Download A processed form of the Spätzle protein defines dorsal

1243
Development 120, 1243-1250 (1994)
Printed in Great Britain © The Company of Biologists Limited 1994
A processed form of the Spätzle protein defines dorsal-ventral polarity in the
Drosophila embryo
David S. Schneider*, Yishi Jin†, Donald Morisato and Kathryn V. Anderson
Genetics Division, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA
*Present address: Cardiovascular Research Institute, University of California, San Francisco, CA 94143-0130
†Present address: Department of Biology, MIT, Cambridge, MA 02139
SUMMARY
Stein et al. (1991) identified a soluble, extracellular factor
that induces ventral structures at the site where it is
injected in the extracellular space of the early Drosophila
embryo. This factor, called polarizing activity, has the
properties predicted for a ligand for the transmembrane
receptor encoded by the Toll gene. Using a bioassay to
follow activity, we purified a 24×103 Mr protein that has
polarizing activity. The purified protein is recognized by
antibodies to the C-terminal half of the Spätzle protein,
INTRODUCTION
Extracellular morphogens are molecules distributed in a
gradient across a field of cells that elicit different cell fates as
a function of their concentration. In biochemical terms, an
extracellular morphogen would be a ligand for a membrane
receptor and graded receptor activation would lead to different
cellular responses. The initial establishment of dorsal-ventral
polarity in the Drosophila embryo appears to rely on such an
extracellular morphogen gradient defined by a set of maternal
effect genes, although the identity of the morphogen and the
mechanism of gradient formation have not yet been determined.
Embryonic dorsal-ventral asymmetry is triggered by ventral
activation of the transmembrane protein encoded by the maternally transcribed Toll gene. The consequence of ventral activation of Toll is the production of a ventral-to-dorsal concentration gradient of Dorsal protein in the nuclei of the
blastoderm embryo (Roth et al., 1989; Rushlow et al., 1989;
Steward, 1989). Seven other maternal effect genes, pipe, nudel,
windbeutel, gastrulation defective, snake, easter and spätzle,
act upstream of Toll and are required for the production of an
extracellular signal that activates Toll (Anderson et al., 1985;
Hashimoto et al., 1988, 1991; Stein et al., 1991; Stein and
Nüsslein-Volhard, 1992). The Toll protein is distributed
uniformly around the embryonic circumference (Hashimoto et
al., 1991), suggesting that its activity is confined to the ventral
side of the embryo by ventral localization of the ligand that
activates Toll.
In a series of elegant experiments in which the extracellular
indicating that this polarizing activity is a product of the
spätzle gene. The purified protein is smaller than the
primary translation product of spätzle, suggesting that proteolytic processing of Spätzle on the ventral side of the
embryo is required to generate the localized, active form of
the protein.
Key words: Spätzle protein, Drosophila, dorsoventral polarity,
polarizing activity, Toll
fluid that lies between the plasma membrane of the syncytial
blastoderm embryo and the egg shell (the perivitelline fluid)
was transferred from one embryo to another, Stein et al. (1991)
identified a soluble factor that has the properties predicted for
a Toll ligand. When this factor, called polarizing activity, was
injected into the perivitelline space of a recipient embryo, it
defined the polarity of the dorsal-ventral pattern, with the most
ventral cell types developing at the injection site. Thus this
polarizing activity activated the Toll-dependent nuclear
translocation of Dorsal near the site of injection. The polarizing activity was detectable only in the perivitelline fluid of
embryos that lacked Toll, and could not be retrieved from
embryos with wild-type Toll. This suggested that the polarizing activity might be bound to Toll, when Toll is present. Based
on these data, Stein et al. (1991) suggested that the polarizing
activity was a ligand that binds to and activates Toll at the site
of injection.
In this paper, we use a biochemical approach to characterize the polarizing activity. By measuring the phenotypic
response to material injected into the perivitelline space as a
bioassay, we found that it was possible to recover polarizing
activity from the supernatant of wild-type embryo extracts
boiled at pH 4.5. From this starting material, we partially
purified a 24×103 Mr protein that has polarizing activity. This
protein is recognized by antibodies to the C-terminal half of
the Spätzle, indicating the spätzle gene encodes the polarizing
activity. Polarizing activity cannot be extracted from embryos
laid by females that lack spätzle, but is present in the lowpH/boiled supernatant of mutant embryo extracts that lack
other genes that act upstream of Toll. We present evidence that
1244 D. S. Schneider and others
the low-pH/boiling treatment partially proteolyzes Spätzle,
thereby bypassing the genes that are normally required for processing of Spätzle from an inactive precursor to an active
product. We conclude that a processed form of Spätzle defines
embryonic dorsal-ventral polarity by activating Toll on the
ventral side of the embryo.
MATERIALS AND METHODS
Polarizing activity preparation
Toll− perivitelline extracts were prepared by vortexing 0-12 hour
dechorionated
embryos
produced
by
Toll−
females
[Df(3R)roXB3/Df(3R)Tl9QRX] in buffer (30 mM NaCl, 50 mM Hepes,
pH 7.5, 1 mM PMSF) with silicon carbide powder (grain size 1200,
VWR) for one minute in 20 mM Hepes, pH 7.5, 30 mM NaCl.
RNAase A bound to Sepharose (Boehringer) was used to test RNAase
sensitivity. Protease sensitivity was assayed by treating extracts with
2.5 mg/ml proteinase K for 30 minutes at 37°C; proteinase K was
inactivated by boiling. Supernatant fractions were concentrated for
injection using a Centricon-10.
Polarizing activity from wild-type embryos was prepared by
douncing 0-4 hour embryos with a type A pestle until it moved freely,
in five volumes of 20 mM sodium acetate pH 4.5, 30 mM NaCl.
Extracts were clarified by centrifugation for 5 minutes at 3000 g at
4°C and the supernatants were placed in boiling water for 5 minutes
and then immediately placed in ice. Boiled extracts were cleared at
10,000 g for 10 minutes. Extracts were prepared for injection either
by replacing the acidic buffer with 20 mM Hepes pH 7.5, 30 mM
NaCl using a Centricon 10 microconcentrator or by precipitation with
20% TCA at 4°C followed by an acetone wash and resuspension in
20 mM Hepes pH 7.5, 30 mM NaCl.
Embryo injections
We used embryos laid by pip664/pip386 females as recipients for perivitelline injections. Polarizing activity was injected into the perivitelline space of dechorionated syncytial blastoderm (stage 4a or 4b)
embryos at 50% egg length on the flat (dorsal) side of the embryo.
Samples were assayed at a series of two-fold dilutions, until the
greatest dilution that could still induce an asymmetric head fold was
determined. We defined 1 unit (u)/ml of polarizing activity as the concentration that led 50% of injected pipe− embryos to develop an asymmetric head fold. At least 15 embryos at each concentration of polarizing activity for each sample and column fraction were scored to
determine the activity of the sample.
Cuticle preparations were made as described (Wieschaus and
Nüsslein-Volhard, 1986). Embryos were assayed for expression of
Twist protein with a rabbit anti-Twist antibody (Roth et al., 1989; the
kind gift of S. Roth); injected embryos were fixed as described by
Stein et al. (1991).
Polarizing activity purification
30 g of 0-4 hour thawed frozen Oregon R embryos were homogenized
in 5 volumes of 20 mM acetate pH 4.5, 30 mM NaCl, 1 mM PMSF
on ice. Extracts were clarified by centrifugation for 10 minutes at 3000
g at 4°C. 10 ml aliquots of the supernatant in 15 ml Falcon tubes
(Fisher) were placed in boiling water for 5 minutes and were then
immediately cooled to 4°C on ice and then cleared by centrifugation
at 10,000 g for 10 minutes. Protein was precipitated by adjusting
extracts to an ammonium sulfate concentration of 95%. Precipitated
material was collected by centrifugation at 10,000 g for 10 minutes.
The particulate matter was resuspended in 5 ml of 20 mM acetate pH
4.5, 30 mM NaCl (Buffer A) and dialyzed overnight at 4°C against 4
liters of the same buffer. The dialysate was clarified by centrifugation
at 10,000 g for 10 minutes. The supernatant was adjusted to 25%
ammonium sulfate at 4°C and cleared by centrifugation at 10,000 g
for 10 minutes. The supernatant was brought to 40% ammonium
sulfate concentration at 4°C and the precipitate was collected by centrifugation at 10,000 g for 10 minutes and resuspended in 1 ml of 20
mM Hepes pH 7.5, 30 mM NaCl (Buffer B). This resuspended
material was cleared by centrifugation for 5 minutes at 10,000 g. The
supernatant was loaded onto a 1.6×68 cm Ultragel AcA 54 sizing
column equilibrated with Buffer B and eluted at a flow rate of 12
ml/hour with the same buffer at 4°C. Undiluted fractions were assayed
for polarizing activity by injection into pipe− embryos as described
above. Pooled active fractions were loaded onto a HiTrap Blue
column (Cibacron Blue 3GA Sepharose) at 1.2 ml/hour, equilibrated
with Buffer B, washed with 10 ml of buffer B at 6 ml/hour and eluted
with 20 mM Hepes pH 7.5, 1 M NaCl. To prepare fractions for injections, each aliquot was adjusted to 20% TCA in the presence of 1 mg
of BSA for carrier. Samples were incubated on ice for at least one
hour. Precipitated material was washed once with −20°C acetone,
resuspended in Buffer B plus 0.3 mg/ml BSA and stored on ice.
Protein from active fractions was precipitated by the addition of TCA
to 20%. Acetone-washed pellets were resuspended in Laemmli
sample buffer without a reducing agent. Samples were run on 15%
acrylamide mini gels. The entire lane was divided into 2 mm gel slices
which were crushed and then eluted in 0.2 ml of Buffer B plus 0.1%
SDS overnight at 4°C. All gel slices were assayed for activity. Protein
was prepared for injection by acetone precipitation by adding 5
volumes of −20°C acetone and incubating −20°C for 2 hours. Acetone
washed pellets were resuspended in Buffer B.
Western blots were performed as described (Morisato and
Anderson, 1994).
RESULTS
Mass preparation of polarizing activity
The original source of the soluble extracellular component that
could activate the Toll pathway (the polarizing activity) was
perivitelline fluid from embryos laid by Toll− females (Stein et
al., 1991). In those experiments, nanoliter drops of the fluid
were recovered with a micropipette after pricking the vitelline
membrane of the embryos (Stein et al., 1991). Any biochemical characterization of the active component required a larger
source of material. To isolate more perivitelline fluid, we used
a procedure that enriched for perivitelline contents by
vortexing dechorionated embryos in buffer with silicon carbide
particles (Chasan et al., 1992; Materials and Methods).
Perivitelline extracts were made from embryos laid by Toll−
mothers and assayed for biological activity. We used the dorsalized embryos laid by pipe− mothers as recipients in this
assay, because it has been shown that injection of polarizing
activity into these dorsalized embryos leads to the production
of ventral structures at the injection site (Stein et al., 1991).
Furthermore, the dorsalized pipe− phenotype cannot be rescued
by wild-type RNA, cytoplasm or perivitelline fluid injected
into the embryo (Anderson and Nüsslein-Volhard, 1984; Stein
and Nüsslein-Volhard, 1992), ruling out the possibility of characterizing a pipe rescuing activity rather than polarizing
activity. When the Toll− perivitelline extract was injected into
the perivitelline space of pipe− embryos, the embryos showed
a partial restoration of dorsal-ventral pattern elements, with the
most ventral pattern elements differentiating near the injection
site. Thus the Toll− perivitelline extracts, like Toll− perivitelline
fluid, defined the polarity of the embryonic pattern. The polarizing activity present in the Toll− perivitelline extracts was
stable to RNAase A, but was destroyed by treatment with pro-
Spätzle protein defines dorsal-ventral polarity in Drosophila 1245
A
D
E
B
F
C
G
Fig. 1. Rescue of dorsal-ventral pattern elements by perivitelline injection of polarizing activity. Cuticular patterns of uninjected (A,B) and
injected (C) embryos and gastrulation patterns of uninjected (D,E) and injected (F,G) embryos. All embryos are shown dorsal side up, anterior
to the left. The dorsal-ventral pattern elements seen in the cuticle of the wild-type larva (A) include ventrally derived denticle belts,
dorsolaterally derived filzkörper and dorsal hairs. Uninjected embryos laid by pipe− females are dorsalized; they differentiate dorsalized cuticle
(B) that is covered with dorsal hairs around its circumference and lacks both dorsolaterally derived filzkörper and ventrolaterally derived
ventral denticles. An embryo from a pipe− mother injected with polarizing activity prepared from wild-type embryo extracts (C) differentiated
ventral denticle belts and filzkörper (arrow) near the injection site. During gastrulation, the wild-type embryo (D) extends its germ band along
the flat (dorsal) side of the embryo (arrowhead) and the head fold begins at a lateral position. Embryos laid by pipe− females do not make a
ventral furrow and do not undergo germband elongation; instead the embryo forms symmetrical dorsal folds that encircle the embryo (E). In an
embryo laid by a pipe− female and injected with polarizing activity at 4 u/ml (F), the head fold was deepest on the injected side of the embryo
(arrow), indicating that the cells near the injection site behaved like the lateral cells of the wild-type embryo. This was the phenotype used to
follow polarizing activity through the purification procedure. The embryo shown in G was also injected with polarizing activity at 4 u/ml and
aged 20 minutes longer than the embryo shown in F. The embryo shown in G extended its germ band toward the curved, normally ventral side
of the embryo (arrowhead), the opposite direction from germ band extension in wild-type embryos, showing that the polarizing activity defined
the polarity of the embryo.
1246 D. S. Schneider and others
Table 1. Response of dorsal group mutant embryos to
injected polarizing activity
Maternal genotype
pip664/pip386
ndl111/ndl133
gd7/gd7
ea4/ea5022rx1
spzD1RPQ/Df(3R)Tl84cRXD
Tl5BREQ/Tl9QRE
n
Asymmetric
head fold
Germ band
extension
92
37
60
137
80
104
92%
90%
83%
92%
96%
0%
80%
30%
42%
41%
55%
0%
Dorsalized embryos laid by females that lack Toll or the dorsal group genes
that act upstream of Toll were assayed for their ability to respond to
polarizing activity. For each gene, the allele combination used produced
strongly dorsalized embryos. Low-pH/boiled extracts were prepared from 0-4
hour wild-type embryos. Embryos were injected with polarizing activity at 4
u/ml. All of the mutants except Toll responded to polarizing activity,
indicating that, of these genes, only Toll is required to respond to polarizing
activity. n indicates the number of embryos injected.
teinase K, indicating that the active component was a protein.
The polarizing activity was very stable: its biological activity
was not destroyed by heating to 100°C at either pH 7.0 or pH
4.5 (data not shown). The stability of the polarizing activity
was useful in the strategy that we developed to purify the
protein.
Perivitelline extracts from Toll− embryos did not provide
enough material for more extensive biochemical characterization of the polarizing activity. Due to female sterility, Toll−
flies cannot be grown as a homozygous population, and
therefore must be generated by crosses. In addition, Toll has
an important zygotic function and 95% of the Toll− zygotes
expected in a cross die during development (Gerttula et al.,
1988). We therefore tried to recover polarizing activity from
wild-type embryos.
Stein et al. (1991) proposed that polarizing activity was
detectable in perivitelline fluid only in Toll− embryos because
the polarizing activity would be bound to the Toll protein in
wild-type embryos. By this logic, polarizing activity should be
present in wild-type embryos, but in a tightly bound complex.
Because the polarizing activity in Toll− perivitelline extracts
was stable to boiling at pH 4.5, we hoped that these harsh conditions would release any polarizing activity present in wildtype embryos from a complex. Wild-type embryo homogenates
were heated to 100°C at pH 4.5. Although 99.5% of embryonic
proteins were precipitated by boiling at pH 4.5, the supernatant
contained polarizing activity: concentrated supernatant
fractions induced ventral structures at the site of injection when
injected into the perivitelline space of pipe− embryos (Fig. 1).
To test whether the polarizing activity isolated from wildtype embryos by this procedure activated Toll, we assayed
which dorsal group genes were required to respond to the
preparations from wild-type embryos (Table 1). As Stein found
for the polarizing activity in perivitelline fluid, the polarizing
activity in low pH/boiled wild-type embryo extracts induced
the formation of ventral structures in embryos lacking the
dorsal group genes that act upstream of Toll (nudel, pipe, gastrulation defective, easter and spätzle), but had no activity in
Toll− embryos. Therefore, like Stein’s activity, the wild-type
polarizing activity worked by activating Toll and bypassed the
requirement for all of the genes upstream of Toll. Thus, the
low-pH/boiled supernatant fraction of wild-type embryo
Fig. 2. Response of embryos to different concentrations of polarizing
activity. To determine the response of embryos to different doses of
polarizing activity, dilutions of polarizing activity were injected into
embryos laid by pipe− females. Crude polarizing activity was
prepared for injection from low-pH/boiled 0-4 hour embryos. Four
pattern elements that arise at different positions on the dorsal-ventral
circumference of the wild-type embryo were used to monitor the
rescue response: an asymmetric position of the head fold at the onset
of gastrulation, a dorsolateral pattern element (closed circles);
filzkörper in the cuticle, a dorsolateral pattern element (open
squares); ventral denticles in the cuticle, a ventrolateral pattern
element (open triangles); ventral furrow at gastrulation, a ventral
pattern element (closed squares). The induction of a head fold on the
injected side of the embryo (closed circles) was the most sensitive
response to injected polarizing activity. We therefore defined 1 unit
(u)/ml of polarizing activity as the concentration that led 50% of
injected pipe− embryos to develop an asymmetric head fold.
Progressively more ventrally derived structures required higher
concentrations of injected polarizing activity: 4.2 u/ml gave 50% of
the embryos with filzkörper; 44 u/ml gave 50% with ventral
denticles; 150 u/ml gave 50% with a ventral furrow. At this highest
concentration of polarizing activity injected, the embryos appeared
ventralized: the ventral furrow looked enlarged and, while the
embryos differentiated the ventrolaterally-derived ventral denticles,
they failed to differentiate dorsolaterally derived filzkörper.
Injected material was deposited in the perivitelline space at 50% egg
length on the flat (dorsal) side of the embryo. Injection of polarizing
activity into these dorsalized embryos defined the orientation of the
dorsal-ventral embryonic axis such that the site of the deposition of
polarizing activity defined the most ventral cell type in the embryo.
All points represent at least 50 gastrulating embryos or cuticle
preparations
extracts provided an abundant source of material for further
characterization of polarizing activity.
Different concentrations of polarizing activity induce
different cell types
With an extract containing polarizing activity in hand, it
became possible to determine a dose-response relationship for
the activity. We injected extracts at various concentrations into
the perivitelline space of dorsalized pipe− embryos and assayed
the effects on the pattern of gastrulation, the cuticle and the
expression of the ventral marker protein Twist.
Different concentrations of the polarizing activity preparation induced the formation of a series of progressively more
ventral cell types over a 150-fold range in concentration (Fig.
2). In all cases, the rescued pattern was dorsoventrally asymmetric, with the ventral-most structures appearing during gastrulation nearest the site where the polarizing activity was
deposited. Uninjected pipe− embryos were completely dorsal-
Spätzle protein defines dorsal-ventral polarity in Drosophila 1247
A
B
Fig. 3. Expression of Twist in pipe− embryos injected with high
concentrations of polarizing activity. The Twist protein is expressed
in the ventral, presumptive-mesodermal cells of the wild-type
embryo and serves as a marker for ventral cell fates (Leptin and
Grunewald, 1990). (A) The expression pattern of Twist in a wildtype embryo at the beginning of germ band extension, showing that
Twist is expressed only in the cells near the curved, ventral side of
the embryo. (B) In a pipe− embryo that was injected with 200 u/ml
polarizing activity into the perivitelline space along the flat, dorsal
side of the embryo at the syncytial blastoderm stage, Twist was
expressed in the cells nearest the injection site. Injection at this site
caused the germ band to extend in a polarity opposite to that of the
wild-type embryo, towards the curved side of the embryo.
ized both at gastrulation and in the differentiated cuticle. The
first detectable response to injection of low concentrations of
polarizing activity into pipe− embryos was the induction of an
asymmetric head fold during gastrulation, with a head fold (a
lateral pattern element) appearing on the side near the injection
site (Figs 1, 2). At progressively higher concentrations, first
dorsolateral, then ventrolateral and finally ventral structures
were induced. High concentrations of polarizing activity led to
the formation of the most ventral pattern element, the
mesoderm, as shown by the invagination of a ventral furrow at
the site of injection and the expression of the mesodermspecific protein Twist (Fig. 3). The production of mesoderm in
50% of the injected embryos required polarizing activity that
was 150-fold more concentrated than the concentration that
produced the weakest detectable response, the production of a
head fold, in 50% of the injected embryos (Fig. 2). Thus, large
differences in the concentration of polarizing activity are
required to produce the full range of dorsal-ventral pattern
elements.
Based on this dose-response relationship, the most sensitive
assay for polarizing activity was the induction of an asymmetric head fold at the time of gastrulation. This assay was also
rapid, because gastrulation occurs 2 hours after injection. In
addition, this assay was sensitive to small differences in polarizing activity concentrations. We therefore used the morphology of the head fold to follow the polarizing activity during
purification.
Purification of polarizing activity
Using the pattern of gastrulation as a bioassay, we used
standard biochemical procedures to purify the polarizing
activity. The starting material for the purification was the
supernatant from 0-4 hour wild-type embryo extracts heated to
100°C at pH 4.5. Ammonium sulfate precipitation, gel filtration chromatography, dye ligand chromatography and elution
from an SDS-polyacrylamide gel were then used as successive
steps to purify the polarizing activity (Table 2). At each step,
fractions were injected into pipe− embryos and the pattern of
gastrulation was monitored in order to identify the peak of
activity. The peak fractions were pooled and used as the
starting material for the next purification step. Based on the
migration of the active fraction on non-reducing gels, the
purified polarizing activity was about 24×103 Mr in size. The
final fraction was enriched more than 1000-fold for polarizing
Table 2. Purification table
Step
Neutral homogenization
Acid homogenization
Boiling at pH 4.5
Ammonium sulfate cut
AcA54 sizing column
Cibacron blue 3GA
Gel elution
Total activity
[units]
<138
<35
128
68
64
9.5
0.6
Total protein
[mg]
2880
72
11
2.5
0.3
0.03
<0.00005
Specific activity
[u/ml]
<0.048
<0.5
11.6
27.2
213
316
>12,000
Purification
1
2.3
18
27
1034
Yield
(%)
100
53
50
7.5
0.46
Each step in the purification was assayed for activity and total protein, to give the specific activity, fold purification and yield at that step. Activity was
determined using an endpoint dilution assay in which the concentration of the sample was decreased in two-fold increments until only 50% of the injected
embryos made an asymmetric head fold; that concentration was defined as 1 u/ml. Purification and yield were calculated from the first step in which the amount
of polarizing activity could be accurately determined (Boiling). No activity was found in total embryo extracts prepared at neutral pH (Neutral homogenization)
or at pH 4.5 (Acid homogenization). The specific activities in these two preparations represent maximum estimates, based on the lowest activity level that could
have been detected and the total protein in the preparation. If we take the specific activity of the neutral homogenate and compare it to the specific activity of the
most purified sample, then the overall purification is more than 250,000-fold. Because the low pH-boiling step leaves only about 1% of the total protein in the
supernatant, this step is a purification step. However since the low-pH/boiling step also appears to generate polarizing activity by proteolytic cleavage, we cannot
accurately calculate the enrichment of polarizing activity at that step. The neutral and acid homogenization data represent two different preparations; all other
samples are from the acid homogenization preparation shown.
1248 D. S. Schneider and others
Table 3. Presence of polarizing activity in extracts from
dorsal group mutant embryos
Source of extract
(Maternal genotype)
Asymmetric head
fold
n
wild type (Oregon R)
pip664/pip386
ea4/ea5022rx1
spzD1RPQ/Tl84cRXD
83%
82%
82%
0%
24
157
100
43
Embryos laid by females carrying strongly dorsalizing alleles of the dorsal
group genes that act upstream of Toll were assayed for the presence of
polarizing activity. Low-pH/boiled extracts were prepared from 0-4 hour
embryos. Extracts at total protein concentrations of 1 mg/ml were assayed for
polarizing activity by injection into pipe− embryos. Of those mutants tested,
only spätzle− embryos lacked polarizing activity. n indicates the number of
pipe− embryos injected.
activity. This was surprising not only because extracts from
pipe mutants rescued the pipe phenotype, but also because
Stein et al. (1991) showed that all of the genes acting upstream
of Toll were required for the presence of polarizing activity in
perivitelline fluid. We conclude that the low-pH/heat treatment
bypassed the requirements for easter and pipe for the production of polarizing activity and that the treatment produced
polarizing activity in a nonphysiological way.
Genetic experiments had indicated that, of the seven genes
that act upstream of Toll, easter and spätzle are most directly
required to activate Toll (Chasan et al., 1992; Morisato and
A
activity relative to the initial low-pH/boiled supernatant.
However, despite this enrichment, there was not enough of the
purified protein to detect on a silver-stained gel, suggesting that
the protein was very rare in embryos. Because we did not have
enough material to determine the N-terminal sequence of the
protein, we asked whether the purified protein was encoded by
a known dorsal group gene.
Polarizing activity is a product of the spätzle gene
To determine which gene products were required for the production of polarizing activity, we tested whether polarizing
activity was detectable in embryos laid by mothers homozygous for dorsal group mutations. Extracts were made from
embryos laid by mutant mothers and the supernatant after
heating to 100°C at pH 4.5 was assayed for the ability to induce
ventral structures (Table 3). Embryos from spätzle mutant
females did not contain detectable polarizing activity, indicating that spätzle was required for the presence of polarizing
activity. However, low-pH/heat-treated extracts from embryos
from easter or pipe mutant females did have polarizing
Fig. 4. Polarizing activity copurifies with Spätzle. (A) Ultragel AcA
54 gel filtration. Upper curve shows absorption at 280 nm monitored
across the column. Each fraction was tested for activity by injection
into embryos laid by pipe− females; active fractions were those that
induced a head fold in more than 50% of the embryos. To assay for
Spätzle, proteins were separated by SDS-PAGE on a 15% gel under
reducing conditions and blots were probed with antibodies directed
against the C terminus of Spätzle (Morisato and Anderson, 1993).
(B) Steps in purification of polarizing activity. Upper panel: silver
stained 15% polyacrylamide gel of samples of the protein at each
step of the purification, run under reducing conditions. 2 µg of
protein from each of the first four steps of the purification; the
described amounts of protein from the last three steps. Protein
concentration was determined by Bradford assay for the first four
steps, A280 for the next two steps and comparative silver staining
intensity for the last step. No protein was detected by silver staining
in the most highly purified fractions, suggesting that the upper limit
of the amount of protein loaded in this lane was 10 ng, because this
was the smallest amount of BSA that we detected under these
conditions. Lower panel: western blot analysis of samples from each
step of the purification, separated on a 15% polyacrylamide gel under
non-reducing conditions. 140 ng total protein was loaded in each
lane, except for the last lane in which an upper limit of 10 ng of
protein was loaded. The increase in the relative abundance of Spätzle
is most clearly seen in the final steps of the purification.
Mr×10−3
B
Mr
×10−3
Mr×10−3
Spätzle protein defines dorsal-ventral polarity in Drosophila 1249
Anderson, 1994). Because in our assay spätzle, but not easter,
was required for the presence of polarizing activity, we
conclude that Spätzle acts downstream of Easter in the pathway
that leads to Toll activation. Thus the spätzle gene is the only
known dorsal group gene that could encode the polarizing
activity.
We tested whether polarizing activity was encoded by
spätzle using antibodies raised against fusion proteins containing the spätzle open reading frame (Morisato and
Anderson, 1994). In extracts prepared at pH 7.5 from wild-type
embryos, there is a family of proteins encoded by Spätzle that
migrates at 45 to 60×103 Mr under reducing conditions
(Morisato and Anderson, 1994). In embryo extracts boiled at
pH 4.5, the Spätzle protein migrated at 14 to 18×103 Mr under
reducing conditions and at approximately 24×103 Mr under
non-reducing conditions. Thus low-pH/boiling apparently
caused partial proteolysis of Spätzle. The protein recognized
by Spätzle antibodies was present in the low-pH/heated
extracts from wild type, but not spätzle−, embryos, indicating
that the low molecular weight protein is a product of spätzle.
Because low pH-boiled material had polarizing activity but
whole embryo extracts did not, we propose that low-pH/boiling
converts an inactive form of Spätzle into an active, cleaved
product.
The protein recognized by the Spätzle antibodies copurified
with the polarizing activity measured by the bioassay through
the gel filtration, dye ligand chromatography and polyacrylamide gel steps (Fig. 4A,B). This Spätzle protein was highly
enriched in the final steps of the purification, as seen when
equal amounts of total protein were loaded on a gel and
immuno-blotted with the Spätzle antibodies (Fig. 4B). Because
the protein recognized by the Spätzle antibodies copurified
exactly with the polarizing activity, we conclude that the polarizing activity is a product of the Spätzle gene.
The purified Spätzle protein that had the polarizing activity
was smaller than the 45-60×103 Mr primary translation
products of Spätzle (Morisato and Anderson, 1994). Antibodies directed against the C-terminal, but not the N-terminal half,
of the spätzle open reading frame recognized the polarizing
activity (data not shown), suggesting the polarizing activity
included C-terminal, but not N-terminal, portions of the
primary Spätzle translation product.
DISCUSSION
We have used a biochemical approach to characterize an extracellular factor that defines dorsal-ventral polarity in the
Drosophila embryo. The factor that we identified biochemically is encoded by the spätzle gene, which by genetic criteria
acts immediately upstream of the receptor encoded by Toll
(Morisato and Anderson, 1994). Thus, biochemistry and
genetics have converged to define a single extracellular
molecule that activates Toll on the ventral side of the embryo
and thereby defines the polarity of the embryo.
A processed form of the Spätzle protein induces
dorsal-ventral polarity
Using a bioassay developed by Stein et al. (1991), we partially
purified a protein from wild-type embryos extracts that induces
an organized dorsal-ventral pattern in the Drosophila embryo.
Like the activity defined by Stein et al. (1991), the purified
polarizing activity induces ventral structures at the site of
injection and produces a set of lateral and dorsolateral pattern
elements in their normal spatial order. Of the genes that act
upstream of the receptor Toll, spätzle, but not pipe or easter,
is required for the presence of polarizing activity in lowpH/boiled extracts, suggesting that spätzle encodes the polarizing activity. A 24×103 Mr form of Spätzle protein copurifies
with the biological activity that induces ventral structures
throughout the entire purification procedure. We conclude that
a form of the Spätzle protein is the extracellular molecule that
induces dorsal-ventral polarity when injected into the extracellular space.
The form of Spätzle that we purified is smaller than the
primary translation products of the spätzle gene (Morisato and
Anderson, 1994) and appears to represent a proteolytic product
of full-length Spätzle protein. The Spätzle protein in wild-type
embryos appears to undergo proteolytic processing to generate
the active form of the protein (Morisato and Anderson, 1994).
The proteolytic processing of Spätzle in wild-type embryos
appears to produce a form of the protein in which the Nterminal and C-terminal halves of the cleaved product
remained disulfide-bonded together (Morisato and Anderson,
1994). In contrast, the low-pH/boiled form of Spätzle that we
purified appears to be a C-terminal domain that is not disulfidebonded to the N terminus of the protein. The polarizing activity
of the isolated C-terminal domain indicates that the protein
sequences required for activation of Toll are localized in the
C-terminal portion of the precursor molecule.
We were fortunate because heating embryo extracts at pH
4.5 processes Spätzle in vitro in a way that converts the fulllength protein to a protein with polarizing activity. While we
had hoped to liberate the polarizing activity from a complex
with Toll in wild-type embryos by the low pH and heat
treatment, instead we generated polarizing activity, apparently
by inducing a proteolytic cleavage event. Although most
peptide bonds are stable under these conditions, some peptide
bonds, aspartate-proline, asparagine-glutamate and asparagineglycine, hydrolyze at high temperature and low pH (Landon,
1977; Voorter et al., 1988; Martin et al., 1990); however,
Spätzle does not contain any of these dipeptides. Therefore we
infer that the proteolysis of Spätzle is either an unusual acid
hydrolysis event or that unfolding of the protein at high temperature and low pH renders it accessible to a protease present
in the extracts. In either case, the biological activity of the
material suggests that the low-pH/boiling treatment creates a
product similar to that produced by the normal proteolytic processing of Spätzle, perhaps because a region of the protein is
exposed or strained and therefore particularly labile.
Processed Spätzle activates Toll on the ventral side
of the wild-type embryo
In order to induce an asymmetric dorsal-ventral pattern, the
form of Spätzle that we purified must remain localized
following injection. We assume that Spätzle binds to the
plasma membrane near the site of injection and, as a consequence of Spätzle binding, Toll is activated at that position in
the embryo. The simplest way to account for these findings is
to infer that processed Spätzle is the ligand that binds directly
to Toll and that triggers Toll to relay a signal to the cytoplasm.
The available genetic and biochemical data indicate that
1250 D. S. Schneider and others
spätzle acts immediately upstream of Toll and no other genes
are known that act at that step in the pathway, which is also
consistent with the hypothesis that processed Spätzle is the Toll
ligand. Now that both the receptor and its putative ligand have
been cloned, it will be possible to test directly whether
processed Spätzle binds to and activates Toll.
Whether or not processed Spätzle acts as a classic ligand for
Toll, our experiments show that processed Spätzle is the limiting
component that determines where Toll is active. The doseresponse relationships that we observed show that different concentrations of processed Spätzle induce different cell types.
Therefore it seems likely that processed Spätzle is distributed in
a gradient in the perivitelline space and that different concentrations of processed Spätzle lead to a gradient of Toll activity
which leads to the nuclear gradient of Dorsal protein.
The dose-response curve for the processed form of Spätzle
that we purified is rather shallow: a 150-fold higher concentration of processed Spätzle is required to induce the most
ventral pattern element, the mesoderm, than is required to
produce a detectable dorsolateral cell fate. Thus if Spätzle acts
as a graded morphogen to organize the dorsal-ventral
embryonic pattern, its concentration gradient would be very
steep, with processed Spätzle two orders of magnitude more
concentrated on the ventral midline than at the lateral positions.
This suggests that the production of a gradient of processed
Spätzle requires stringent mechanisms to insure a high ventral
concentration of the processed protein without that protein
spreading to more lateral positions.
We thank Sylvia Sanders, David King and Tohru Yoshihisa for
advice with protein purification procedures. We thank Siegfried Roth
for the kind gift of Twist antibodies. We also thank Lisa Molz, Sylvia
Sanders and the members of the Anderson lab for reading this work.
D. M. was a Fellow of the Miller Institute for Basic Research in
Science and the Dupont Fellow of the Life Sciences Research Foundation. The work was supported by National Institutes of Health grant
GM 35437 and National Science Foundation Faculty Awards for
Women DCB-9023672 to K. V. A.
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(Accepted 7 February 1994)