Fly 5:3, 242-246; July/August/September 2011; © 2011 Landes Bioscience
242 Fly Volume 5 Issue 3
Extra View to: Liu J, Ma J. Fates-shifted is an F-box
protein that targets Bicoid for degradation and
regulates developmental fate determination
in Drosophila embryos. Nature Cell Biol 2011;
13:22–9; PMID: 21170036; DOI:10.1038/ncb2141.
Key words: morphogen gradient, Bicoid,
half-life, diffusion constant, steady state,
positional information, robustness,
Drosophila, fates-shifted, ubiquitination
*Correspondence to: Jun Ma;
shifted (fsd), that plays a role in targeting
Bcd for ubiquitination and degradation.
Our analysis of mutant Drosophila
embryos suggests that Bcd protein deg-
radation is important for proper gradi-
ent formation and developmental fate
specification. Here we describe further
experiments that lead to an estimate of
Bcd half-life, <15 min, in embryos dur-
ing the time of gradient formation. We
use our findings to evaluate different
models of Bcd gradient formation. With
this new estimate, we simulate the Bcd
gradient formation process in our own
biologically realistic 2-D model. Finally,
we discuss the role of Bcd-encoded posi-
tional information in controlling the
positioning and precision of developmen-
n a recent publication,1 we identified a
novel F-box protein, encoded by fates-
Models of Bcd Gradient Formation
Bcd is a morphogenetic protein that
forms a concentration gradient along
the anterior-posterior (A-P) axis in early
Drosophila embryos.2 It controls embry-
onic patterning by activating its tar-
get genes in a concentration-dependent
manner.3-7 Despite extensive studies, it
currently remains controversial how the
Bcd concentration gradient is formed.
There are two broad, contrasting mod-
els. A simple diffusion model8 has been
widely used to explain the exponential
gradient of Bcd,9-11 where Bcd protein is
synthesized at the anterior, diffuses and
decays throughout the embryo (hence
also referred to as the SDD model). The
diffusion model produces a steady state
Morphogen gradient formation and action
Insights from studying Bicoid protein degradation
Junbo Liu,1 Feng He1 and Jun Ma1,2,*
1Division of Biomedical Informatics; 2Division of Developmental Biology; Cincinnati Children’s Research Foundation; Cincinnati, OH USA
exponential profile of the Bcd concentra-
tion: B = Ae-x/λ, where A is the amplitude,
x is distance from the anterior and λ is
the length constant.9 However, since bcd
mRNA, the source for Bcd production,
is not restricted to a single point in the
actual embryo,12,13 the idealized version
of this model is inadequate for Bcd. In
fact, based on the observed redistribution
of bcd mRNA, a contrasting model was
proposed recently in reference 14. In this
model, the process of Bcd gradient forma-
tion and its final shape are dictated by the
redistribution process of bcd mRNA.14,15
We refer to this model as the mRNA-
dictated-gradient model. Since the mea-
sured bcd mRNA profile differs from the
exponential Bcd protein profile,14 the pure
form of the proposed mRNA-dictated-
gradient model also appears inadequate
for explaining fully how the Bcd protein
gradient is formed.12,16
In addition to these two contrasting
models, several other models have also
been proposed for Bcd.17-19 Since Bcd dif-
fusion remains a component of all these
models, they as a group are more related to
the diffusion model than the mRNA-dic-
tated-gradient model. Each of these models
was proposed to explain specific properties
of the Bcd gradient system. For example,
an intriguing property of the Bcd gradient
is the stability of its nuclear concentrations
as a function of developmental time.20
While the nuclear number undergoes an
exponential increase after each nuclear
division, the profiles of nuclear Bcd con-
centration remain relatively stable at the
interphase of nuclear cycles 10–14. This
observation led to the proposal of a nuclear
trapping model,17 where Bcd protein is
www.landesbioscience.com Fly 243
according to this model, the final shape
of the protein gradient is determined
exclusively by the bcd mRNA gradient,
rather than Bcd protein degradation and
diffusion.12,14-16 Second, our estimated
Bcd t1/2 sheds light on an outstanding
controversy over the diffusion constant
D of Bcd in the cytoplasm of embryos.
While an earlier study20 suggested a D
value of ~0.3 μm2s-1, a more recent study
in reference 21 revealed a value that is >20
times larger, ~7.4 μm2s-1. In a simple dif-
fusion model,8,9 the length constant λ of
the steady state exponential profile is a
function of the diffusion constant D and
decay rate ω, λ2 = D/ω. Under this frame-
work, our estimated half-life of Bcd (t1/2
= 15 min corresponds to ω = 0.0008 s-1)
and a consensus λ estimate of ~100 μm
are more consistent with a large D value.
We note that, since our estimated Bcd
t1/2 represents an upper bound, a formal
possibility exists that the actual D value
for Bcd could be even larger than the
reported ~7.4 μm2s-1.
degradation activities than later embryos
(2–3 hr). Since the 2–3 hr embryos con-
tain those that are actively clearing up
the Bcd gradient, we used the previous
estimate obtained from such embryos2
to calibrate our biochemical data. Using
such calibration, we estimate that Bcd t1/2
in embryos at the time of Bcd gradient
formation is <15 min. While we are aware
of the inherent limitations of biochemical
assays for quantifying biophysical proper-
ties inside an embryo, this value represents
the best estimate currently available.
Our estimated Bcd t1/2 of <15 min has
important implications for the mecha-
nisms of Bcd gradient formation. First,
this value is not consistent with the
assumptions made in the nuclear trap-
ping and pre-steady-state models,17,18
where Bcd is proposed not to decay or to
decay slowly during the gradient forma-
tion process. Our reported finding that
Bcd degradation is important for gradient
formation1 is also inconsistent with the
mRNA-dictated-gradient model because,
reversibly trapped by the nucleus to allow
the formation of a stable nuclear Bcd con-
centration gradient. The nuclear trapping
model proposes that Bcd does not decay
during the period of gradient formation.17
Another model, a pre-steady-state model,18
was proposed to explain the precision of
the expression patterns of Bcd target genes
among different embryos. This model
proposes that the positional information
provided by the Bcd gradient is decoded
before the gradient reaches its steady state.
It also requires Bcd to be a stable protein
with a half-life comparable to the proposed
decoding time (60~90 min).18
An Estimate of Bcd Half-Life
to Evaluate Different Models
An important finding reported in our
recent study is that perturbed Bcd deg-
radation, in embryos from fsd females
(referred to as fsd embryos), led to an
altered Bcd gradient profile.1 These results
suggest that Bcd degradation is important
for the gradient formation process. To
evaluate different models of Bcd gradient
formation, it is critical to have an estimate
of the Bcd half-life, t1/2, in embryos at the
time of Bcd gradient formation. Such a
value is currently unavailable. Based on
the kinetics of the disappearance of Bcd
after cellularization,2 it was estimated that
Bcd t1/2 in cellularized embryos is <30 min.
This value is for embryos that are actively
“clearing up” the Bcd gradient that is no
longer needed. In our reported study in
reference 1, we used 0–3 hr embryonic
extracts to assay Bcd degradation. These
extracts reflect, collectively, the proper-
ties of embryos that are both undergo-
ing Bcd gradient formation and actively
clearing up the Bcd gradient (i.e., before
and after cellularization, respectively).
To gain further insights into Bcd degra-
dation properties in embryos as a func-
tion of developmental time, we generated
extracts from staged 0–1, 1–2 and 2–3 hr
embryos, with all experiments performed
side-by-side to allow direct comparisons.
Our Bcd degradation assays in these
extracts revealed an estimated t1/2 of 19.7,
18.9 and 43.6 min, respectively (Fig. 1).
These results show that embryos undergo-
ing the process of Bcd gradient formation
(0–1 and 1–2 hr) actually have higher Bcd
Figure 1. Bcd degradation and estimation of Bcd half-life. (A) Extracts generated from 0–1, 1–2
and 2–3 hr w1118 embryos were used to assay Bcd degradation.1 As shown previously in reference 1,
Bcd degradation in embryonic extracts is inhibited by MG132, indicating that proteasome-depen-
dent activities, as opposed to some non-specific activities, are responsible for Bcd degradation.
All experiments shown here were performed side-by-side. As discussed previously in reference 1,
data from experiments that are not done side-by-side cannot, and should not, be compared with
each other. (B) The plot shows the percentage of Bcd protein remaining at different time points
of the degradation reaction. Solid lines represent exponential fitting to experimental data. (For
further details, see text and ref. 1).
244 Fly Volume 5 Issue 3
two separate cases where mutant embryos
exhibit increased variability in the hunch-
back (hb) expression boundary, we were
able to trace the origin of such variability
directly to perturbed Bcd gradient prop-
erties.23,35 To our knowledge, these results
represent the only experimental evidence
that a precise Bcd gradient is necessary
for precise developmental decisions. Our
finding1 that the shift in Bcd-encoded
positional information in fsd embryos
matches the shift in hb expression bound-
ary further underscores a direct and
dominant role of Bcd in instructing hb
expression (see also ref. 22). Exactly how
the positional information provided by
the Bcd gradient feeds into precise pat-
terning decisions is a subject of intense
theoretical investigations.34,35,37-39 In a
recent experimental study,40 we investi-
gated the role of Bcd in the actual tran-
scriptional events of its target genes in
developing embryos. Our results suggest
that Bcd acts as a direct and sustained
input for these transcriptional events.
In addition, a comparison between the
noise in Bcd-dependent transcriptional
events and the noise in Bcd-dependent
transcriptional products provides a first
experimental demonstration of the effect
of time/space averaging in reducing the
useful framework in guiding our think-
ing and analyses of the Bcd gradient for-
Interpretation of Bcd-Encoded
One of the fundamental questions regard-
ing the actions of morphogens relates to
the precision of the positional information
provided by a morphogen gradient and
the extent to which such information can
influence the precision of developmental
decisions.25,26 For the Bcd gradient profile,
embryo-to-embryo variations were ini-
tially reported to be very large, suggesting
that the positional information provided
by the Bcd gradient is imprecise.9,27,28
These findings prompted the proposal of
different models that can correct embryo-
to-embryo fluctuations in Bcd-encoded
positional information.18,29-33 However,
more recent studies revealed that the Bcd
gradient profile is highly precise among
different embryos.23,34,35 But whether a
precise Bcd gradient is important for pre-
cise patterning remains controversial. It
was suggested that, based on thermal per-
turbations, a precise Bcd gradient is not
required for precise patterning.36 Our own
studies based on genetic perturbations
have led to a contrasting proposal.23,35 In
Bcd Gradient Formation
Simulated in a Biologically
We have developed recently a biologically
realistic 2-D model for the Bcd gradient
formation process.12,22 This model is dif-
ferent from the simple diffusion model
because it has incorporated several key
biological features relevant to the Bcd
gradient system, including the loca-
tion and amount of bcd mRNA and the
exponential increase in nuclear numbers
after each nuclear division (reviewed in
ref. 12). Using this model and our newly
estimated Bcd t1/2, we conducted simu-
lation studies to evaluate Bcd gradient
properties (for details, see Fig. 2 legend).
Figure 2A shows the simulated profiles
of nuclear Bcd concentration, [Bn], at
nuclear cycles 10–14, which exhibit an
experimentally observed stability.20 As
further discussed in reference 12, an
interaction between Bcd and the genome
within the nucleus in our model plays an
important role in maintaining the stabil-
ity of nuclear Bcd concentrations dur-
ing the time when the nuclear number
is increasing exponentially (see also ref.
17). Figure 2B shows that our simulated
[Bn] profile and experimentally mea-
sured Bcd profile,23 both at early nuclear
cycle 14, exhibit strong resemblance to
each other. These and additional results
(below) show that our biologically realis-
tic model can recapitulate key properties
of the Bcd gradient. For example, it also
readily explains the differences in Bcd
gradient profiles caused by the geometric
asymmetry between the dorsal and ven-
tral sides of the embryo.22 In addition, it
recapitulates the scaling properties of the
Bcd gradient23 by simply assuming a cor-
relation between the Bcd production rate
and embryo volume.12 This hypothesized
correlation was recently observed experi-
mentally13 through quantitative measure-
ments of bcd mRNA in large and small
embryos from selected Drosophila lines.24
Since our model is based on Bcd diffu-
sion and degradation while incorporating
relevant, biologically realistic features of
the embryo, our results suggest that the
diffusion model—despite the inadequa-
cies of its idealized form in fully captur-
ing Bcd gradient properties—remains a
Figure 2. Simulated nuclear Bcd gradient profiles. (A) Nuclear Bcd concentration [Bn], in arbitrary
units, within the cortical layer of a simulated embryo was obtained in a biologically realistic 2-D
model.12 Here the simulated [Bn] profiles as a function of fractional embryo length x/L are shown
for nuclear cycles 10 to 14. These simulated [Bn] profiles, as seen experimentally,20 differ by <10%,
with a calculated g value of -0.09 (for details see ref. 12). This simulation was performed using ω
= 0.0008 s-1 and D = 8 μm2s-1. All other parameters used here were the same as in the main model
described in reference 12, except the center coordinate of the bcd mRNA sphere (55 μm in current
work). (B) Comparison between simulated [Bn] profile and the experimentally measured mean Bcd
profile (with standard deviation shown).23 Both profiles are from early nuclear cycle 14 and each
has a length constant λ of ~100 μm.
www.landesbioscience.com Fly 245
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Recent studies of the role of the termi-
nal system in the expression of Bcd target
genes have led to the proposal of a mor-
phogen network model.41-44 This model
emphasizes the integration of mater-
nal inputs of both the terminal system
and the Bcd gradient; cross-regulation
between the zygotic products of gap genes
is also a hallmark feature of the proposed
gene regulatory network models.18,33,45,46
genes respond to distinct Bcd concentra-
tion thresholds remains an important and
challenging problem.41-43,47,48 Meanwhile,
how hb is expressed in response to Bcd
has attracted most extensive studies in
the field.9,22,23,33-35,46,49,50 Unlike genes that
are expressed near the head region, the hb
expression boundary does not appear to
be influenced strongly by the terminal sys-
tem.41 Thus, the positioning and precision
of the hb expression boundary provides a
best readout of Bcd-encoded positional
information and properties intrinsic to
the Bcd gradient system. In addition, the
hb expression boundary is located near
the middle of the embryo, where the scal-
ing properties can influence the pattern-
ing landscape along the entire A-P axis.51
Importantly, the scaling properties of hb
can also be traced directly to the scaling
properties of the Bcd gradient.12,13,23 These
and other results have led us to propose in
reference 23 that the Bcd gradient itself is
a robust system (see also refs. 34 and 49).
Understanding the precise mechanisms of
Bcd-activated hb transcription will fur-
ther expand our knowledge of not only
how Bcd works in particular, but also how
morphogens work in general.
This work was supported in part by grants
from NIH and NSF (to Jun Ma) and an
AHA postdoctoral fellowship (to Feng He).
1. Liu J, Ma J. Fates-shifted is an F-box protein that
targets Bicoid for degradation and regulates devel-
opmental fate determination in Drosophila embryos.
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51. de Lachapelle AM, Bergmann S. Precision and scal-
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