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First published online January 23, 2009
doi: 10.1242/10.1242/dev.031195
1 Center for Developmental Genetics, Stony Brook University, Stony Brook, NY
11794-5140, USA.
2 Department of Experimental Medical Sciences, Lund University, BMC B13, S-22184
Lund, Sweden.
3 Institut für Molekularbiologie, Universität Zürich,
Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
Authors for correspondence (e-mails:
markus.noll{at}molbio.uzh.ch;
stefan.baumgartner{at}med.lu.se)
Accepted 25 November 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: bicoid, Morphogenetic gradient, Staufen, ARTS model
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). In textbooks, the Bcd protein serves
as paradigm as the first identified morphogen, the concentration gradient of
which provides the initial positional information in the anterior half of the
embryo where it differentially activates segmentation genes, particularly gap
genes. The activity of the morphogen is expressed in a spectacular gradient of
the Bcd protein, the concentration of which approximates an exponential
decline with distance from the anterior pole in the nuclei of
syncytial-blastoderm embryos (Driever and
Nüsslein-Volhard, 1988;
Houchmandzadeh et al., 2002).
This gradient and its activity provided the first experimental proof of the
French Flag model of pattern formation proposed by Wolpert
(Wolpert, 1969). In this
model, Wolpert reasoned that the spatial concentration gradient of a
morphogen, with its maximum at one end of a uniaxial field of cells, could
provide the positional information that uniquely specifies cellular fates.
The bcd gene was first isolated and characterized at the molecular
level in an initial test of the gene network hypothesis, which states that
genes belonging to a network with an integrated function are structurally
linked by a relatively small number of protein-coding and cis-regulatory
domains that are characteristic of the network
(Frigerio et al., 1986)
(reviewed by Noll, 1993). Two
crucial properties of bcd were identified: (1) bcd encodes a
transcription factor that includes a homeodomain, and (2) its maternal mRNA
forms a concentration gradient along the anteroposterior axis of the embryo at
stages preceding cellular blastoderm
(Frigerio et al., 1986). These
properties explained, for the first time, the ability of the bcd
product to act as a maternal morphogen. Changes in the spatial distribution of
maternal bcd mRNA, visualized in tissue sections by in situ
hybridization with a 3H-labeled bcd cDNA, were documented
at three stages: (1) bcd transcripts accumulate at the anterior
margin of oocytes in the female abdomen; (2) after fertilization transcripts
move posteriorly, forming a concentration gradient along the anteroposterior
axis in cleavage-stage embryos; and (3) during nuclear cycle 14, transcripts
display an extended gradient along the apical cortex of the
syncytial-blastoderm embryo (Frigerio et
al., 1986).
Later, another paper appeared that showed the bcd mRNA patterns of
an early cleavage-stage and a syncytial-blastoderm embryo
(Berleth et al., 1988).
Although both patterns were entirely consistent with those published earlier
(Frigerio et al., 1986), this
paper mentioned only that bcd transcripts were "becoming
concentrated in the cortical cytoplasm in the form of an anterior cap",
but disregarded the presence of the mRNA gradient. Soon thereafter, the first
publication appeared demonstrating that Bcd protein forms an exponential
concentration gradient with its maximum at the anterior pole, reaching
background levels in the posterior third of embryos at early nuclear cycle 14
(Driever and Nüsslein-Volhard,
1988). The authors proposed that the exponential gradient is
generated from a bcd mRNA source, localized in the anterior-most
portion of the embryo, by diffusion and dispersed degradation of the Bcd
protein. However, they provided no evidence to support a crucial feature of
their model, namely their contention that bcd mRNA, the source of Bcd
protein, remains localized at the anterior pole. In a later attempt to
compensate for this shortcoming, St Johnston et al.
(St Johnston et al., 1989)
described the spatial distribution of bcd mRNA. They indicated that
it became localized to the periphery of the embryo in a movement that
"sometimes resulted in a slight posteriorwards shift in the RNA
distribution". Still, the Bcd protein diffusion model was not
questioned.
Since then, this model has become anchored in textbooks as a fundamental
paradigm of developmental biology. Its importance and implications have been
described in a review (Ephrussi and St
Johnston, 2004), supplemented with personal perspectives of the
authors who proposed it (Driever,
2004; Nüsslein-Volhard,
2004). Recently, however, this SDD model - the naming of which
refers to the localized synthesis, diffusion and spatially uniform degradation
of the Bcd protein - has been subjected to a critical test
(Gregor et al., 2007). The
model assumes a Bcd protein source that is fed by translation of the
bcd mRNA localized at the anterior pole, diffusion of Bcd away from
its source, and spatially uniform degradation by a first-order reaction
(Gregor et al., 2007), which
predicts an exponential decay of the Bcd concentration with increasing
distance from the anterior pole (Wolpert,
1969). Although this prediction was verified, a series of
ingenious experiments that measured the diffusion constant of Bcd in the
cortex of embryos uncovered a serious difficulty with the model: the diffusion
constant was two orders of magnitude too low to explain the observation that
the steady state of the Bcd gradient profile is reached within 1.5 hours
(Gregor et al., 2007).
Here, we propose a simple solution to this dilemma by reinforcing and
extending results that documented a bcd mRNA gradient, in agreement
with the first molecular characterization of the bcd gene
(Frigerio et al., 1986).
Revisiting our published results and using a sensitive fluorescent in situ
hybridization (FISH) method and confocal microscopy, we demonstrate that (1) a
bcd mRNA concentration gradient is formed along the cortex of the
embryo by nuclear cycle 10 or the beginning of syncytial blastoderm; (2) the
gradient falls off exponentially with distance from the anterior pole and
persists unchanged during nuclear cycles 10-13; (3) bcd mRNA is
transported to the apical nuclear periplasm during syncytial blastoderm; and
(4) bcd transcripts are degraded rapidly during the first third of
nuclear cycle 14. In addition, we show that the bcd mRNA and protein
patterns behave very similarly. These results exclude the SDD model, which
needs to be replaced by a new model in which the Bcd protein gradient is
dictated by the bcd mRNA gradient and its subsequent translation. In
contrast to the SDD model, this ARTS (active RNA transport and synthesis)
model explains the formation of the Bcd protein gradient by an active
transport of its mRNA on microtubules, followed by synthesis of the
protein.
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MATERIALS AND METHODS |
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MATERIALS AND METHODS
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DISCUSSION
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;
Berleth et al., 1988). This
cDNA was used in the first demonstration of a bcd mRNA gradient
(Frigerio et al., 1986), and
served as the source of Bcd antigen for the preparation of an anti-Bcd
antiserum in the first publication demonstrating a Bcd protein gradient
(Driever and Nüsslein-Volhard,
1988) as well as in subsequent publications
(Kosman et al., 1998). In situ
hybridization of digoxigenin (DIG)-labeled probes and their immunochemical
detection by alkaline phosphatase or horseradish peroxidase
(Fig. 1D-F) followed standard
protocols (Tautz and Pfeifle,
1989).
To optimize the sensitivity of FISH, we modified a standard in situ
hybridization protocol (Tautz and Pfeifle,
1989) (detailed protocol available on request). Care was taken to
keep the parameters in different in situ hybridization experiments constant.
To ensure that the apical periplasm of embryos was not affected during
devitellinization and hybridization, embryos were fixed up to 1 day before
devitellinization. During devitellinization, only short and mild vortexing was
applied. To maintain the integrity of the apical periplasm during
hybridization, embryos were fixed for 1 hour in 1% formaldehyde before
treatment with 10 µg/ml proteinase K for 5 minutes, which is five times
shorter than recommended by current protocols. Hybridized probes were detected
with an anti-DIG monoclonal antibody (Roche) diluted 1:200, and Alexa 555-
(Invitrogen) or Cy3-coupled goat anti-mouse secondary antibodies diluted
1:1500. To determine the developmental stage, embryos were counterstained with
DAPI or TOTO3 (Invitrogen).
An anti-Bcd antiserum, raised in rabbits against amino acids 112-396 of Bcd (including the homeodomain), was affinity-purified and used at 1:20 dilution. To visualize bcd mRNA as well as Bcd protein, Bcd protein was detected by the sequential use of anti-Bcd and Alexa 555-coupled goat anti-rabbit antisera, followed by FISH and the detection of bcd mRNA by the sequential use of an anti-DIG monoclonal antibody and Alexa 647-coupled goat anti-mouse antiserum. Alexa 555 (Bcd protein) and Alexa 647 (bcd mRNA) were chosen as fluorochrome coupled to secondary antibodies to minimize their cross-channel activity. Anti-Stau antibodies (a generous gift of Daniel St Johnston) were used at 1:2500 dilution.
When only proteins (Stau or Bcd) were visualized, consistently better fluorescent signal intensities (extremely low background combined with a wide range of sensitivity) were obtained with heat-fixed than with formaldehyde-fixed embryos. Moreover, heat-fixed embryos showed much better preservation of the apical periplasm.
Data acquisition
Confocal images (1024x1024 pixels, 8 bits) were taken with a Leica
TCS SP or a Zeiss LSM Pascal microscope. Image stacks were acquired as
sagittal sections through entire embryos, and fluorescence intensities were
taken from midsagittal planes. For collection of fluorescent signals, the
parameters of the confocal microscope were kept the same in all experiments.
Intensity graphs were obtained from a circular area moved along the dorsal
cortex of midsagittal sections (Alexandrov
et al., 2008) (a detailed description of the algorithms, scripts
and tools used is available on request).
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RESULTS |
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SUMMARY
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MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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)]. The
most striking of these pictures, illustrating a midsagittal section of an
embryo at nuclear cycle 14, is shown again here
(Fig. 1A,B). Though not
presented at the time, we had further analyzed the shape and extent of this
bcd mRNA gradient by counting the number of silver grains in the
apical periplasm of each dorsal nucleus
(Fig. 1B) and plotting this as
a function of the position of the nucleus along the anteroposterior axis. The
resulting curve of bcd mRNA concentration
(Fig. 1C) decreases
monotonically from a maximum at the anterior pole to background levels at
35% egg length (EL; anterior pole is 0%), as pointed out previously
(Frigerio et al., 1986).
Although more sensitive methods permit detection of bcd mRNA at more
posterior positions (see below), Fig.
1A-C demonstrates unequivocally that (1) bcd mRNA is not
strictly localized to the anterior pole of the embryo but forms a
concentration gradient that decreases with distance from the anterior pole,
and (2) bcd mRNA is localized to the apical periplasm at this stage
of nuclear cycle 14. Both results have been emphasized previously
(Frigerio et al., 1986).
Several years after our original publication, a non-radioactive and hence
faster method of detecting mRNA became available, using probes labeled with
DIG and alkaline phosphatase coupled to an anti-DIG antibody
(Tautz and Pfeifle, 1989).
This method, previously used to analyze bcd mRNA distributions
(St Johnston et al., 1989),
was tested by exposing the embryos for 30 minutes to the color substrate of
alkaline phosphatase in order to detect the full extent of the gradient. This
approach is indeed capable of detecting the bcd mRNA gradient, as
evident from an embryo at nuclear cycle 10
(Fig. 1D) and its ImageJ scan
(Fig. 1G). Considerably shorter
exposures, however, generated the false impression that transcripts are
trapped at the anterior pole of the embryo, in apparent agreement with the SDD
model. Nevertheless, even in these faintly stained embryos, the bcd
mRNA gradient was readily detectable by use of optical measurements combined
with ImageJ, which are more sensitive than the eye.
|
). As
is evident from Fig. 1F and its
ImageJ scan (Fig. 1I),
nos mRNA was clearly restricted to the posterior tip, excluding
artifacts due to substrate diffusion.
These results raise the question of why others have not observed a
bcd mRNA gradient as we do. As argued, the color reaction might have
been too short or the stained images were not subjected to a quantitative
analysis. Such an analysis of bcd mRNA concentrations has been shown
in a graph representing the average of five embryos at nuclear cycle 13
(St Johnston et al., 1989).
Although this graph was corrected for non-linearity of the staining method, it
is not consistent with our analysis using modern experimental and analytical
tools (see below). In addition, in some cases a bcd mRNA gradient can
indeed be observed but was missed or ignored (T. Berleth, PhD thesis,
University of Tübingen, 1988) (Berleth
et al., 1988; St Johnston et
al., 1989; Schnorrer et al.,
2002; Song et al.,
2007).
Analysis by FISH and confocal microscopy of bcd mRNA gradient formation
To visualize the bcd mRNA patterns, we modified a well-established
in situ hybridization protocol (Tautz and
Pfeifle, 1989) to raise its sensitivity. Moreover, we used FISH
and confocal microscopy, which combine high sensitivity and high spatial
resolution (Lécuyer et al.,
2007). In unfertilized eggs, bcd transcripts are
localized at the anterior tip of the embryo
(Fig. 2A), where they are
tightly associated with the cortex, as is evident from more-peripheral
confocal sections (Weil et al.,
2008) (data not shown). Their localization is very similar to that
observed in mature (Frigerio et al.,
1986) and live-imaged oocytes
(Weil et al., 2006). A
confocal stack (not shown) revealed a small `cap' of bcd mRNA with a
posterior limit at 7-9% EL and a skew to the dorsal side
(Fig. 2A). In 4-nuclei embryos,
the cap extended posteriorly (Fig.
2B). In 32-nuclei embryos, bcd transcripts continued to
move posteriorly along the periphery of the embryo
(Fig. 2C). It is important to
emphasize that transcripts do not diffuse as their movement is restricted to
the cortex of the embryo. This movement continued during subsequent nuclear
divisions (Fig. 2D) until the
nuclei reached the periphery at nuclear cycle 10
(Fig. 2E). Little change in the
transcript pattern was observed between nuclear cycles 10 and 13, although by
this time two bcd mRNA gradients were evident: one along the basal,
the other along the apical, periplasm of nuclei. Mitosis, as exemplified by an
embryo at the end of nuclear cycle 11, did not affect the distribution of
bcd mRNA (Fig. 2F).
During nuclear cycle 13, basal and apical bcd gradients were
prominent, though basal bcd transcripts covered a much wider layer of
the cortex than did apical transcripts
(Fig. 2G and inset). During
early nuclear cycle 14, a striking change in pattern was observed:
bcd transcripts began to disappear from the basal periplasm, while
their apical concentration appeared unchanged
(Fig. 2H and inset). The
extended gradient was obvious from the monotonically decreasing bcd
mRNA concentration (Fig. 2K).
Subsequently, basal bcd transcripts disappeared within minutes, but
apical transcripts remained (Fig.
2I,L). A few minutes later, the apical transcripts had also
disappeared (data not shown).
|
).
In unfertilized eggs and cleavage-stage embryos, where nuclei have not yet
migrated to the periphery (Fig.
2A-C), basal and apical bcd transcript levels were
measured along lines located at the same distance from the surface of the
embryo as those employed at later stages
(Fig. 3L). In unfertilized eggs
and early cleavage-stage embryos, basal transcripts formed a steep gradient
with a bend at 20% EL (Fig.
3A-C, blue) and thus might appear to be "strictly localized
to the anterior cytoplasm" (Ephrussi
and St Johnston, 2004). By contrast, apical transcripts began to
form a shallow gradient (Fig.
3B,C, pink). Clearly, basal as well as apical bcd mRNAs
already extended to posterior regions, as was apparent from a progressive
increase in their concentration posterior to the bend
(Fig. 3B,C).
During nuclear cycles 7-9, the slope of the basal gradient decreased,
moving the bend to 30% EL, whereas the slope of the apical gradient
increased (Fig. 3D). By the
time the nuclei reached the periphery, similarly shaped gradients of basal and
apical bcd mRNAs were obvious
(Fig. 3E). During the
subsequent nuclear cycles, the appearance of these gradients did not change
substantially (Fig. 3F,G).
During early nuclear cycle 14, basal transcripts were first reduced, forming a
shallow gradient, and then disappeared, whereas apical transcript levels were
still high but also began to decrease (Fig.
3H,I). Before nuclear cycle 14, bcd mRNA must be stable
because little, if any, degradation was apparent, as previously observed in
activated unfertilized eggs (Surdej and
Jacobs-Lorena, 1998).
The close similarity between the bcd mRNA
(Fig. 3E-I) and protein
gradients during syncytial blastoderm
(Driever and Nüsslein-Volhard,
1988; Houchmandzadeh et al.,
2002; Gregor et al.,
2007) raised the question of whether the Bcd protein gradient, as
analyzed by our method (Fig.
3L), displayed the same profile. Indeed, we found that the Bcd
protein gradient (Fig. 3J) was
in excellent agreement with published results
(Houchmandzadeh et al., 2002):
it decreased exponentially with increasing distance from the anterior pole to
65% EL (Fig. 3K), beyond
which Bcd intensities no longer differed significantly from background.
|
;
Gutjahr et al., 1993). At all
stages, considerable variability in the bcd mRNA gradient profiles
was observed, which was largest at the most anterior positions, in excellent
agreement with previously published variability in Bcd protein profiles among
embryos at similar stages (Houchmandzadeh
et al., 2002; Holloway et al.,
2006). Basal bcd transcript profiles of 15 embryos at cycle 13 displayed a monotonic decrease with increasing distance from the anterior pole, with a minor deviation from exponentiality that was visible as a small bend at 30% EL when plotted on a logarithmic scale (Fig. 4A and inset). This deviation was not apparent in apical transcript profiles (Fig. 4B and inset). At this stage, gradients of apical bcd transcripts were shallower and anterior intensities lower than those of basal transcripts. Basal transcript profiles of 46 embryos at 0-4 minutes of nuclear cycle 14 (Fig. 4C) showed no significant difference to those of embryos at nuclear cycle 13 (Fig. 4A). By contrast, apical transcript profiles exhibited a marked increase in intensity (Fig. 4D) compared with those of cycle 13 embryos (Fig. 4B), which suggests a movement of bcd transcripts from basal to apical periplasm. At this time, basal and apical transcripts showed very similar profiles (Fig. 4C,D and insets). Finally, bcd transcript profiles of nine embryos at 8-12 minutes after onset of nuclear cycle 14 revealed the nearly complete absence of basal transcripts (Fig. 4E), while apical transcripts had decreased considerably (Fig. 4F). This drastic decrease in both basal and apical bcd transcripts reflects a rapid degradation of bcd mRNA during early cycle 14. Both the apical and basal profiles remained close to exponential (Fig. 4E,F, insets).
Transport of bcd mRNA from basal to apical periplasm and degradation of bcd mRNA during early nuclear cycle 14
An intriguing feature of the bcd mRNA patterns is the increase in
apical, at the expense of basal, bcd mRNA levels during early nuclear
cycle 14. Most likely, this results from an active transport of basal
bcd mRNA to the apical periplasm
(Fig. 3G,H)
(Bullock and Ish-Horowicz,
2001; Wilkie and Davis,
2001). We analyzed this apical migration in detail in embryos from
the same batch as in Fig. 4. To
facilitate the analysis, fluorescence intensities, reflecting bcd
mRNA concentrations, were converted to a color scale
(Fig. 5). During nuclear cycle
13, bcd transcripts were still uniformly distributed between basal
and apical periplasm (Fig. 5A),
and no net apical migration of bcd mRNA was apparent up to this stage
(Fig. 3E-G). During the
following nuclear division, no changes in the patterns or ratio of basal to
apical transcripts were apparent (Fig.
5B,C), a theme that continued up to 4 minutes after onset of
nuclear cycle 14 at 25°C, although there was a slight decrease in the
relative amount of basal versus apical bcd transcripts, while their
relative maximum concentrations appeared unaltered
(Fig. 5C,D). However, by
10 minutes after onset of nuclear cycle 14, most basal bcd
transcripts had disappeared, while the concentrations of apical transcripts
were slightly diminished (Fig.
5E). This drastic loss of basal transcripts results from the onset
of bcd mRNA degradation. Apical transcripts are only mildly reduced
because they have been replenished by an efficient apical transport of basal
transcripts. By 16 minutes after onset of nuclear cycle 14, nearly all of
the apical transcripts have disappeared as well
(Fig. 5F). Thus, all
bcd mRNAs are degraded within 15-20 minutes.
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;
Braakman et al., 1991).
Congruence of protein and mRNA gradients at all times therefore implies that
the Bcd protein gradient does not depend on protein diffusion but is dictated
by the bcd mRNA gradient. As is evident from
Fig. 6A-I, the bcd
mRNA (green) and protein (red) patterns were virtually identical at all times,
except that the protein was localized in the nucleus and the mRNA in the
nuclear periplasm. Particularly striking is the similarity of the
cleavage-stage patterns (Fig.
6A-C). During nuclear cycle 13, the Bcd protein is presumably
translated from both basal and apical bcd mRNAs
(Fig. 6D-F), whereas Bcd
protein is imported into the nucleus only from the apical periplasm when the
basal bcd mRNA has disappeared
(Fig. 6G-I). Thus, these
results demonstrate, in clear contradiction to the SDD model
(Driever and Nüsslein-Volhard,
1988), that the Bcd protein gradient arises by translation of its
mRNA, which forms the gradient.
Similar localization and gradient of Staufen protein and bcd mRNA
Localization of bcd mRNA during late oogenesis and early
embryogenesis has been shown to depend on the product of the staufen
(stau) gene (St Johnston et al.,
1989; Ferrandon et al.,
1994; Weil et al.,
2006; Weil et al.,
2008). Stau protein binds to the 3' UTR of bcd mRNA
and is thought to be required to localize bcd mRNA to the anterior
pole of the early embryo (Ferrandon et
al., 1994). We tested whether Stau might also be associated with
bcd mRNA in embryos by visualizing its distribution. In early
cleavage embryos, Stau was localized to the posterior pole, but was also
present in an anterior cap (Fig.
6J), similar to that of bcd mRNA
(Fig. 6A). The similarity
between the bcd mRNA and anterior Stau patterns persisted during
subsequent stages (data not shown) through nuclear cycle 13
(Fig. 6D,K). In particular,
Stau also formed an apical gradient (Fig.
6L) when basal bcd mRNA had disappeared
(Fig. 6G). In contrast to
bcd mRNA, however, some Stau remained in the basal periplasm, which
indicates a function of Stau that is not associated with bcd mRNA.
These results strongly suggest that bcd mRNA is bound to Stau protein
in the embryo.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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) by
demonstrating a gradient of bcd mRNA that is very similar to that of
the Bcd protein. Therefore, our results contradict the SDD model
(Driever and Nüsslein-Volhard,
1988). Although the SDD model correctly predicts an exponential
Bcd protein gradient, the diffusion coefficient of Bcd, as measured in
syncytial-blastoderm embryos, is two orders of magnitude too low to account
for the fact that the steady state of its gradient is reached at syncytial
blastoderm (Gregor et al.,
2007), a finding that is in serious conflict with this model. Our
results strongly suggest that the bcd mRNA gradient forms the protein
gradient and is established by transport of the mRNA along the cortex of the
embryo.
|
). The posterior transport of bcd mRNA is driven by
its concentration gradient. (3) This transport is arrested by the breakdown of
the cortical microtubular network when the nuclei reach the cortex
(Foe and Alberts, 1983). At
this time, a new microtubular network of astral microtubules forms. These
extend from the centrosomes, located between the plasma membrane and each
nucleus, and surround each nucleus with apical-basal polarity
(Karr and Alberts, 1986).
Thus, the bcd mRNA gradient is established by nuclear cycle 10 and
does not change until the end of nuclear cycle 13. (4) During syncytial
blastoderm, the bcd mRNP particles are transported apically on astral
microtubules by the minus end-directed dynein/dynactin motor complex
(Wilkie and Davis, 2001), a
process that depends on the maternal proteins Bicaudal D (BicD) and
Egalitarian (Egl) (Bullock and
Ish-Horowicz, 2001). (5) Whereas bcd mRNA is stable until
nuclear cycle 13, it is rapidly degraded during early nuclear cycle 14. We
assume that this degradation is mediated by Stau and is triggered by apical
factors and signals. This model combines our results with those reported by others. Below, we discuss in detail the evidence that led us to this ARTS (active RNA transport and protein synthesis) model. Despite its speculative aspects, it should serve as a useful hypothesis for future experiments that test its predictions. In addition, the model postulates a new principle to explain the formation of the bcd mRNA gradient: a quasi-random transport through a cortical microtubular network that is driven by a high initial concentration of bcd mRNA at the anterior pole.
Release of bcd mRNA from the anterior cortex upon fertilization
Stau protein binds to the 3'UTR of bcd mRNA in oocytes and
colocalizes with bcd mRNA at the anterior pole of freshly laid eggs
(Ferrandon et al., 1994)
(Fig. 6A,B). Additional
proteins probably stabilize the interaction of Stau with bcd mRNA in
the embryo, as in oocytes (Irion and St
Johnston, 2007). Localization of bcd mRNA to the anterior
pole is established by continual active transport of the Stau-bcd
mRNA complex on microtubules, mediated by the minus end-directed motor dynein,
when nurse cells empty their content into stage 10B-13 oocytes
(Pokrywka and Stephenson,
1991; Weil et al.,
2006). Subsequent anchoring of the Stau-bcd mRNA complex
to the actin cytoskeleton stabilizes its anterior localization in mature
oocytes (Weil et al., 2006;
Weil et al., 2008). This
anchoring step depends on swallow (swa), the product of
which interacts with the dynein light chain and 
Tub37C, which is part
of the MTOC at the anterior end of oocytes
(Schnorrer et al., 2000;
Schnorrer et al., 2002). Upon
fertilization, calcium signaling releases the Stau-bcd mRNA complex
from the actin cytoskeleton, which depends on the product of the
sarah (sra) gene, an inhibitor of the calcium-dependent
phosphatase calcineurin (Weil et al.,
2008). Swa protein is no longer required and is degraded
(Schnorrer et al., 2000).
A network of microtubules, in which the MTOCs are closely spaced (separated
by a few microns), occupies the cortical region of embryos during nuclear
cycles 1-9 (Karr and Alberts,
1986; Callaini and Riparbelli,
1997). Consistent with this observation is the pattern of cortical
staining of early embryos for several Dgrips (S.B., unpublished), proteins of
the -tubulin ring complex that caps the minus ends of microtubules at
MTOCs (Gunawardane et al.,
2000). Evidently, these microtubules nucleate from MTOCs that are
established in late oocytes (Schnorrer et
al., 2002; Vogt et al.,
2006). To our knowledge, no function has been reported for this
cortical microtubular network, which breaks down at the end of nuclear cycle
9. We propose that its existence is crucial for the formation of the
bcd mRNA gradient.
Posterior cortical transport of bcd mRNA mediated by a nonpolar microtubular network
A mechanism based on diffusion of the bcd mRNA cannot explain the
gradient observed because bcd mRNA is restricted to the cortex of the
embryo. Diffusion of bcd mRNA to the interior would dramatically
reduce its concentration along the cortex, where its function is required,
because unlike for Bcd protein in the SDD model, there is no source
replenishing the lost bcd mRNA. Active transport of a
Stau-bcd mRNA complex on microtubules, similar to that observed in
late-stage oocytes (Weil et al.,
2006), is suggested by the striking colocalization of Stau and
bcd mRNA until the latter disappears
(Fig. 6). However, the
microtubules with MTOCs located at the anterior pole are disassembled in late
oocytes (Theurkauf et al.,
1992; Weil et al.,
2008). Indeed, the cortical microtubular network in embryos at
nuclear cycles 1-9 shows no sign of an overall polarity, but appears to be
nonpolar, with its plus ends growing in all directions from MTOCs closely
spaced throughout the cortex (Karr and
Alberts, 1986; Callaini and
Riparbelli, 1997).
How can such a nonpolar microtubular network establish a bcd mRNA
gradient by active transport of the Stau-bcd mRNP particles? Because
the network exhibits no polarity, it supports only random transport as would
occur by diffusion. The only restriction to the random transport is its
confinement to the cortex of the embryo. Like diffusion, it is driven by the
concentration gradient of the transported molecules, here by the high initial
concentration of bcd mRNA at the anterior pole
(Fig. 2B). Average transport
velocities of Stau-bcd mRNA complexes on microtubules, as measured in
stage 10B-13 oocytes, range from 0.36 to 2.15 µm/second
(Weil et al., 2008). Such a
non-random transport in the embryo would move bcd mRNA molecules
within minutes from the anterior to the posterior pole and thus destroy its
function as an anterior morphogen. Therefore, it seems crucial that
bcd mRNA transport in the embryo occurs through a nonpolar
microtubular network. It is additionally important that the time of 90 minutes
that is required to establish the bcd mRNA gradient at 25°C is
tuned finely with the time required for the first nine nuclear divisions,
after which the nuclei reach the cortex.
The efficiency of a system employing random transport can be estimated from
the average posterior drift velocity of bcd mRNAs along the cortex.
When, 90 minutes after fertilization, syncytial blastoderm is reached,
bcd mRNA has moved posteriorly on average by 50 µm (from 5%
EL at fertilization to 15% EL), which corresponds to an average drift velocity
of 0.01 µm/second. This is 100 times slower than the average transport
velocity on a microtubule in the oocyte
(Weil et al., 2008) and is
thus rather inefficient. Since this transport of bcd mRNA occurs on a
microtubular network with randomly oriented microtubules, it is irrelevant
whether transport is mediated by the minus end-directed dynein/dynactin or the
plus end-directed kinesin motors. In the oocyte, Stau-bcd mRNP
particles are transported exclusively by dynein
(Weil et al., 2006) in a
process that depends on the presence of Exuperantia (Exu) in nurse cells
(Cha et al., 2001). Since Exu
disappears from late oocytes (Macdonald et
al., 1991), this might permit Stau-bcd mRNPs to interact
with dynein or kinesin upon their release from the actin cytoskeleton.
Just before the present study was submitted, transport in oocytes through a
microtubular network exhibiting only a slight directional bias (57% of plus
ends oriented posteriorly) was shown to localize Stau-oskar
(osk) mRNA particles to the posterior pole
(Zimyanin et al., 2008).
Although it is conceivable that the bcd mRNA gradient is established
through such a biased microtubular network, the net average posterior velocity
in oocytes of 0.03 µm/second (Zimyanin
et al., 2008) would displace the bcd mRNA on average by
162 µm towards the posterior pole of the embryo by the time the
bcd RNA gradient is established. This is more than twice the observed
average posterior displacement of bcd mRNA
(Fig. 3E). Nevertheless, the
bcd mRNA gradient might be established through such a biased
microtubular network if transport is mediated by both minus- and plus-end
motors. In such a case, however, the average posterior drift velocity would
also depend on the availability of both motors. If the probability of
Stau-bcd mRNP interacting with either motor is the same, transport by
the microtubular network becomes independent of its directional bias, and the
network behaves like the nonpolar microtubular network. However, we favor a
nonpolar microtubular network in the embryo because it seems more robust to
disturbances.
Apical transport of bcd mRNA during syncytial blastoderm
An intriguing feature of the bcd mRNA gradient during nuclear
cycles 10-13 is the maintenance of a constant apical gradient similar to the
basal gradient (Figs 3 and
5). It has been noted
previously that bcd transcripts are localized to the narrow apical
periplasm at late syncytial blastoderm
(Frigerio et al., 1986)
(Fig. 1A,B), and that this
localization depends on a signal in their 3'UTR
(Davis and Ish-Horowicz, 1991).
Apical transport of bcd mRNA becomes evident during nuclear cycle 14,
when the excess of basal bcd mRNA disappears more rapidly than its
apical counterpart (Fig.
5C-F).
Although no net apical transport of bcd mRNA is apparent before
its degradation during nuclear cycle 14, the establishment of an astral
microtubular network during nuclear cycle 9
(Karr and Alberts, 1986)
suggests that it might occur as early as nuclear cycle 10. Such a system,
capable of transporting Stau-bcd mRNA particles to the apical
periplasm, might be important to stabilize the bcd mRNA gradient
against disturbances by the strong periplasmic flow that is observed in the
cortex during nuclear cycles 10-13 (Foe
and Alberts, 1983). Nevertheless, if Stau-bcd mRNA
complexes detach when they reach the minus ends at the apical MTOCs, some
apically localized bcd mRNAs might be subject to the periplasmic
flow. Such a disturbance would be minor, as it would be corrected immediately
by rapid apical transport of Stau-bcd mRNA particles, which occurs at
a velocity of 0.5 µm/second (Bullock and
Ish-Horowicz, 2001; Wilkie and
Davis, 2001).
Why is it important to localize bcd mRNA not only to the basal,
but also to the apical, nuclear periplasm? An answer is probably provided by
elegant studies that have demonstrated that the nuclear concentration of Bcd
protein remains approximately constant at a certain position along the
anteroposterior axis during syncytial blastoderm
(Gregor et al., 2007). This
finding was surprising in view of the fact that the number of nuclei double
after each nuclear division, their volume increases by 30% during interphase
of each nuclear cycle, and the Bcd concentration drops fourfold when nuclear
membranes disappear during mitosis. It was explained by measurements revealing
that nuclear import of Bcd is sufficiently rapid to maintain a high and
constant nuclear Bcd concentration. Hence, it might be crucial that Bcd can be
imported through the entire nuclear surface
(Gregor et al., 2007).
Consistent with an accelerated nuclear import of Bcd by the product of the
lesswright (lwr) gene
(Epps and Tanda, 1998), we
found Lwr in cleavage-stage and syncytial-blastoderm nuclei (K.F. and S.B.,
unpublished).
Is degradation of bcd mRNA also mediated by Stau?
Whereas bcd mRNA is stable before nuclear cycle 14, basal
bcd mRNA disappears owing to its degradation and transport to the
apical periplasm within 10 minutes of early nuclear cycle 14
(Fig. 5C-F). Thus, the
estimated half-life of basal bcd mRNA is 2 minutes. Apical
bcd mRNA decreases only when basal bcd mRNA becomes limiting
(Fig. 5E,F). At this time, the
estimated half-life of apical bcd mRNA is also 2 minutes
(Fig. 5E,F). Therefore,
bcd mRNA is degraded in the basal and apical periplasm, or only in
the latter. This degradation is presumably mediated by a bcd
instability element (BIE) located within a 43-nucleotide sequence following
the stop codon (Surdej and Jacobs-Lorena,
1998). In mammals, Stau may trigger the degradation of an mRNA by
binding to its 3'UTR and to the nonsense-mediated decay (NMD) factor
Upf1, in a process that is different from NMD and is called Staufen-mediated
mRNA decay (SMD) (Kim et al.,
2005). As the Drosophila genome encodes a Upf1 homolog,
Stau might well function not only in the transport of bcd mRNA but
also in its degradation.
Since Bcd protein disappears 25 minutes after bcd mRNA, a lag
during which its level decreases at least tenfold (S.B., unpublished), its
half-life is less than 8 minutes at this time. The presence of a conserved
PEST sequence in Bcd (Berleth et al.,
1988; Gregor et al.,
2008) might be responsible for its short half-life, presumably
also during earlier stages, a hypothesis that is consistent with the
similarity between the slopes of the bcd mRNA and protein
gradients.
A fundamental difference between the ARTS and SDD models
There are many ways to generate a morphogenetic gradient. The original
proposal of how the Bcd protein gradient forms
(Driever and Nüsslein-Volhard,
1988) closely followed Wolpert's model of generating a
morphogenetic gradient by a localized source synthesizing the morphogenetic
molecules that are subject to diffusion and spatially uniform degradation
(Wolpert, 1969). This model
predicts a steady state at which the Bcd concentration decays exponentially
along the anteroposterior axis (Gregor et
al., 2007). We now see that the Bcd protein gradient is generated
by an entirely different mechanism. Since there is no source of bcd
mRNA, its posterior transport from the anterior pole must be arrested when the
optimal gradient is reached. This arrest is triggered by the breakdown of the
cortical microtubular network and is well timed with the arrival of the nuclei
at the cortex, when gap genes are activated by the Bcd protein
(Bergmann et al., 2007). At
this time, the gradient is established and remains constant until nuclear
cycle 14, when bcd mRNA is rapidly degraded. Thus, the bcd
mRNA gradient is not established as a steady state, but by a process that is
terminated by the breakdown of the microtubular network required for its
formation.
Compared with the diffusion-based mechanism of the SDD model, a random
active transport system has several advantages for the formation of the
bcd mRNA gradient. The microtubular network is able to confine the
movement of the bcd mRNA to the space where its function is required.
The final shape of the gradient depends on several parameters: the initial
concentration of bcd mRNA at the anterior pole, the transport
velocity along microtubules, the average travel time per microtubule, the time
between discharge from one and reloading onto another microtubule, and the
time when transport is arrested by the breakdown of the microtubular network
that supports the random transport. In addition, the availability of minus
end- and plus end-directed motors might further influence the generation of
the bcd mRNA gradient by random transport. Therefore, perhaps the
greatest advantage of random active transport is that variations in these
parameters during evolution permit the adaptation of the gradient to its
optimal shape at the time when its function is required during development
(Gregor et al., 2005). For
these reasons, we suspect that random active transport represents a general
mechanism that might have found wide application during evolution.
|
Footnotes |
|---|
-tubulin; David Holloway and Nina Golyandina for
discussions; and Hans Noll and a reviewer for comments on the manuscript. S.B.
thanks the Wenner-Gren Foundations for support
during a sabbatical in Zürich. This work was supported by the
Swedish `Vetenskapsrådet', the
Swedish Cancer Foundation, the
Medical Faculty of Lund (S.B.), by the Joint
NSF/NIGMS BioMath Program
Grant R01-GM072022 (A.S.), and by the
Swiss National Science Foundation and the
Kanton Zürich (M.N.). A.S. is supported by
NIH NIGMS BioMath Program
Grant R01-GM072022. Deposited in PMC for release after
12 months.
* Present address: Department of Genetics, Faculty of Agriculture, Ain Shams
University, Cairo, Egypt 
Present address: University of Copenhagen, Institute of Biology,
Universitetsparken 15, Building 12, DK-2100 Copenhagen, Denmark
|
REFERENCES |
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|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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