A gradient of bicoid protein in Drosophila embryos.
ABSTRACT The maternal gene bicoid (bcd) organizes anterior development in Drosophila. Its mRNA is localized at the anterior tip of the oocyte and early embryo. Antibodies raised against bcd fusion proteins recognize a 55-57 kd doublet band in Western blots of extracts of 0-4 hr old embryos. This protein is absent or reduced in embryonic extracts of nine of the 11 bcd alleles. The protein is concentrated in the nuclei of cleavage stage embryos. It cannot be detected in oocytes, indicating temporal control of bcd mRNA translation. The bcd protein is distributed in an exponential concentration gradient with a maximum at the anterior tip, reaching background levels in the posterior third of the embryo. The gradient is probably generated by diffusion from the local mRNA source and dispersed degradation.
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ABSTRACT: In pattern-forming developmental systems, cells commonly interpret graded input signals, known as morphogens. Morphogens often pattern tissues through cascades of sequential gene expression steps. Such a multi-tiered structure appears to constitute suboptimal use of the positional information provided by the input morphogen because noise is added at each tier. However, the conventional theory neglects the role of the format in which information is encoded. We argue that the relevant performance measure is not solely the amount of information carried by the morphogen, but the amount of information that can be accessed by the downstream network. We demonstrate that quantifying the information that is accessible to the system naturally explains the prevalence of multi-tiered network architectures as a consequence of the noise inherent to the control of gene expression. We support our argument with empirical observations from patterning along the major body axis of the fruit fly embryo.01/2015;
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ABSTRACT: In pattern-forming developmental systems, cells commonly interpret graded input signals, known as morphogens. Morphogens often pattern tissues through cascades of sequential gene expression steps. Such a multi-tiered structure appears to constitute suboptimal use of the positional informa-tion provided by the input morphogen because noise is added at each tier. However, the conventional theory neglects the role of the format in which information is encoded. We argue that the relevant performance measure is not solely the amount of information carried by the morphogen, but the amount of information that can be accessed by the downstream network. We demonstrate that quantifying the information that is accessible to the system naturally explains the prevalence of multi-tiered network architectures as a consequence of the noise inherent to the control of gene expression. We support our argument with empirical observations from patterning along the major body axis of the fruit fly embryo.arXiv:1501.07342. 01/2015;
Cell, Vol. 54, 83–93, July 1, 1988, Copyright 1988 by Cell Press
A Gradient of bicoid Protein in Drosophila Embryos
1959; Kalthoff, 1979; Lehmann and Nu ¨sslein-Volhard,
1986; Frohnho ¨fer and Nu ¨sslein-Volhard, 1986; Weeks and
Melton, 1987; and see below). The graded distribution of
substances with biological activity has been demon-
strated in the cases of the “head activator” in hydra
(Schaller and Gierer, 1973; Schaller and Bodenmu ¨ller,
1981) and retinoic acid in chick limb buds (Maden, 1982;
Thaller and Eichele, 1987), yet no morphogen gradient
has been demonstrated in any early embryo. During Dro-
sophila embryogenesis, the products of the genes caudal
(cad) (Mlodzik et al., 1985; Macdonald and Struhl, 1986;
Mlodzik and Gehring, 1987) and hunchback (hb) (Tautz,
1988) are transiently distributed in shallow concentration
gradients. However, the functions of these gradients are
For the Drosophila embryo, evidence from experimental
embryology (Frohnho ¨fer et al., 1986) as well as genetic
analysis (Nu ¨sslein-Volhard, 1979; Nu ¨sslein-Volhard et al.,
1987) indicates that the anteroposterior pattern is deter-
mined by two opposing gradients, with sources at the an-
terior and posterior egg poles, respectively (Lehmann
and Nu ¨sslein-Volhard, 1986; Frohnho ¨fer and Nu ¨sslein-
Volhard, 1986). Several lines of evidence indicate that the
gene bicoid (bcd) is responsible for the anterior gradient.
In embryos from bcd?females, head and thorax are lack-
ing and are replaced by a posterior telson. Transplantation
of cytoplasm from the anterior tip of wild-type embryos into
bcd?embryos can restore a near-normal pattern as well
as induce anterior structures at ectopic positions. The size
and quality of the induced anterior structures depend on
the amount (concentration) of the transplanted bcd?ac-
tivity, which itself is determined by the number of wild-type
bcd?gene copies in the donor female (Frohnho ¨fer and
Nu ¨sslein-Volhard, 1986). The bcd gene has been cloned
and sequenced. It codes for an mRNA that is localized at
the anterior tip of the oocyte and early embryo (Frigerio
et al., 1986; Berleth et al., 1988).
A striking property of the bcd?activity is its long-range
effect on neighboring regions. In bcd?embryos not only
are the structures normally formed at the site of mRNA lo-
calization deleted, but the anlagen of the entire anterior
egg half are also lacking. Furthermore, the posterior anla-
gen are enlarged and spread toward the anterior (Frohn-
ho ¨fer and Nu ¨sslein-Volhard, 1986). In transplantation ex-
periments using bcd?activity, the polarity and pattern of
the embryo along more than half of its length can be
changed (Frohnho ¨fer et al., 1987). These extraordinary
features of the bcd gene can best be explained by invok-
ing a gradient mechanism in which different concentra-
tions of the bcd gene product determine the series of
different structures along the anterior pattern (Frohnho ¨fer
and Nu ¨sslein-Volhard, 1986, 1987; Nu ¨sslein-Volhard et al.,
1987). Since the bcd mRNA is strictly localized at the an-
terior tip of the wild-type embryo, the RNA itself cannot ful-
fill the role of the anterior morphogen. The bcd protein,
however, is a good candidate for the anterior gradient mol-
ecule. In addition, the presence of a homeobox in the cod-
Wolfgang Driever and Christiane Nu ¨sslein-Volhard
Max-Planck-Institut fu ¨r Entwicklungsbiologie
Abteilung III Genetik
7400 Tu ¨bingen, Federal Republic of Germany
The maternal gene bicoid (bcd) organizes anterior de-
velopment in Drosophila. Its mRNA is localized at the
anterior tipof the oocyte andearly embryo. Antibodies
raised against bcd fusion proteins recognize a 55–57
kd doublet band in Western blots of extracts of 0–4 hr
old embryos. This protein is absent or reduced in em-
bryonic extracts of nine of the 11 bcd alleles. The pro-
tein is concentrated in the nuclei of cleavage stage
embryos. It cannot be detected in oocytes, indicating
temporal control of bcd mRNA translation. The bcd
protein is distributed in an exponential concentration
gradient with a maximum at the anterior tip, reaching
background levels in the posterior third of the embryo.
The gradient is probably generated by diffusion from
the local mRNA source and dispersed degradation.
Gradients in development have been invoked as mecha-
nisms for creating spatial diversity from seemingly uni-
form states since the beginning of this century (Morgan,
1901; Child, 1915). They were postulated on the basis of
transplantation and isolation experiments performed in a
number of embryonic systems, such as sea urchins
(Runnstro ¨m, 1929; Ho ¨rstadius, 1939), amphibians (Dalcq
and Pasteel, 1938), and insects (Sander, 1959, 1976). In
these experiments it appeared that the differentiation
properties of the tissue along the embryonic axes
changed in a quantitative rather than qualitative manner,
and could best be explained by the gradual change of the
concentration of a morphogenetic substance.
According to the concept of positional information (Wol-
pert, 1969), the concentration of a morphogen instructs
cells within an embryonic field of their position. The cells
then interpret this information by an appropriate program
of differentiation. Several models have been proposed
that describe the generation of stable gradients of mor-
phogens which could specify subregions of the embryo in
a concentration-dependent manner. The simplest gra-
dient models use the property of diffusion to describe the
distribution of the morphogen, starting with an initial
asymmetry in the form of a local source (Lawrence, 1966;
Stumpf, 1966; Crick, 1970; Lewis et al., 1977). In models
involving autocatalysis and lateral inhibition, stable con-
centration gradients can result from very slight, random
fluctuations (Gierer and Meinhardt, 1972). In several in-
stances, localized entities have been found that are good
candidates for sources of morphogen gradients (Sander,
Figure 1. Structures of the bcd Transcripts and bcd Fusion Proteins, and Specificity of the Anti-bcd Antibody
(A) Structure of the most abundant, 2.6 kb bcd mRNA (a) and of the species with a shorter second intron (b). Junction of the splice donor
site of the first intron and the splice acceptor site of the last intron results in a 1.6 kb mRNA (c; see also Berleth et al., 1988). (B) Putative
proteins derived from the longest open reading frames in the above transcripts. The PRD repeat and the homeobox are indicated by shaded
areas. Protein type b would contain five additional amino acids just in front of the homebox domain (black box). Type c protein (149 amino
acids, 16.4 kd) lacks the homeobox. (C) Structures of the bcd fusion proteins: (a) LacZ–bcd fusion protein containing amino acids 112 to 489
of a type b protein (see Experimental Procedures); (b) TrpE–bcd fusion protein. (D) Specificity of anti-bcd antibodies. Coomassie blue–stained
SDS-PAGE gels (lanes 1, 2, 5, 6) and Western blots (lanes 3, 4, 7, 8) showing total protein from induced bacteria containing the unfused lacZ
gene (lanes 1 and 3), the lacZ–bcd fusion gene (lanes 2 and 4), the unfused trpE gene (lanes 5 and 7), or the trpE–bcd fusion gene (lanes 6
and 8). Bands in lanes 4 and 8 with lower apparent molecular weights than the bcd fusion proteins are due to breakdown products of unstable
fusion proteins. Arrows indicate the sizes of the fusion and nonfusion proteins.
ing region of the bcd gene (Frigerio et al., 1986; Berleth
et al., 1988) suggests that the protein may regulate the ex-
pression of zygotic genes.
In this paper we show that the bcd gene product is a
55 kd protein translated soon after egg deposition. It
is distributed in a steep concentration gradient whose
maximum is at the anterior egg pole. We measured the
distribution of bcd protein in wild-type embryos and
found an exponential decay in protein concentration, a
distribution pattern which can be readily explained by
models involving a local source, diffusion, and dispersed
decay. The protein is detectable at up to 30% egg length
(where 0% isthe posterior pole), providingan explanation
for the long-range effects observed in bcd?embryos.
The accompanying paper (Driever and Nu ¨sslein-Volhard,
1988) provides evidence for the hypothesis that the posi-
tions of the embryonic anlagen in the anterior half of the
embryo are determined by the bcd product in a concen-
1C). Antibodies specific for the bcd gene product were
purified by affinity chromatography on a TrpE–bcd fusion
Procedures). We also raised monoclonal antibodies
against LacZ–bcd and TrpE–bcd fusion proteins (see Ex-
We extracted protein from staged embryos, separated
them by SDS–polyacrylamide gel electrophoresis (SDS-
PAGE), and analyzed Western blots (Figure 2) by probing
with monoclonal anti-bcd antibodies. The immunostain
ing intensity between 2 and 4 hr after egg deposition.
These proteins were not detectable in ovaries or at later
stages of development. They correspond to an apparent
doublet of proteins with molecular masses between 55
and 57 kd. The same proteins are recognized by rabbit
polyclonal antibodies (data not shown). Other protein
bands are due to immunological cross-reactivity of the
second antibody, as judged from the control lane pro-
cessed without first antibody (Figure 2).
The electrophoretic mobility of the immunoreactive
bands (55 and 57 kd) is in good agreement with the pre-
dicted molecular mass of the bcd protein (53.9 kd).
Slightly higher apparent molecular weights than calcu-
lated have been reported for proteins extremely rich in
proline content, such as fushi tarazu (Carroll and Scott,
1986) and Kru ¨ppel (Gaul et al., 1987). The bcd protein
contains 10% proline. The 57 kd band might be a protein
derived from the minor splicing variant (cDNA type b,
Figure 1), although the five additional noncharged amino
acids (545 daltons) would not be expected to result in a
2–3 kd rise in molecular mass. We were not able to detect
Identification and Characterization of bcd Protein
in Drosophila Embryonic Extracts
Schematic structures of bcd transcripts and their longest
open reading frames (Frigerio et al., 1986; Berleth et al.
1988) are shown in Figures 1A and 1B. To analyze the
bcd product in Drosophila, we raised rabbit polyclonal
antibodies against a LacZ–bcd fusion protein containing
the carboxy-terminal 378amino acids of thelongest open
reading frame from the most abundant transcript (Figure
Figure 3. Detection of bcd Protein in bcd Mutants
Embryonic extracts from2–3 hr old mutantembryos were separated
by SDS-PAGE. The Western blot was subsequently developed as
in Figure 2. The maternal genotypes (bcd alleles) of the embryos
are indicated above the lanes; all females were heterozygous for
Df(3R)LIN. Maternal bcd alleles are strong(s), intermediate (i), or
weak (w), as indicated (Frohnho ¨fer and Nu ¨sslein-Volhard, 1986).
Apparent molecular masses of marker proteins in kd are indicated
Figure 2. Developmental Profile of the bcd Protein in Drosophila
Proteins from staged embryos were separated by SDS-PAGE and
transferred to nitrocellulose. The Western blot was incubated with
monoclonal anti-bcd antibodies. Ages of the embryos are indicated
at the top in hours after egg deposition (21?C). Apparent molecular
masses of marker proteins in kd are indicated at left. Lane c shows
a blot incubated with second antibody only.
The bcd Protein Distribution in Early Embryos
We stained whole mount preparations of early embryos
with anti-bcd antibody (Figure 4). Soon after egg deposi-
tion the bcd protein first becomes detectable (Figure 4A)
at the anterior tip of the egg, at or near the site of localiza-
tion of the bcd mRNA (Berleth et al., 1988). At the time
of pole cell formation (Figure 4B), bcd protein is already
distributed in a concentration gradient with a maximum
at the anterior tip. The basic shape of this gradient ap-
pears to be stable during the syncytial blastoderm stage,
with the posterior limit of detection at about 30% egg
length (Figures 4C and 4D). The total level of bcd protein
appears to increase slightly until the onset of cellulariza-
tion. It decreases slowly during cellularization and then
more rapidly during gastrulation (Figures 4E and 4F).
Traces of bcd protein can still be detected in the nuclei
at the end of germ band elongation (data not shown).
There is no indication of dorsoventral asymmetry in bcd
protein distribution during early development.
Although bcd protein is present in the cytoplasm, its
predominant location is in the nuclei, where it is strongly
concentrated. Nuclear localization is expected for a ho-
meobox-containing, putative DNA-binding protein. Yolk
nuclei as well as peripheral nuclei are stained. In contrast
to the cad protein (Macdonald and Struhl, 1986) and
Kru ¨ppel protein (Gaul et al., 1987), the bcd protein is
not associated with distinct subnuclear structures during
interphase. During mitosis the staining disappears com-
pletely from nuclear structures, and for a short time the
embryos display a protein gradient in which no cellular
substructures are preferentially stained (Figure 4G).
To determine the onset of bcd translation, we stained
frozen sections of ovaries for the bcd protein. bcd protein
was not detectable at any stage during oogenesis (data
oogenesis (Frigerio et al., 1986; Berleth et al., 1988), this
indicates that bcd mRNA translation is blocked during
bicoid protein with antibodies against a peptide of nine
amino acids including the five appearing in cDNA b (un-
published data). It is more likely that the appearance of
two bands is due to a posttranslational modification such
as phosphorylation,which would be expectedto produce
a considerablechange in electrophoreticmobility. Similar
banding patterns for other developmentally regulated
found to be due to phosphorylations (e.g., Ultrabithorax;
L. Gavis and D. Hogness, personal communication). In
the case of bcd, the pattern of protein species changes
during early development in that the protein forms with
higher apparent molecular weight appear at later stages.
at their highest abundance (2–4 hr after egg deposition)
has been shown to correspond to the temperature-sensi-
tive period of the bcdE3allele (Frohnho ¨fer and Nu ¨sslein-
To confirm that the 55 kd protein we have identified is
the bcd gene product, we used monoclonal antibodies
to probe protein extracts from embryos derived from fe-
induced bcd alleles (Figure 3). The immunologically de-
tectable protein pattern was altered only in the 55–57 kd
range, and none of the strong alleles (E1, E2, GB, 23–16,
33–5) gave rise to a protein in the bcd size range. How-
ever, the sensitivity of the method may preclude the de-
tection of a severely truncated or very unstable protein
with the antibody. Among the hypomorphic bcd alleles
the pattern was diverse. Two of the intermediate alleles
(E3, E4) showed normal or almost normal bcd protein
levels. The bcd protein was reduced in the weak mutants
2–13 and 111–3 and could not be detected at all in two
additional alleles, one of which shows only a very weak
mutant phenotype (E5; Frohnho ¨fer and Nu ¨sslein-Vol-
Figure 4. bcd Protein in Whole Mounts of Wild-Type Embryos
Wild-type embryos of different ages were stained for bcd protein as described in Experimental Procedures. Whole mount preparations were
(stage 4c), focused on the surface. (D) Syncytial blastoderm, focused on the plane of the pole cells. (E) Cellular blastoderm (stage 5b). (F)
Beginning of germ band extension (stage 7). (G) Embryo of stage 4 during mitosis. Note the lack of staining of nuclear structures. (H) Embryo
of a bcdE1/bcdE1female. In all cases anterior is at left, dorsal at top. Staging is according to Campos-Ortega and Hartenstein (1985).
oogenesis. In unfertilized eggs establishment of the bcd
protein gradient proceeds in a normal fashion. However,
at about 2 to 4 hr after egg deposition, levels of bcd
protein exceed the ones in fertilized eggs (see Figure 6E).
This suggests that either the transcript or the protein is
more stable in unfertilized eggs. The finding of a higher
than normal bcd protein level in unfertilized eggs explains
the results of transplantation experiments. When unfertil-
ized eggs were used as donors, the bcd?activity was
found to be both more concentrated and more stable
than in fertilized eggs (Frohnho ¨fer and Nu ¨sslein-Volhard,
1986). We conclude that the event of fertilization is not
required for the onset of bcd translation. Similar results
have been obtained for translation of the maternal cad
transcript (Macdonald and Struhl, 1986).
bcd Protein Distribution in bcd Mutant Embryos
In whole mounts of embryos from files carrying strong
bcd mutant alleles, no bcd protein could be detected,
confirming the data of the Western blot analysis (Figure
Table 1. bcd Protein in bcd Mutant Embryos
bcd Protein on
Whole Mount EmbryosAlleleStrength
4H, Table 1). This might be due to nonsense mutations
stability, or ethyl methanesulfonate–induced small dele-
tions (as has been shown in the case of the alleles bcdE1
and bcdE2; see Berleth et al., 1988). In contrast to the
results of the Western analysis, bcd protein can be de-
tected in whole mount immunostainings of mutant em-
bryos from flies carrying each of six hypomorphic alleles.
In the case of the weak alleles the level is reduced, while
it appears to be normal for the intermediate alleles. Thus
the phenotypes observed for the weak alleles may be
caused by reduced levels of active protein (due to greater
instability or a lower rate of synthesis), while for the inter-
mediate alleles normal levels of protein with reduced ac-
tivity may prevail. The failure to detect a 55 kd band in
the bcd085mutant in Western blots (with a normal level in
whole mount embryos) suggests a truncated protein that
is not recognized by the monoclonal antibody.
Figure 6F, which shows bcd immunostain intensity in
bcdE1mutant embryos and control embryos. By visually
comparing the range of staining intensities measured in
the wild-type embryos with that obtained from serial dilu-
tions of bcd protein on nitrocellulose (Figure 5), one can
roughly estimate that the bcd protein concentration
changes by 2 to 3 orders of magnitude within the anterior
half of the embryo.
A plot of the logarithm of the concentration (Figure 6B)
is near-linear over the largest portion of the egg length,
indicating an exponential decay of the protein concentra-
tion toward the posterior. In the anteriormost 15% of the
embryo, the slope is steeper. This is probably due to the
fact that in this region the more intensely stained nuclei
enter the plain of focus (see below). Furthermore, this is
the region where the bcd mRNA, the source of the bcd
ground levels are reached at about 30% egg length (Fig-
ures 6B and 6F).
The intensity of the anti-bcd immunostain was also
determined along the dorsal and ventral periphery of the
egg, at the positions of the nuclei in early nuclear cycle 14
the nucleus, the bcd protein concentration in the nuclei
is higher than in the surrounding cytoplasmic regions.
Because of ambiguities in the positioning of the line of
measurement and a strong influence of egg curvature on
the measurement (underestimation of terminal values),
we did not use this method of determining the protein
distribution in subsequent analyses. At first sight both
methods seem to yield principally different curves. How-
the anteriormost values, the shapes of the graphs be-
comes quite similar apart from different absolute values.
To assess the influence of egg curvature and nuclear
density on the measurements, we examined the stain
intensity of an evenly distributed protein with both cyto-
plasmic and nuclear localization. Such a situation is pro-
vided in the distribution of the cad protein in bcdE1mutant
embryos during early nuclear cycle 14. Figure 6D shows
the distribution of cad protein in mutant bcdE1and in wild-
type embryos during early nuclear cycle 14. In wild-type
embryos, cad protein is distributed in an approximately
linear gradient from 0% to 100% egg length with a high
point at the posterior pole (Macdonald and Struhl, 1986);
this gradient is less steep than the bcd protein gradient.
In bcdE1mutant embryos, the cad protein immunostain
The Shape of the bcd Protein
We measured the intensity of the bcd immunostain either
directly in whole mount embryos by using a sensitive
photodiode scanning about 3 ?m in area, or by digitizing
a high-resolution video image obtained directly from the
gram (see Experimental Procedures). Both methods give
similar results. Using these techniques we avoided many
of the problems caused by the nonlinearity of signals in
densitometric analyses of photographic prints. Under the
applied conditions the signal of the horseradish peroxi-
dase (HRP) reaction is proportional to the bcd protein
sities of serial dilutions of the bcd protein in dot blots
on nitrocellulose (see Experimental Procedures). Figure
5 shows the intensity of immunostaining obtained for
different amounts of fusion protein applied to nitrocel-
The intensity of bcd protein immunostain measured
along a line from the anterior to the posterior of the em-
bryo is displayed in Figure 6A, including the 2-fold stan-
dard deviation. The bcd protein is distributed in a sharp,
nonlinear concentration gradient. There is a very steep
decline in bcd protein concentration in the region close
to the anterior pole, while at positions posterior to 50%
egg length further decreases in concentration are barely
detectable. The background level can be evaluated from
cellularization, we estimate a half-life for the bcd protein
of less than 1/2 hr.
In the syncytial Drosophila embryo, no cell boundaries
limit the diffusion of a protein the size of the bcd protein.
Diffusion constants for proteins in the cytoplasm have
been calculated to be in the range of 0.3–1.0 ? 10?8cm2/
sec (Mastro et al., 1984; Wojcieszyn et al., 1981). These
estimates derive from fluorescence recovery after spot
photobleaching of fluorescent conjugates as well as from
electron spin resonance of spin labels. The diffusion con-
stants are reported to be relatively independent of the
molecular weights of the proteins (Kreis et al., 1982; Mas-
tude are capable of generating a gradient over a 200 ?m
distance, starting at a local source, within about 1 hr. The
calculated time corresponds well to the time between the
onset of bcd translation after egg deposition and the
earliest time when the mature-shaped gradient can be
detected in the egg of 500 ?m length.
These calculations show that the bcd protein gradient
may simply be generated by diffusion and nonspecific
proteolytic activities. However, more complex processes
involving specific interactions with other compounds
cannot be excluded. For example, active wave-like con-
tractions of the cytoplasm observed during each cleav-
age cycle (Foe and Alberts, 1983) may influence the for-
mation and shape of the gradient. Furthermore, the
degradation of bcd protein may (but need not) involve
specific proteases. Secondary modifications of the pro-
tein might interfere with its stability and thus influence
the shape of the gradient.
Figure 5. Determination of the Linear Range of the Immunostaining
For details, see Experimental Procedures.
reveals a density profile that is even except for two small
terminal peaks. These peaks are due to the fact that at
terminal positions the more intensely stained nuclei are
at the plane of focus, while they are out of focus between
10% and 90% egg length. This implies that the measure-
ments of bcd protein distribution as displayed, for exam-
ple, in Figures 6A and 6B are not significantly affected
by the egg shape except at positions between 90% and
100% egg length.
The shape of the bcd protein gradient in unfertilized
eggs (Figure 6E) is similar to that in fertilized eggs. The
ization of bcd mRNA in very early fertilized embryos (Ber-
leth et al., 1988). Between young (0–1 hr) and old (1–7
hr) unfertilized eggs, there is a distinct increase in bcd
Translational Control of bcd
Although bcd transcription starts early in oogenesis and
the mRNA is localized long before egg deposition, we
could not detect bcd protein in oocytes. This implies that
bcd mRNA translation either is blocked during oogenesis
or might require translational activation as a part of the
general activation of the maternal program of the oocyte
(Mahowald et al., 1983). Translation begins, independent
of fertilization, soon after egg deposition. For bcd mRNA
ponents that trap the bcd mRNA at the anterior of the
oocyte and early embryo. On the other hand, the cad
message, which is also translated only after egg deposi-
tion, is evenly distributed throughout the egg (Macdonald
and Struhl, 1986). The biological significance of the trans-
lational control is not entirely obvious. Since the bcd pro-
tein concentration increases during the early hours of
embryogenesis, the final shape of the gradient might ulti-
mately depend on the total time of bcd mRNA translation.
Alternatively, together with other maternal messages, the
bcd mRNA may be subject to a more general translational
control mechanism, allowing for a concerted action of
several gene products in early development.
Establishment of the bcd Protein Gradient
The source of the bcd protein is the localized bcd mRNA.
Comparison of the distributions of bcd mRNA and protein
reveals a striking incongruity. bcd mRNA is localized at
the anterior tip of the embryo (80%–100% egg length)
length.In contrast,bcdproteinconcentration reachesthe
limit of detection at about 30% egg length (Figure 6B).
A simple conceivable mechanism for the formation of
the bcd protein concentration gradient is diffusion start-
ing from the local mRNA source, and degradation
throughout the embryo. An appropriate balance between
the rates of bcd mRNA translation, of diffusion, and of
dispersed proteolytic degradation would generate a rela-
tively stable, nonlinear, graded distribution of the bcd
protein. Such a model would require low stability of the
bcd protein. The bcd protein contains several PEST se-
quences, which are present in proteins of short half-life
and are thought to be signals for degradation (Rogers et
al., 1986; Rechsteiner et al., 1987). The most significant
PEST sequence spans from amino acid 170 to 203 (for
the sequence, see Berleth et al., 1988). From the observa-
tion that the last traces of the protein can be detected
after gastrulation whereas the mRNA disappears during
Molecular Mechanisms of bcd Function
The presence of a homeobox domain (McGinnis et al.,
1984; Laughon and Scott, 1984; Desplan et al., 1985) in
bcd suggests that the bcd protein binds specifically to
DNA and thereby regulates the transcription of zygotic
target genes. The predominant nuclear location of the
bcd protein in early embryos supports this hypothesis.
Figure 6. Quantitation of Immunostain Intensity in Whole Mount Preparations of Embryos
Video images were obtained from whole mount preparations and were analyzed by digitizing images as described in Experimental Procedures.
(A) Anti-bcd protein immunostain intensity measured along the anteroposterior axis in wild-type early nuclear cycle 14 embryos. Data from
ten embroys were averaged and are displayed with 2-fold standard deviations. (B) Natural logarithms of measurements in (A). (C) Anti-bcd
immunostain intensity measured along the anteroposterior axis (open squares) versus along the line of nuclei at the dorsal or ventral periphery
(filled squares) during early nuclear cycle 14. (D) Expression of cad protein in wild-type (open squares) and bcdE1mutant embryos (filled
squares) as revealed from anti-cad whole mount immunostainings of early nuclear cycle 14 embroys. (E) bcd protein distribution in 0–1 hr
(filled squares) and 1–7 hr (open squares) unfertilized eggs. Staining was done in two different batches. Posteriormost values were assumed
to be background levels and were used for relating the two separate stainings. (F) Immunostain intensity in an embryo from a bcdE1/bcdE1
female (closed squares). Open squares show normal bcd protein distribution in osk?embryos stained in the same batch. osk?embryos have
the same bcd protein distribution as wild-type embroys (Driever and Nu ¨sslein-Volhard, 1988).
There is no precedent for how the protein might act in
regulating different target genes in a concentration-
dependent manner during early development. The con-
centration of bcd protein in the nuclei, from which it must
changes from the anteriormost position to 50% egg
length by 2 or 3 orders of magnitude in an exponential
fashion. This large range of concentration would allow
differential binding, in a concentration-dependent man-
ity for the bcd protein. Multiple binding sites for the bcd
protein might exist in a promoter region, and a monomer–
oligomerequilibriumof bcdproteincouldlead todifferen-
tial binding behaviors. Dimer formation of DNA-binding
proteins has been shown in prokaryotes as well as eu-
karyotes (e.g., GCN4 in yeast; Hope and Struhl, 1987).
The properties of the bcd protein therefore might provide
a molecular basis for a transition of the continuously
changing bcd concentration into a series of discrete
states of activated genes.
development) rather than exerting a discrete morphoge-
In contrast, in the case of the bcd protein gradient the
phenotypic effects can be detected in mutant embryos
correlate very well. Furthermore, the bcd mRNA and
protein distribution can explain in detail the results of
transplantation experiments (Frohnho ¨fer and Nu ¨sslein-
Volhard, 1986, 1987; Nu ¨sslein-Volhard et al., 1987). The
accompanying paper (Driever and Nu ¨sslein-Volhard,
1988) presents extensive evidence that the bcd protein
gradient determines anterior pattern in the Drosophila
Gradients in Early Development
During the last decades a number of models explaining
the formation and interpretation of morphogenetic gradi-
ents have been proposed (Lawrence, 1966; Wolpert,
1969; Crick, 1970; Gierer and Meinhardt, 1972; Lewis et
al., 1977; Meinhardt, 1977, 1982, 1986; MacWilliams,
1978). According to our results, it is likely that the bcd
protein gradient is generated simply by diffusion from a
local source and dispersed degradation. So far there is
no evidence for more elaborate regulatory mechanisms
such as autocatalysis as proposed by Gierer and Mein-
hardt (1972). In Meinhardt’s (1977, 1986) models for early
insect development, only one maternally derived gradient
with a high point at the posterior end determines four
cardinal (central) and two marginal (terminal) zones in a
concentration-dependent manner. The borders between
ing zones for the initiation of the first periodic pattern,
the double segments. In a similar fashion, for the anterior
pattern the bcd protein gradient might regulate some of
the earliest zygotically transcribed genes, the gap genes,
each of which is necessary for the development of broad
regions along the anteroposterior axis (Nu ¨sslein-Volhard
and Wieschaus, 1980). The possible influences of bcd on
the expression of the gap genes are discussed in detail
by Driever and Nu ¨sslein-Volhard (1988).
bcd might interact with other maternal gene products.
The cad gene is expressed both maternally and zygoti-
cally (Macdonald and Struhl, 1986). In wild-type embryos
the cad protein derived from the maternal transcript
shows a graded distribution at early nuclear cycle 14, but
is homogeneously distributed in the very early stages of
embryonic development. In bcd?embryos at early nu-
clear cycle 14, cad protein derived from maternal mRNA
is evenly distributed (Figure 6D). These results suggest
that the bcd protein negatively regulates cad translation
or cad transcript stability directly or indirectly, thereby
generating the cad protein gradient. The fact that in the
wild type cad protein gradient formation precedes cad
RNA degradation favors an involvement of bcd in transla-
tional control. The effects of bcd on the abdominal region
might therefore also result from its interaction with the
cad gradient. Similar to cad, the gene hb (in addition to
its zygotic expression) gives rise to maternal transcripts
and protein that forms a gradient, but in this case with
activity (Tautz, 1988). Both the maternal cad and maternal
hb gradients have in common that their genetic deletion
does not result in a mutant embryonic phenotype; in their
absence embryonic development is largely normal (Mac-
donald and Struhl, 1986; Lehmann and Nu ¨sslein-Volhard,
1987). Thereforecad and hbmay have moregeneral func-
The wild-type stock was Oregon R. The bcd alleles and Df(3R)LIN,
bcd?, have been described (Frohnho ¨fer and Nu ¨sslein-Volhard,
1986). All mutant chromosomes carried suitable visible markers.
Flies were grown and eggs collected under standard conditions
(Nu ¨sslein-Volhard et al., 1984). Staging of embryos was according
to Campos-Orlega and Harlenstein (1985).
The bcd cDNA clones have been described previously (Frigerio et
al., 1986; Berleth et al., 1988). The lacZ–bcd fusion gene was ob-
tained by cloning the 1898 bp Pstl–HindIII fragment coding for 77%
of the largest open reading frame into the Pstl and HindIII sites of
pUR291 (Ru ¨ther and Mu ¨ller-Hill, 1985). To obtain the trpE–bcd fu-
sion, the 1910 bp BamHI–HindIII fragment containing the bcd se-
quences was isolated from the lacZ–bcd fusion and cloned into the
BamHI and HindIII sites of pATH11, thereby extending the trpE open
reading frame (Ko ¨rner, unpublished; Kla ¨mbt and Schmidt, 1986).
Cloning was carried out in JM83 host strains as described in Ma-
niatis et al. (1982). The lacZ–bcd fusion construct (pURbcd) was
transformed into E. coli 71-18 hosts, and the trpE–bcd fusion con-
struct (pATHbcd) was transformed into an E. coli C-600 host.
Production and Purification of Polyclonal Antibodies
to be efficient in producing antibodies with little or no cross-reactiv-
ity to the carrier protein (see Kla ¨mbt and Schmidt, 1986; Gaul et
For preparation of LacZ–bcd fusion protein, bacteria were grown
in L broth (50 ?g/ml ampicillin) to an OD800of approximately 0.4 and
were subsequently induced for 2 hr by addition of 1 mM isopropyl
thiogalactoside. Isolation of the LacZ–bcd protein was according
to a protocol modified from Rio et al. (1986). Bacteria from 2 liter
induced cultures were thawed on ice in 50 ml of buffer A (50 mM
Tris [pH 7.9], 200 mM NaCl, 2 mM EDTA, 2 mM ?-mercaptoethanol,
1 mM phenylmethylsulfonyl fluoride, 1 ?M pepstatin, 1 ?M leupep-
tin), and lysozyme was added (final concentration 0.2 mg/ml). After
a 20 min incubation on ice, Triton X-100 was added to 1%; after a
further 10 min on ice, NP-40 was added to 0.5%. After an additional
incubation for 10 min, the suspension was sonicated (three times
for 30 sec; Branson Sonifier with microtip, setting 4) on ice. Ten
milliliters of the lysatewas layered on top of a10 ml sucrose cushion
(40% sucrose, 10 mM Tris [pH 7.5], 1 mM EDTA, 200 mM NaCl) and
centrifuged (30 min; 15000 ? g, 4?C). The pellet was resuspended
in 5 ml of PBS, and 20 ml of extraction buffer B (8 M urea, 0.5 M
1 ?M pepstatin, 1 ?M leupeptin) was added. The suspension was
dissolved by extensive vortexing and then dialyzed against dialysis
buffer C (50 mM Tris [pH 7.9], 0.5 M NaCl, 10% glycerol, 1 mM
phenylmethysulfonyl fluoride). Precipitated material was sedi-
mented, and supernatant and precipitate were analyzed by SDS-
PAGE. About 80% of the LacZ–bcd fusion protein was in the pellet,
and it was approximately 20% pure. LacZ–bcd fusion protein was
dissolved in sample buffer (Laemmli, 1974) and subjected to prepar-
ative SDS-PAGE. Protein bands were visualized by incubating the
gel with 0.25 M KCl in H2O. The LacZ–bcd fusion protein band was
cut out. Gel slices were pressed through very fine metal sieves and
suspended in 2 vols of complete Freund’s adjuvant (incomplete for
booster injections). Rabbits were injected at multiple subcutaneous
and intramuscular sites, boosted after 4 weeks, and after an addi-
tional 2 weeks bled on a weekly schedule. Two out of four rabbits
produced high titers of antibodies against the bcd part of the fu-
Bacteria carrying the trpE–bcd constructs were grown in M9 me-
dium (Maniatis et al., 1982), supplemented with 5 g/l tryptophan-
free Casamino acids (Difco), to an OD800of 0.4. Fusion protein syn-
thesis was induced by addition of 5 ?g/ml indolylacrylic acid (Serva)
for 4 hr. The fusion protein was isolated according to Rio et al.
0.5 M NaCl (three times for 2 hr, once overnight), and precipitated
material was sedimented 10000 ? g, 10 min). The supernatant (1.4
mg/ml protein) was used to prepare an affinity column (Affigel 10/
15, 3:1 ratio; Biorad) according to the manufacturer’s protocol (14.5
mg of protein coupled to 10 ml of Affigel 10/15). The column was
washed with elution buffer (4 M MgCl2in PBS) and equilibrated
Ten milliliters of antiserum was diluted 1:1 with PBS, 300 mM
NaCl, 0.1% Triton X-100, and loaded onto the column. The column
was washed (PBS, 300 mM NaCl, 0.1% Triton X-100), and the anti-
body was eluted with 4 M MgCl2 (pH 3.8). Fractions containing
ual anti-?-galactosidase antibodies, the antibodies were passed
through a ?-galactosidase–Affigel column.
were absorbed by passing the antibodies over an Affigel column
as well as affinity-purified antibodies was checked for activity
against bcd-specific sequences as described by Gaul et al. (1987).
Whole Mounts of Embryos
Immunological staining of whole mount embryos with biotinylated
tor Laboratories, Avidin Biotin ABC system) was carried out as de-
scribed by Macdonald and Struhl (1986) except that during the
washes we added 100 mM NaCl to the solutions. Antibodies against
cad protein were obtained from Paul Macdonald (Macdonald and
Microphotographs were taken on Agfapan 25 (Agfa) with a Zeiss
photomicroscope using Nomarski optics.
Measurement of Whole Mount Staining
With embryos used for quantitative analysis, the HRP staining reac-
tion was done for a relatively short time (4 min, versus ca. 8 min for
range. To test the linearity of the ABC-HRP staining reaction within
a given concentration range, we spotted serial dilutions of the TrpE–
bcd fusion protein on nitrocellulose (150 pg to 10 ?g in a volume
of 10 ?l) and performed an immunostaining reaction identical to the
whole mount immunostaining. The nitrocellulose was dissolved in
tometer (Joyce and Loebel). The ABC-HRP staining intensity was
whole mounts showed that the signal intensity in the embryos was
well within the linear range posterior to 90% egg length.
Video pictures were obtained from early nuclear cycle 14, whole
mount–stained embryos using a Zeiss photomicroscope with bright
field optics and a Panasonic WV-1850/G video camera. The image
was digitized onan IBM-AT equipped with anFG100 board (Imaging
Technologies Inc.) and analyzed using Imagepro software (Media
Cybernetics). First, a background picture was taken, and the immu-
nostain image wascorrected for background bythe difference func-
tion supplied by the multiimage operation submenu. By this proce-
dure we can exclude an influence of slightly inhomogeneous
illumination or other technical factors on the image analysis.
The computer assigned gray values between 1 and 256 to the
pixels (image points). We measured these gray values along an
axis from the anterior to the posterior pole (except where stated
otherwise) in a central plane of focus (pole cells in focus). Thirty
equidistant points (correction for egg length) were measured and
analyzed for five to ten embryos of each genotype. Mean values
and standard deviations were calculated (see Figure 6A). The arbi-
trary units displayed on the y-axis of the graphs correspond to
the gray values 1–256 assigned by the computer. Using a different
approach of quantifying the staining (scanning the whole mounts
with sensitive photodiodes through a microscope; equipment sup-
plied by T. Bonhoeffer and V. Braitenberg, Tu ¨bingen) gave approxi-
mately the same results.
Generation of Monoclonal Antibodies
Female BALB/cJ mice were injected intraperitoneally with about 50
?g of LacZ–bcd fusion protein (purified by SDS-PAGE as described
above) in complete Freund’s adjuvant (GIBCO). They were boosted
several times with the LacZ–bcd fusion protein (in incomplete
fusion protein (in PBS); the latter injections were at 3 days and at
12 hr before cell fusion. This scheme should especially stimulate
proliferation of cells producing antibodies against the bcd part of
the fusion proteins. Fusion with P3-NC1/1-Ag4-1 myeloma cells
(Ko ¨hler et al., 1976) using polyethylene glycol, selection, screening,
and subcloning were done according to standard procedures
(Ko ¨hler and Milstein, 1976; Fazekas de St. Groth and Schneidegger,
1980; Saumweber, 1980).
Western Blot Analysis
Staged embryos were collected on agar plates, washed, dechorio-
nated in 50% bleach, and washed again (10 mM Tris [pH 7.2], 300
mM NaCl, 0.5% Triton X-100). One hundred microliter samples of
settled embryos were frozen in liquid nitrogen and thawed while
being homogenized with 300 ?l of 2? sample buffer, 8 M urea. After
incubation for 5 min at 95?C, undissolved material was sedimented.
Fifteen microliters of the supernatant was applied to each slot on
a 3 mm thick 10% SDS–polyacrylamide gel. Following SDS-PAGE,
protein was blotted to nitrocellulose; complete transfer at high field
was obtained within 3 hr as checked by the transfer of prestained
molecular weight markers (Sigma). Blots were incubated for 30 min
in 5% low-fat dry milk in PBS, 0.1% Tween 80, and then overnight
four times for 15 min each and incubated with affinity-purified, alka-
son Immunoresearch) for 3 hr at room temperature. After washing,
staining was developed with bromochloroindolyl phosphate (5 mg/
ml in dimethyl sulfoxide; 1 part) and nitro blue tetrazolium (1 mg/ml
in H2O; 10 parts) at pH 10.2 in 50 mM Na2CO3, 2 mM MgCl2(100
parts), for several hours (Leary et al. 1983).
We thank T. Berleth, U. Gaul, S. Henke-Fahle, and S. Richstein for
various valuable contributions and advice with the preparation of
antibodies; P. Macdonald for cad antibody; T. Bonhoeffer and B.
Stolze for help with the measurements of bcd protein distribution;
and M. Kingler, S. Roth, H. and R. Schnabel, and L. Stevens, for
stimulating discussion and suggestions on the manuscript. M.
Schorpp took excellent care of the rabbits, R. Groemke-Lutz pre-
pared the photographs, and K. Ralinofski and V. Koch typed the
The costs of publication of this article were defrayed in part by
the payment of page charges. This article must therefore be hereby
marked “advertisement” in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
Received February 18, 1988; revised April 15, 1988.
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