ventral domain defined by twist expression
(Fig. 2A,B; Table 1)
(François et al.,
1994). In embryos produced by ea125.3 females,
each domain was expanded, such that the total sog domain occupied 50%
of the embryo circumference (Fig.
2C,D; Table 1). In
embryos produced by ea83l and ea5022
females, the sog domain was expanded across the dorsal midline
(Fig. 2E-H), although in some
of these embryos, the staining became weaker on the dorsal side; this region
of lower expression might correspond to a concentration of nuclear Dorsal
protein normally found in one or two nuclei at the dorsal sog
boundary. Because the dorsal boundary was sometimes difficult to determine
(see below), only the ventral unstained domain was quantitated in these
embryos.
In embryos laid by females carrying the lateralizing alleles, sog staining was significantly expanded. Consistent with the observations described above, a mixed population was observed among the embryos laid by ea20n/+ females; some embryos showed uniform sog staining, while others maintained a ventral unstained domain (Fig. 2I). By comparison, all embryos laid by ea20n/ea- females showed uniform sog expression (Fig. 2J). Similarly, embryos produced by ea5.13/+ and ea5.13/ea- females showed sog staining across the dorsoventral axis (Fig. 2K,L).
The quantitation of the sog domain showed that the slope of the Dorsal gradient was flattened in all of these mutant embryos. In addition, a small but significant change in the size of the presumptive mesoderm was detected by quantitating the size of the ventral unstained domain. Embryos laid by ea83l/ea- and ea5022/ea- females showed a decrease in the size of the ventral domain, when compared with the wild-type size seen in embryos laid by ea83l/+ and ea5022/+ females (Table 1).
rho
The rho gene is transcribed in two ventrolateral stripes that
total 18.5% of the wild-type embryo circumference
(Fig. 3A,B;
Table 1) (Bier et al., 1990). In
contrast to the weak sog expression observed across the dorsal side
in some mutant embryos, dorsal boundaries for the rho domain were
well defined. The size of each rho domain was modestly expanded in
embryos produced by females carrying the ventralizing alleles
ea125.3, ea83l and
ea5022 (Fig.
3C-H; Table 1). The
size of each rho domain was further increased in embryos laid by
ea20n/+ females, while the two stripes were fused into a
single domain in embryos produced by
ea20n/ea- females
(Fig. 3I,J). Embryos produced
by ea5.13/+ and
ea5.13/ea- females expressed
rho in all cells along the dorsoventral axis
(Fig. 3K,L). As noted above for
the quantitation of sog expression, the expansion of rho
expression observed in embryos produced by
ea83l/ea- and
ea5022/ea- females were each
accompanied by a decrease in the ventral unstained domain.
twist
The twist gene is transcribed in cells that give rise to mesoderm.
The Twist protein, visualized by anti-Twist antibodies, is expressed in the
ventral 21.5% of the wild-type embryo circumference
(Table 1) (Thisse et al., 1988). The
size of the Twist domain was nearly the same as in wild type in embryos
produced by females carrying the ventralizing alleles
ea125.3, ea83l and
ea5022 (Table
1). The Twist domain was slightly reduced in embryos laid by
ea20n/+ females, while no Twist expression was detected in
embryos laid by ea20n/ea- females
(Table 1). A very faint,
reduced Twist domain was observed among some embryos laid by
ea5.13/+ females (data not shown), while no Twist
expression was detected in embryos laid by
ea5.13/ea- females. In the wild-type
embryo, the size of the Twist domain was in good agreement with the ventral
domain also defined by the absence of sog and rho
expression. In the eaD mutant embryos, the Twist domain
appeared slightly larger than the ventral domain when determined by examining
sog and rho transcription; some cells could be expressing
low levels of both rho and Twist, as a consequence of a reduction in
the slope of the Dorsal gradient. In no case was the ventral domain expanded
in any of the embryos produced by the eaD alleles.
Summary
A comparison of changes in the Dorsal gradient shape, as inferred from the
expression domains of marker genes, is depicted in
Fig. 4. First, analysis of
zen expression showed that the dorsal domain, defined by the absence
of nuclear Dorsal, was lost in all of the mutant embryos. Second, the domain
of low nuclear Dorsal, reflected by the activation of sog
transcription, was expanded dorsally in all mutant embryos. This decrease in
the slope of the Dorsal gradient was further characterized by the analysis of
rho expression, a marker corresponding to intermediate levels of
nuclear Dorsal. Finally, a reduction in the size of the ventral domain of high
nuclear Dorsal, inferred from Twist staining and the absence of sog
and rho transcription, was observed in all mutant embryos, except for
the weakest ventralizing allele ea125.3. In moderately
ventralized embryos, this reduction was small but significant; in lateralized
embryos, the ventral domain was completely absent.
Detection of Easter catalytic domain in eaD
embryo extracts
In order to address the mechanism underlying these changes in the Dorsal
gradient, we asked if the eaD mutations were affecting the
production or inhibition of active Easter. Easter zymogen activation requires
cleavage at a site between an N-terminal pro-domain and a C-terminal catalytic
domain (Chasan et al., 1992).
However, the cleaved protease domain is never detected in wild-type embryos.
Instead, activated Easter is found in a stable complex called Ea-X that
migrates as a 80-85 kDa band (Misra et
al., 1998
).
We prepared extracts from embryos produced by
ea83l/ea- and
ea5.13/ea- females, as representative
of the ventralizing and lateralizing alleles. In order to generate a size
marker for the C-terminal catalytic domain, we prepared extracts from embryos
laid by eaN/+ and
ea8/ea- females. In the N-terminal
deletion mutant ea
N, the pro-domain is deleted and
the signal sequence is fused directly to the catalytic domain
(Chasan et al., 1992
). The
embryos laid by females carrying a P[ea
N] transgene
are weakly ventralized. In embryos carrying the recessive
ea8 allele, production of the Easter catalytic domain is
observed rather than the Ea-X complex
(Misra et al., 1998
).
Easter forms were detected on an immunoblot probed with anti-Easter
antibodies (Fig. 5). The amount
of Easter zymogen correlated with easter dose; a higher level was
observed in wild type compared with embryos laid by +/ea-
females (data not shown). As expected, the Ea-X band was present in the
wild-type extract, appeared more prominent in the eaN/+ embryo
extract and was absent in the ea8/ea- embryo
extract. In embryos produced by the eaD mutations, the
Ea-X band was reduced in the ea83l/ea- extract
and virtually absent in the ea5.13/ea- extract.
Significantly, there was a corresponding increase in the level of C-terminal
catalytic domain. The amount of the catalytic domain in the
ea5.13/ea- extract appeared comparable with the
level in the ea8/ea- extract. These findings
suggest that in the eaD mutations, zymogen activation
produces a catalytic domain that fails to be or is only partially inactivated
by the inhibitor X.
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Detection of EaD protease activity in embryos
The production of the C-terminal catalytic domain had previously been
observed only with catalytically inactive easter mutants, exemplified
by the ea8 allele, a missense mutation located in the
presumptive substrate binding pocket, and the eaS338A
allele, in which the active site serine-338 was replaced with alanine
(Misra et al., 1998). We
hypothesized that the C-terminal catalytic domain in the
eaD alleles, in contrast to the recessive mutations,
retained protease activity after zymogen activation. In order to test this
model, we assessed EaD protease activity in two different
experiments.
First, we determined the level of processed Spätzle in
eaD embryo extracts, because Easter protease activity
generates processed Spätzle (Morisato
and Anderson, 1994; DeLotto and
DeLotto, 1998
). No processed Spätzle is produced in
ea- extracts; a higher level of processed Spätzle is
observed in a transformant line carrying the ea
N mutation
(Morisato and Anderson, 1994
).
Extracts were prepared from embryos laid by eaD/+ and
eaD/ea- females. Precursor and processed forms
of Spätzle were detected on an immunoblot probed with antibodies specific
to the Spätzle C-terminal domain (Fig.
6). As expected, a lower level of processed Spätzle was
observed in the +/ea- embryo extract compared with the
wild-type embryo extract. Similarly, the level of processed Spätzle was
lower in the eaD/ea- embryo extract compared
with the corresponding eaD/+ embryo extract. In general,
the amount of processed Spätzle in each of the
eaD/ea- lanes appeared roughly comparable with
the level in the +/ea- lane. The exception to this
generalization was observed with the lateralizing ea5.13
allele. The amount of processed Spätzle observed in extracts prepared
from embryos laid by ea5.13/+ and
ea5.13/ea- females was reproducibly higher than
in wild type.
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Second, we used an injection assay to ask whether we could detect wild-type
and eaD activated Easter in the embryo. By transferring
perivitelline fluid from one embryo to another, Stein et al.
(Stein et al., 1991)
characterized a ventralizing activity exhibiting properties expected of the
Toll ligand. When this `polarizing activity', later identified to be processed
Spätzle, was injected into the perivitelline space of recipient embryos
laid by pipe- females, the site of injection defined the
ventral pole of a new axis (Stein et al.,
1991
; Stein and
Nüsslein-Volhard, 1992
;
Schneider et al., 1994
). We
reasoned that by using donor embryos laid by spz- females
(and thereby removing the presumptive Toll ligand), we might be able to detect
the activity responsible for generating processed Spätzle, i.e. active
Easter.
We carried out perivitelline fluid transfer experiments from gastrulating donor embryos laid by different mutant females into stage 4 recipient embryos laid by pipe- females. We did not detect axis-inducing activity from donor embryos laid by either spz- or spz- Toll- females (Table 2), suggesting that active Easter is either rapidly inactivated or sequestered in a non-transplantable complex. Furthermore, we did not detect activity from donor embryos laid by ea83l spz- females. By contrast, we observed axis-inducing activity from donor embryos laid by ea5.13 spz- females. This observation not only provided functional evidence for Ea5.13 protease activity, but also demonstrated that the activity remained stable until gastrulation, many hours after zymogen activation.
|
Analysis of EaD protease activity in cultured
Drosophila S2 cells
In order to study the Spätzle cleavage reaction, we co-expressed
precursor Spätzle and wild-type EaN proteins in cultured
Drosophila S2 cells. We observed production of processed Spätzle
in the conditioned medium, with the level of cleavage dependent on the amount
of Ea
N expressed (Fig.
7A, lanes 3-5; Fig.
7B, lanes 2-4). We did not detect the formation of the inhibited
Ea
N-X form in these transfected S2 cells.
|
We analyzed the effect of the ea83l and
ea5.13 mutations on the cleavage reaction. The level of
Spätzle processing carried out by Ea83lN was
indistinguishable from wild-type Ea
N
(Fig. 7A, lanes 6-8),
suggesting that the ea83l mutation does not affect Easter
catalytic activity. By comparison, Ea5.13
N exhibited weaker
protease activity (Fig. 7A,
lanes 9-11), although it appeared to be expressed at higher levels
(Fig. 7B, lanes 8-10). This
result suggests that the lateralized phenotype produced by the
ea5.13 mutation arises from two separate effects: (1) lack
of proper inhibition following zymogen activation leads to loss of the dorsal
zen domain, and (2) reduced Easter catalytic activity prevents
formation of the ventral twist domain (see Discussion).
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DISCUSSION |
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Ventralized embryos produced by grk- and
Egfr- females exhibit an expansion of the ventral Twist
domain at the expense of the dorsal domain, while maintaining a wild-type
slope of the Dorsal gradient, as assessed by the size of the rho and
sog domains (Schüpbach,
1987; Roth and Schüpbach,
1994
; Morisato,
2001
). By contrast, in the ventralized embryos produced by
ea83l/ea- and
ea5022/ea- females, the slope of the Dorsal
gradient is flattened, leading to broader domains of rho and
sog expression. This change is accompanied by a decrease, rather than
an increase, in the size of the ventral Twist domain. The
eaD ventralized phenotype thus appears to arise from a
redistribution of the ventral signal. This change in the shape of the Dorsal
gradient is even more dramatic in lateralized embryos, leading to a loss of
detectable dorsoventral polarity.
Monitoring the expression of target genes enabled the analysis of the Dorsal gradient, but also placed a limit on resolution. For example, the phenotypes of embryos laid by ea20n/ea- and ea5.13/ea- females initially appeared identical when assessed by sog RNA expression (Fig. 2J,L). A more refined image of the shape of the Dorsal gradient emerged after monitoring rho RNA expression (Fig. 3J,L), which responds to a narrower concentration range of nuclear Dorsal. Although embryos laid by ea5.13/ea- females appear symmetric, it remains formally possible that residual polarity could be detected if a marker corresponding to an even narrower range of nuclear Dorsal were available.
Genetic dominance of eaD alleles
Embryos produced by ea125.3/ea-,
ea83l/ea-, ea5022/ea- and
ea20n/ea- females show varying degrees of
dorsal-ventral asymmetry. The presence of a wild-type dose of easter
causes a slight expansion of the Twist domain and a slight reduction in the
rho domains, thereby producing a shift towards the normal shape of a
Dorsal gradient. The Dorsal gradient in embryos laid by
eaD/+ females reflects a partial contribution from each
easter allele, rather than a simple superimposition of the gradient
shapes observed in embryos laid by eaD/ea- and
+/ea- females. This behavior could be explained if only a
set amount of Easter zymogen (wild type and mutant combined) could be cleaved
by a limiting amount of activated Snake.
Although embryos laid by ea5.13/+ and ea5.13/ea- females can be distinguished by their cuticles (Fig. 1K,L), the expression of marker genes in these embryos is quite similar. In particular, formation of the Twist domain is largely inhibited in embryos laid by ea5.13/+ females. It remains to be determined whether the stronger dominance exhibited by ea5.13 can be explained by the simple dose argument presented above, or whether Ea5.13 is interfering with the proper formation of the Dorsal gradient by a more active mechanism.
Easter functions affected by ventralizing and lateralizing
eaD mutations
The experiments described above suggest how the spectrum of phenotypes
observed in ventralized and lateralized embryos can be explained by separately
considering two distinct properties of the Easter protein: (1) inactivation by
inhibitor X; and (2) Easter protease activity.
In both ventralized and lateralized embryos, the shape of the Dorsal
gradient is altered by the absence of the dorsal zen domain, which is
replaced by an expanded sog domain. Our studies suggest that this
phenotype arises from the failure of activated Easter to be properly regulated
after zymogen cleavage: the protease domain fails to form a complex with X and
remains active (Fig. 5). This
interpretation is consistent with earlier studies that characterized the
effects of changing eaD dose. Injection of
ea83l and ea125.3 RNA into
ea- embryos produced a ventralized phenotype, while lower
levels of the same RNA rescued to hatching, suggesting that the Easter
produced by these ventralizing alleles were defective in negative regulation
(Jin and Anderson, 1990).
The stability of the Ea-X complex suggested that X might be a serpin,
reacting with the active site serine of Easter
(Misra et al., 1998). Most
mutations that map in the Easter catalytic domain would be expected to affect
both protease activity and the interaction with inhibitor X. We suggest that
the ventralizing eaD alleles (as exemplified by
ea83l) form a special class of mutations that retain
catalytic activity, but affect inhibition by X. These studies imply that
regulation of Easter following zymogen activation is required for maintaining
polarity during formation of the Dorsal gradient. If activated Easter were
capable of diffusion, X may primarily play a kinetic role to maintain the
initial asymmetry of zymogen activation, by inhibiting activated Easter before
its diffusion to the dorsal side.
In lateralized embryos, the Dorsal gradient appears even less polar. Both
the dorsal zen and the ventral twist domains are absent,
with the lateral sog domain expanded along the entire dorsoventral
axis. The experiments described above suggest that in addition to their
failure to be inactivated by X, the lateralizing alleles also partially reduce
Easter protease activity. As the eaD mutations map to
conserved regions within the protease domain, it is not surprising that some
of these alleles show effects on catalytic activity. This point is most
clearly observed for the case of the Ea5.13N protein, which
exhibits less Spätzle processing activity than wild-type Ea
N upon
expression in cultured S2 cells (Fig.
7). In the embryo, this weaker Ea5.13 protease is
apparently unable to generate the level of processed Spätzle required for
nuclear translocation of Dorsal that leads to twist
transcription.
The final amount of processed Spätzle generated in the embryo depends on both Easter specific activity and the length of time the enzyme remains active. In the case of embryos produced by ea5.13 females, perivitelline transfer experiments detected stable Ea5.13 activity many hours after cellularization (Table 2). Despite the lower specific activity of Ea5.13 suggested by the S2 experiments (Fig. 7), the prolonged time of Easter action results in a higher level of processed Spätzle in embryos (Fig. 6). Similarly, the ea20n mutation appears to cause a significant decrease in specific activity, as suggested by a reduction in the combined size of the domains expressing rho RNA and Twist in embryos produced by ea20n/ea- females compared with embryos laid by other eaD/ea- females (Fig. 4 and Table 1). Yet, nearly wild-type amounts of processed Spätzle are observed in ea20n embryo extracts (Fig. 6), probably because Ea20n fails to be quickly inactivated by inhibitor X.
At a broader level, the analysis of eaD mutations underscores the important relationship between timing of signal production and generation of spatial pattern. Previous studies demonstrated that cleavage of Spätzle is required by Easter for Toll activation. The experiments described here refine this model by showing that the wild-type shape of the Dorsal gradient requires high levels of Easter protease activity during a brief period of time. Failure to properly inactivate Easter leads to a loss of the dorsal domain of the axis, thereby leading to a ventralized phenotype. When this defect is coupled with a reduction in Easter protease function, wild-type levels of processed Spätzle are eventually produced in the embryo, but the shape of the Dorsal gradient becomes more symmetric.
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ACKNOWLEDGMENTS |
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