REVIEW
Dynamic patterning by morphogens illuminated by cis-regulatory
studies
Jihyun Irizarry and Angelike Stathopoulos
*
ABSTRACT
Morphogen concentration changes in space as well as over time
during development. However, how these dynamics are interpreted
by cells to specify fate is not well understood. Here, we focus on two
morphogens: the maternal transcription factors Bicoid and Dorsal,
which directly regulate target genes to pattern
Drosophila
embryos.
The actions of these factors at enhancers has been thoroughly
dissected and provides a rich platform for understanding direct input
by morphogens and their changing roles over time. Importantly,
Bicoid and Dorsal do not work alone; we also discuss additional
inputs that work with morphogens to control spatiotemporal gene
expression in embryos.
KEY WORDS:
Drosophila melanogaster
, Bicoid (Bcd), Dorsal (Dl),
Morphogen, Thresholds, Cis-regulatory mechanism, Gradient,
Chromatin, Repressor, Activator, Enhancer, Embryonic patterning
INTRODUCTION
Morphogens are molecules distributed in a graded manner in order
to provide positional cues for cell fate specification during
development (Rogers and Schier, 2011; Wolpert, 1996). In
general, morphogens are proteins that are produced from localized
sources and transported in the extracellular space toward
neighboring cells. Once the gradients are formed in this way, the
receiving cells interpret their respective position within the tissue by
sensing the concentration of morphogens. In response, cells activate
different sets of target genes, thus initiating the patterning processes
and ultimately defining cell fate. This phenomenon is observed for a
number of morphogens acting in various organisms, such as
sonic hedgehog (Shh) in chick neural tube patterning (Briscoe
et al., 2001), Wingless (Wg) in
Drosophila
appendage
development (Neumann and Cohen, 1997), squint in zebrafish
germ layer patterning (Chen and Schier, 2001) and activin in
Xenopus
mesoderm and ectoderm induction (McDowell and
Gurdon, 1999). Owing to the importance of morphogens for
patterning, dissecting the molecular mechanisms by which they
control gene activation has been an intense field of study for
decades.
One of the core issues investigated with regards to morphogens is
the mechanism by which their concentrations are translated into
gene expression outputs. One of the earliest models of patterning
proposed that differences in target gene spatial expression
domains are directly related to morphogen concentration
(Wolpert, 1969) (Fig. 1A). In this threshold-dependent model,
target gene transcription is initiated only when the morphogen
concentration is present above a certain level, whereas
transcription is not initiated in domains where the morphogen
concentration is present below this level. In this manner,
gradients of morphogens regulate patterning in a field of cells
by differentially controlling gene expression in response to the
concentration change present in space.
In addition, recent studies have shed light on the importance of
time, both duration and timing of exposure to morphogens, for
target gene expression (Rogers and Schier, 2011; Sagner and
Briscoe, 2017). Target gene expression is influenced by morphogen
input as well as by the current gene expression state of the receiving
cells. Responses to a given morphogen will differ between receiving
cells with different extant gene expression programs. For example,
during dorsal-ventral (DV) patterning in gastrulating zebrafish
embryos, cell fate is specified in a temporally progressive manner
due to changes in target tissue sensitivity to BMP signaling inputs.
BMP signaling during early gastrulation supports patterning of
anterior ventrolateral domains, whereas later the same signal
promotes patterning of posterior ventrolateral domains (Tucker
et al., 2008). Despite accumulating data supporting temporal
specificity of morphogens in development, it remains unclear
whether changes in levels of morphogens over time (i.e. morphogen
dynamics) generally control dynamics in target gene expression
directly. Target gene dynamics may relate instead to a function of
the underlying gene regulatory networks (i.e. an indirect response).
The highly context-specific response highlights the importance of
understanding how time can influence the effects of morphogens on
target genes.
In
Drosophila
embryos, two morphogens, Dorsal (Dl) and Bicoid
(Bcd), control patterning along two body axes: the dorsal-DV and
anterior-posterior (AP), respectively. How Dl and Bcd morphogens
control patterning across the developing embryo has been and
remains an active area of investigation. These specific morphogens
are unusual because they act directly in nuclei before cellularization
of the
Drosophila
embryo has occurred; therefore, the responses of
their target genes do not rely on extensive intracellular signaling
cascades. For this reason, and because
Drosophila
embryos develop
very quickly (morphogen-mediated patterning occurs within only a
few hours), it is often assumed that increases in morphogen
concentration correlate directly with expression of target genes. In
this Review, we compare what is known about Bcd- and Dl-
dependent patterning mechanisms, with a particular emphasis on
the functions of these morphogens over time. We also discuss what
is known about additional inputs that contribute to spatiotemporal
regulation of target gene expression. Modeling efforts aimed at
understanding Dl and Bcd functions have been highlighted in recent
reviews (Huang and Saunders, 2020; Schloop et al., 2020) and are
therefore not discussed here, but suggest that morphogen
concentration alone is not sufficient to predict target gene
expression and reinforce the view that additional inputs are
important.
California Institute of Technology, Division of Biology and Biological Engineering,
1200 East California Blvd., Pasadena, CA 91125, USA.
*Author for correspondence (angelike@caltech.edu)
A.S., 0000-0001-6597-2036
1
© 2021. Published by The Company of Biologists Ltd
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Development (2021) 148, dev196113. doi:10.1242/dev.196113
DEVELOPMENT
Drosophila
morphogens Bicoid and Dorsal: gradient
formation and dynamics
Upon fertilization,
Drosophila
embryos undergo 13 rounds of rapid
DNA replication and mitoses in the absence of cytokinesis. These
divisions, referred to as nuclear cycles, occur in a syncytium in
which there are no cellular membranes and the embryo is essentially
one cell. The first nine nuclear cycles last about 8 min each; whereas
starting at nuclear cycle (nc) 10, the duration lengthens. At this
point, widespread zygotic gene expression is initiated. It is at this
early stage of the developmental process of the
Drosophila
embryo
that patterning along the AP and DV body axes is established
(reviewed by Stathopoulos and Newcomb, 2020). Two maternally
deposited morphogens, Bcd and Dl, are key factors that orchestrate
this patterning process.
Bicoid: a morphogen that controls patterning along the AP axis
Maternally expressed
bcd
mRNA contains localization sequences
that result in the concentration of transcripts at the anterior pole of
Drosophila
embryos. Upon fertilization, translation of these
localized transcripts results in a gradient of protein along the AP
axis (Fig. 1B) (Driever and Nüsslein-Volhard, 1988). Synthesis in
the anterior pole is followed by diffusion, as well as protein
degradation, to extend the range and adapt the shape of the Bcd
gradient, respectively (Durrieu et al., 2018; Gregor et al., 2007;
Little et al., 2011). Once the gradient forms, Bcd, a homeodomain
transcription factor (TF), binds to specific DNA sequence motifs,
and differentially activates genes along the AP axis (Struhl et al.,
1989). Bcd can affect target gene expression even in posterior
regions, where the gradient is more diffuse, by forming local hubs
within nuclei that are concentrated enough to facilitate Bcd binding
to enhancers (Mir et al., 2017). Furthermore, despite the short time
that Bcd molecules are bound to DNA (Mir et al., 2018), this input is
translated into accurate and fast gene expression decisions at target
enhancers that occur on the timescale of a few minutes (Desponds
et al., 2020). Together, these mechanisms that regulate the
distribution of Bcd in the embryo and its interaction with target
enhancers determine the ability of this morphogen to differentially
affect gene expression across the AP axis.
Although most studies of Bcd target gene expression are focused
on nc14, nuclear Bcd is observed as early as nc6 and exhibits
spatiotemporal dynamics (reviewed by Huang and Saunders, 2020;
Little et al., 2011). Bcd levels rise quickly within nuclei at the onset
of each nuclear cycle (Fig. 1B). Furthermore, although peak levels
increase slightly from one nuclear cycle to the next, compared with
Dl dynamics (discussed below) Bcd changes are relatively small
when measured from the perspective of a single nucleus at the
anterior of the embryo (Fig. 1B) (Gregor et al., 2007; reviewed by
Sandler et al., 2018). In addition, studies of specific target genes,
such as
hunchback
(
hb
), have suggested that Bcd input becomes
dispensable by late nc14. At this point, the expression
boundaries of many targets, including
hb, knirps
(
kni
)and
Krüppel
(
Kr
)
,
are supported by cross-regulation of gap genes that
function as repressors (Jaeger et
al., 2004; Liu et al., 2013; Manu
et al., 2009).
These particular studies have suggested that early nc14 is the
pivotal time when Bcd provides positional information for
patterning (Liu et al., 2013). However, another recent study has
shown that removal of Bcd at any time between nc10 and
gastrulation causes a loss of anterior-most embryonic structures
(Huang et al., 2017). Whereas integration of Bcd over an
∼
1.5 h
period is required for a subset of target genes expressed in anterior
cells, cells located more posteriorly only require Bcd input for a
short time to establish the correct fates. In this way, cell fates along
the AP axis are determined from posterior to anterior and may
facilitate boundary formation, because nuclei that have reached their
final fates may no longer be influenced by fluctuations in local
concentration of Bcd (reviewed by Huang and Saunders, 2020).
These results support the view that there is not one particular time
point at which Bcd acts, but rather that it acts at different times
depending on the AP localization of expression of its targets.
Morphogen concentration
t
1
t
2
Normalized nuclear intensity
A
B
Dl-Venus
P
D
V
A
A
B
D
D
l-
l
V
V
enu
enu
V
V
V
V
s
s
Bcd-GFP
20
40
60
80
100
(min)
nc
10 11 12
13
14
t
1
t
2
0.2
0.4
0.6
0.8
1
Fig. 1. Comparison of Bicoid and Dorsal morphogens in space and over time in
Drosophila
embryos.
(A) The French flag model for patterning
(Wolpert, 1969) includes morphogen production from a source (green) along with its diffusion to neighboring cells, generating a morphogen gradient
. A cell
exposed to morphogen concentration above threshold 1 (t
1
) adopts one cell fate (pink), whereas cells at a distance adopt distinct cell fates (gray versus yellow) in
response to lower morphogen concentrations above or below threshold 2 (t
2
). (B) A conceptual representation of the AP patterning morphogen Bicoid (Bcd; blue)
and the DV patterning morphogen Dorsal (Dl; red) from nuclear cycle (nc) 10 to 14. Fluorescence intensity was measured by monitoring Bcd-GFP or Dl-Venus.
For Bcd-GFP, the measurements were taken at a single nucleus located 10% of the way along the AP axis. For Dl-Venus, the measurements were taken at the
ventral-most region. The intensities were normalized to the maximum value (Gregor et al., 2007; Reeves et al., 2012). Scheme representing gradients of Bcd
along the AP axis (blue) and Dl along the DV axis (red) in
Drosophila
blastoderm embryos, and a hypothetical target gene threshold responses graph.
A, anterior pole; P, posterior pole; D, dorsal region; V, ventral region.
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DEVELOPMENT
Dorsal: a morphogen that controls patterning along the DV axis
dl
mRNA, like
bcd
, is maternally deposited; unlike
bcd
,
dl
mRNA
and protein that is synthesized upon fertilization are uniformly
distributed in precellular embryos (Anderson and Nüsslein-
Volhard, 1984; Roth et al., 1989). Dl cannot enter the nucleus on
its own, however, and the regulated transport of protein into ventral
nuclei establishes a gradient that allows Dl to function as a
morphogen to support DV axis patterning (Roth et al., 1989;
Rushlow et al., 1989; Steward, 1989). Toll signaling pathway
activation regulates this nuclear translocation of Dl protein. In the
absence of Toll signaling pathway activation, Dl is bound by the I
κ
B
homolog Cactus and is therefore sequestered in the cytoplasm
(Belvin and Anderson, 1996). Cactus facilitates diffusion, or
shuttling, of Dl within the syncytial blastoderm by forming a Dl/
Cactus complex (Carrell et al., 2017). This mechanism also helps to
ensure that patterning is robust to changes in Dl morphogen
concentration (Al Asafen et al., 2020). Activation of the Toll
receptor leads to a downstream intracellular cascade that ultimately
results in phosphorylation and subsequent degradation of Cactus.
The Toll receptor is ubiquitously expressed throughout embryos;
however, its ligand Spätzle is activated in a graded manner along the
DV axis within the extracellular space, such that highest Toll
receptor activation occurs in ventral regions (reviewed by Reeves
and Stathopoulos, 2009). In this way, Dl is freed from the Dl/Cactus
complex in a ventrally biased manner and is then imported into
nuclei, where it activates over 50 genes (Belvin et al., 1995;
Stathopoulos et al., 2002). The result of Toll signaling is a nuclear-
cytoplasmic Dl gradient with the highest levels of nuclear Dl in
ventral regions and progressively lower levels dorsally (Fig. 1B).
In contrast to the fast nuclear import displayed by Bcd at the
onset of each nuclear cycle, levels of nuclear Dl rise slowly
throughout the nuclear cycle. This difference likely relates to Toll-
dependent signaling being required for Dl nuclear import, whereas
Bcd is free to enter nuclei immediately once they reform after each
division. Nuclear Dl levels rise during each nuclear cycle,
plummet upon nuclear envelope breakdown at division and are
slowly re-established upon nuclei reformation following division
(Reeves et al., 2012). In addition, Dl levels rise between nuclear
cycles, never reaching a steady state; such that, from the
perspective of a nucleus located in ventral regions, input exhibits
a saw-tooth trend (Fig. 1B).
Cis
-regulatory interpretation of morphogen concentration
and importance of additional inputs
The dynamics associated with the morphogens Bcd and Dl suggest
that both provide not only positional cues, but also impart temporal
information toward target gene expression during the
Drosophila
body patterning processes. Enhancer sequences provide information
about the cis-regulatory logic of inputs that function to support gene
expression outputs and provide insight into the mechanisms of
action of these morphogens.
Interactions between morphogen transcription factors and pioneer
factors initiate gene activation during embryonic patterning
Enhancers are short regions of DNA that control transcription of
genes, presumably, by making direct contact with gene promoters
(reviewed by Furlong and Levine, 2018). In general, enhancers are
DNA sequences of a few hundred base pairs long (average
∼
500 bp)
located in
cis
to target genes, and include short, specific TF-binding
sites. Enhancer activity is context dependent and perturbing its
action can lead to developmental defects and disease (Bhatia et al.,
2013; Lupiáñez et al., 2015; Uslu et al., 2014).
During early
Drosophila
development, TFs distributed in
gradients serve as morphogens to support cell fate specification
by controlling enhancer activity. Quantitative
in vivo
measurements
of animal transcription, including recent innovations in live
imaging, are widely used to extend understanding of morphogen
responses (Ferraro et al., 2016). Specific, quantitative features of
gene transcription, such as precision, accuracy, robustness,
plasticity and stability can now be reliably assayed (Bentovim
et al., 2017). For example, a study of the
hb
proximal enhancer, a
750 bp region regulated by Bcd, has demonstrated that this
enhancer, together with its associated promoter, P2, is sufficient
to properly capture the spatiotemporal dynamics of establishment of
the
hb
pattern (Lucas et al., 2018). However, in this particular case,
mathematical modeling suggested that the six Bcd-binding sites
present in this enhancer sequence are insufficient to support
expression that is as steep as the
hb
gene expression pattern.
Additional input has been invoked to explain this discrepancy,
because either additional cryptic, possibly low-affinity, Bcd-
binding sites and/or other TFs distributed in gradients could also
affect the delineation of the boundary. For example, maternal Hb
protein is also present in an anterior-to-posterior gradient, and
supports zygotic
hb
expression by facilitating expression at a lower
Bicoid concentration threshold and by making activation faster
(Porcher et al., 2010; Simpson-Brose et al., 1994). Although Bcd
activates more than 40 target genes to initiate AP patterning in an
apparently concentration-dependent manner, it is recognized that
input from other factors is necessary to support proper target gene
expression (reviewed in in Briscoe and Small, 2015).
In particular, during early embryonic development, enhancer
regions must become free of nucleosomes, in order to be accessible
to binding of morphogen TFs. This process is supported by a
particular class of TFs called pioneer factors. Pioneers are the first
sequence-specific DNA-binding factors to engage target sites
within chromatin during development. A pioneer factor can bind
to target enhancers in
‘
closed
’
or inaccessible chromatin and
facilitate chromatin remodeling processes, which allows other TFs
to subsequently bind enhancers to control target gene activity (Zaret
and Carroll, 2011). At the early blastoderm stage, maternally
deposited Zelda (Zld) acts as a crucial pioneer factor to increase
chromatin accessibility by supporting local depletion of
nucleosomes with the effect being dependent on the number and
position of Zld motifs (Li et al., 2014; Schulz et al., 2015; Sun et al.,
2015). Furthermore, Zld functions as a global activator to initiate
zygotic gene expression, including early body axis patterning genes
in embryos (Harrison et al., 2011; Nien et al., 2011).
Zld associates with enhancer sequences for patterning genes that
are subsequently also bound by Bcd and Dl. A recent study has
examined occupancy of Zld and Dl on DNA throughout the genome
with fine temporal resolution, revealing that although Zld binding
can be detected as early as nc8, little to no Dl binding is observed at
that time (Li and Eisen, 2018 preprint). These results suggest that
Zld input precedes that of Dl and possibly potentiates Dl binding at
target gene enhancers. In
zld
mutants, chromatin accessibility at
enhancers decreases, and Bcd or Dl binding is significantly reduced
(Li and Eisen, 2018 preprint; Xu et al., 2014). Thus, Bcd- or Dl-
dependent enhancers lose their activity when
zld
is absent.
Similarly, when the number of Zld-binding sites in enhancers of
target genes is reduced, reporter expression can be delayed and
exhibits domain shifts (Yamada et al., 2019). In contrast, addition of
more Zld-binding sites to enhancers can result in precocious
expression (Bosch and Bosch, 2006; Foo et al., 2014) or expansion
of the expression domains (Ozdemir et al., 2014). Furthermore,
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Development (2021) 148, dev196113. doi:10.1242/dev.196113
DEVELOPMENT
introducing Zld-binding sites into an inactive enhancer can convert
it into a morphogen-dependent responsive enhancer (Foo et al.,
2014; Xu et al., 2014). Zld does not remain detectably associated
with mitotic chromosomes, although sub-nuclear dynamic hubs
form where transient Zld binding likely occurs (Dufourt et al.,
2018). It is possible that Zld potentiates transcriptional outputs by
the Dl and Bcd morphogens by modulating the nuclear environment
to support their ability to exist in hubs (Mir et al., 2018; Yamada
et al., 2019), by influencing the activity of specific promoters (Ling
et al., 2019) and/or by supporting poised RNA polymerase at target
genes pre-activation (Boija and Mannervik, 2016; Koenecke et al.,
2017). Taken together, these studies indicate that Zld likely
functions by priming the genome to potentiate morphogens to
activate target gene enhancers in space but also by influencing gene
expression timing.
Although, to date, no studies have demonstrated whether Dl can
directly regulate chromatin accessibility, a recent study using
ATAC-seq has analyzed the ability of Bcd to support chromatin
accessibility (Buenrostro et al., 2015; Hannon et al., 2017).
Chromatin accessibility associated with particular Bcd target
genes active along the AP axis is sensitive to levels of this
morphogen (Hannon et al., 2017). In particular, the results are
consistent with a model in which Bcd operates at high
concentrations at the anterior of embryos to establish chromatin
accessibility at target sites. When levels of Bcd are artificially
increased, enhancers associated with some Bcd target genes most
sensitive to Bcd levels exhibit increases in chromatin accessibility.
These results suggest that high levels of Bcd promote remodeling of
chromatin structure.
Relationship of affinity of binding sites for Dl and Bcd within target
gene enhancers to threshold outputs
Once enhancer regions of target genes become accessible,
morphogens also play a role in promoting activation of transcription
to support the patterning process. As discussed above, in the
threshold-dependent model, morphogen concentration provides
positional information to drive cells to differentiate into distinct
fates by activating domain-specific genes. In the context of
Drosophila
DV patterning, three Dl-dependent thresholds have been
characterized, high, intermediate and low (Fig. 2A), that correspond to
three categories of genes: types I, II and III expressed in distinct
domains (reviewed by Reeves and Stathopoulos, 2009). Type I genes
are expressed in the ventral region of the embryo, where the highest
levels of Dl are established due to Toll signaling. In this region,
presumptive mesodermal target genes including
snail
(
sna
)and
twist
(
twi
) are activated. In the ventrolateral region of the embryo,
intermediate levels of Dl activate type II genes, including
ventral
nervous system defective
(
vnd
)and
vein
(
vn
), which specify the
presumptive neurogenic ectoderm. Type III genes are active in more
dorsal regions and have two modes of expression in response to low
levels of morphogen: those activated by low Dl that are present in
lateral regions, such as
short gastrulation
(
sog
); and those repressed
by low Dl with expression limited to dorsal regions, such as
decapentaplegic
(
dpp
). These subsets of type III genes support
neurogenic ectoderm and dorsal ectoderm fates, respectively
(Fig. 2A). Furthermore, mutant backgrounds with uniformly
high, intermediate or low levels of Dl exhibit broad expression of
either type I, II or III genes, respectively; these genotypes
facilitated the identification of over 50 Dl target genes and
miRNAs using gene expression profiling (Biemar et al., 2006;
Stathopoulos et al., 2002).
One molecular model proposed to explain threshold responses is
that Dl-binding site affinity sets the Dl level required for enhancer
activation (reviewed by Hong et al., 2008b; Reeves and
Stathopoulos, 2009). In general, ventrally expressed genes (i.e.
type I) have enhancers with low-affinity Dl-binding sites, so it
follows that their activation requires high Dl levels. In contrast,
ventrolaterally expressed genes (i.e. type II) generally have
enhancers with high-affinity Dl-binding sites, so their activation is
possible at lower levels of Dl (Fig. 3A). A study of 18 enhancers
active along the DV axis found evidence that affinity of Dl-binding
sites correlates with DV position of gene expression (Papatsenko
A
50
10
1
0.5
90
Anterior
2x Bcd (wt)
6x Bcd (flat)
2x Bcd (flat)
2x Bcd (wt)
bcd
mutant
6x Bcd (flat)
2x Bcd (flat)
hb
Bcd concentration
B
otd
2x Bcd (wt)
6x Bcd (flat)
otd
hb
EL (%)
0.2 0.4
0.6
0.8
1
Dl concentration (A.U)
1
0.5
t
1
t
2
t
3
0
Ventral
Dorsal
Posterior
Type I (
sna
and
twi
)
Type III
(
sog
)
Dl
Type II
(
vnd
)
Fig. 2. Morphogen target genes are activated in
a threshold-dependent manner.
(A) Dl
threshold-dependent gene activation along the
DV axis of
Drosophila
embryos. The graph shows
how three different Dl thresholds (t
1
,t
2
and t
3
)
establish three gene expression domains along
the DV axis. Schematic of an embryo cross-
section showing three gene expression domains
(types I-III) dictated by threshold-dependent
responses to graded nuclear Dl levels (red).
(B) A set of Bcd target genes is controlled by Bcd
in a threshold-dependent manner. In wild-type
embryos with two copies of
bcd
, Bcd exists in a
graded manner along the AP axis [2x BCD (wt),
black line] and also supports
hb
expression (pink)
at the anterior of embryos. In a
bcd
mutant,
hb
expression is lost. When the Bcd gradient is
flattened by assay in specific mutant
backgrounds, resulting in intermediate [6x BCD
(flat), mid-gray line] or low [2x BCD (flat), light-gray
line] levels of Bcd throughout the embryo,
hb
expression expands to the posterior pole region.
Another Bcd-target gene,
otd
(blue), also is
expressed at the anterior in wild-type embryos, but
6x BCD embryos exhibit only posterior expansion
of
otd,
not the ubiquitous expression seen for
hb
(Ochoa-Espinosa et al., 2009). EL, egg length.
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DEVELOPMENT
and Levine, 2005). Since this analysis was conducted, many
additional enhancers active along the DV axis have been
characterized (Ozdemir et al., 2011; Zeitlinger et al., 2007),
including a number of secondary (shadow) enhancers associated
with genes (Hong et al., 2008a), bringing the current enhancer tally
closer to 50. As these additional enhancers have yet to be thoroughly
analyzed with respect to TF binding, it remains to be seen whether
Dl-binding site affinity does indeed correlate with position of target
gene boundaries along the DV axis in general.
Additional evidence in support of the threshold model comes
from genes whose boundaries of expression along the DV axis shift
over time in concert with Dl gradient dynamics (Reeves et al.,
2012). For example,
sna
is expressed in the ventral region of
embryos by nc13, at which point it can be deduced that the Dl
concentration has risen above the threshold required to support its
activation. Subsequently, during early nc14, as Dl levels continue to
increase,
sna
expression dorsally expands, indicating that the level
of Dl necessary to support its expression changes spatially over time
(Reeves et al., 2012). Presumably,
sna
exhibits a real-time response
to dynamic morphogen input.
However, some studies reveal additional complexities that
highlight the limitations of the threshold model alone for
explaining morphogen-dependent gene expression. For example,
twist
(
twi
) is an early Dl target gene that encodes a bHLH TF that
functions in a presumed feed-forward mechanism along with Dl to
support ventral gene expression (Kosman et al., 1991). Many
enhancers associated with genes expressed along the DV axis are
bound by both Dorsal and Twist (Zeitlinger et al., 2007). When Twi is
ectopically expressed at high levels, the Dl threshold responses are
spatially reversed (Stathopoulos and Levine, 2002), suggesting that,
although Dl threshold responses exist, they also likely receive input
from other factors and can be influenced by Twi levels in particular.
Furthermore, as many of the target genes of Dl are co-regulated by
multiple enhancers, the affinity of Dl-binding sites to one enhancer
may not be a good predictor of the role of Dl in the context of multi-
enhancer cis-regulatory systems, as discussed below.
Similar to the DV patterning target genes, a set of genes
expressed along the AP axis directly respond to Bcd in a
concentration-dependent manner (Driever et al., 1989). In
embryos in which the gradient has been flattened using specific
genetic backgrounds that produce uniform Bcd levels across the
embryo, AP target genes
hb
,
Kr
and
giant
(
gt
) are expressed in an
on/off fashion (Fig. 2B) (Ochoa-Espinosa et al., 2009). Like
ubiquitous expression of Dl target genes in mutant backgrounds that
contain one level of Dl (e.g. Stathopoulos et al., 2002), the genes
hb
and
gt
are responsive to specific levels of Bcd and are expressed
broadly in embryos that contain low or intermediate levels of Bcd
throughout, respectively (Ochoa-Espinosa et al., 2009). In contrast,
the genes
orthodenticle
(
otd
),
empty spiracles
(
ems
) and
buttonhead
(
btd
) are not ubiquitously expressed in embryos with flattened Bcd
gradients but instead exhibit shifts only in the positions of their
posterior boundaries of expression (Ochoa-Espinosa et al., 2009)
(Fig. 2B). This result suggests that
otd
,
ems
and
btd
boundaries are
positioned by other mechanisms, not simply in response to Bcd
levels. However, a more recent study has suggested that the
approach of flattening of the Bcd gradient using mutants does not
completely flatten Bcd levels and that a shallow gradient is still
present. This other group, using a transgenic approach to flatten the
Bcd gradient, has found that some target genes (e.g.
btd
) respond
with all or none responses to Bcd levels, whereas others (e.g.
kni
)
exhibit only a shift (Hannon et al., 2017). Taken together, the results
suggest that, similar to Dl target genes, some AP patterning genes
High affinity to Dl
Low affinity to Dl
A Binding-site affinity
Low Bcd levels
High Bcd levels
B Chromatin accessibility
On
On
Dl
High affinity to Dl
Low affinity to Dl
Twi
On
On
C Activator synergy
[ ]
D Dorsal boundaries set by combinatorial inputs
Off
On
Su(H)
90
50
10
EL (%)
Bcd
Run
Concentration
slp1
Wild type
run
mutant
run, cic
mutant
E Posterior boundaries set by combinatorial inputs
Bcd
Key
Fig. 3. Factors influencing threshold outputs.
(A) Affinity of Dl-binding sites
(high affinity, dark purple; low affinity, light purple) on target enhancer
regions dictates Dl threshold levels (red). (B) Regulation of chromatin
accessibility by Bcd (blue). In nuclei with a high Bcd concentration,
chromatin is more accessible compared with the low Bcd concentration
domain. (C) Cooperative i
nputs of Dl and its activator Twi (b
rown rect
angle)
synergistically support target gene expression (shown in b
rown). (D) Dorsal
boundaries of DV patterning genes are correctly set by a repressive
input, e.g. from Su(H) (green triangle), which acts to limit Dl-dependent
activation (Ozdemir et al., 2014). Without repressive Su(H) input, the
boundaries of
sna
(brown) are imprecise. In comparison, combinatorial i
nput
from Dl and Su(H) supports precise
sna
dorsal boundaries with clear on/off
domains of expression. (E) Posterior bo
undaries of AP patterning genes are
controlled by Run (pink). In the
run
mutant,
slp1
expression (blue) slightly
expands posteriorly, and expands even more posteriorly in a
run
and
cic
double mutant. EL, egg length.
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DEVELOPMENT
are activated and positioned in a Bcd concentration-dependent
manner, but others are not; it is certain that additional factors also
play roles during the patterning process along the AP axis.
In contrast to a preliminary analysis of a subset of Dl target
enhancers (Papatsenko and Levine, 2005), an analysis of Bcd target
gene enhancers has found little correlation between the affinity of
binding sites within target gene enhancers and the expression
domains of many AP-patterning genes. This finding does not
support a model in which Bcd threshold response relates simply to
binding-site affinity (Briscoe and Small, 2015; Ochoa-Espinosa
et al., 2005; Segal et al., 2008). Surprisingly, however, chromatin
accessibility of Bcd target genes is sensitive to Bcd concentration
and suggests that gene expression may be threshold dependent, even
if the affinity of specific binding sites does not always follow a
straightforward correlation (Fig. 3B) (Hannon et al., 2017). It is
possible that not only the binding-site affinity, but also the length of
exposure to a morphogen, can influence threshold outputs.
Combinatorial regulation by multiple factors in the establishment of
expression boundaries along the AP and DV axes
While Dl is a pivotal input to target genes expressed in ventral
regions along the DV axis, these genes also receive input from an
early Dl target gene, Twi (Ozdemir et al., 2011; Sandmann et al.,
2007), as noted above. During DV axis patterning, boundaries of
ventral target genes are defined by synergistic interactions between
Dl and Twi (Szymanski and Levine, 1995). For example, a 57 bp
sequence within the
twi
proximal element (
twi_
PE) enhancer,
which is located adjacent to the promoter, has two low-affinity Dl-
binding sites and drives expression within a 12- to 14-cell width
ventral domain (Jiang and Levine, 1993). The dorsal boundaries of
the expression supported by this element are expanded to
encompass a domain of 20 cells in width upon addition of two
Twi-binding sites (E-box sequences) to the
twi
_PE sequence
(Fig. 3C).
In addition to synergistic input by morphogens and other
activators, repressors are also crucial regulators of the spatial
limits of target gene expression. The Dl gradient establishes the
initial expression pattern, but the early domains of expression of Dl
target genes are, in general, broader than the final patterns. Dl target
genes expressed in the presumptive neurogenic ectoderm are
repressed by Sna to specify the ventral boundaries of these stripes
of gene expression (Ip et al., 1992; e.g. Kosman et al., 1991). Sna
repressor is expressed in ventral regions and functions by inhibiting
initiation, not release, of paused RNA Polymerase II (Bothma et al.,
2011). Positioning of the dorsal boundaries of Dl target genes
requires additional repressive inputs, including those from the
maternal proteins Suppressor of Hairless [Su(H)] (Ozdemir et al.,
2014; Schweisguth and Posakony, 1992) and Capicua (Cic) (Ajuria
et al., 2011; Garcia and Stathopoulos, 2011). In the case of
sna
, the
Su(H) TF sets the dorsal boundary through its binding at the
sna
distal enhancer (Fig. 3D). When the Su(H)-binding sites are mutated
in this enhancer, expression is expanded dorsally. Other Dl target
genes (e.g.
sog
) also exhibit dorsally expanded expression in
Su(H)
mutants, suggesting a more general role for this factor (Ozdemir
et al., 2014).
Boundaries of AP patterning genes are also set by combinatorial
input from activators, pioneer factors, as well as repressors. Bcd-Hb
cooperativity is important for activation of a subset of Bcd target
genes (Porcher et al., 2010; Simpson-Brose et al., 1994). In
particular, maternal and zygotic Hb is required for specification of
anterior structures (Simpson-Brose et al., 1994). Alternatively, Zld
pioneer factor has been shown to support activation of enhancers in
response to low concentrations of Bcd in the posterior of embryos
(Xu et al., 2014). Repressors also support AP patterning. For
example, Runt (Run) TF is expressed in the embryonic trunk and
excluded from the poles, and acts as a repressor to limit the posterior
boundaries of particular Bcd-dependent target genes, such as
otd
(Chen et al., 2012; Gergen and Butler, 1988). Similarly, enhancers
of
ems
,
sloppy paired 1
(
slp1
) and
sloppy paired 2
(
slp2
) also exhibit
a posterior shift in the
run
mutant. At these enhancer regions, high-
affinity Run-binding sites are enriched, and their mutation results in
a posterior shift of the expression patterns of the target gene.
Furthermore, posterior shifts become more severe when the
enhancers are expressed in embryos mutant for multiple
repressors, such as Run, Cic and the gap protein Kr (Chen et al.,
2012; Löhr et al., 2009) (Fig. 3E).
Taken together, the studies described in this section expand our
understanding of how morphogens support expression of patterning
genes. Although these transcription factors can activate target gene
enhancers in a concentration-dependent manner, input from
morphogens alone is usually not sufficient to explain many
aspects of target gene expression responses. Input from additional
effectors at enhancers is key, where synergistic activation
contributes to specificity and robustness of target gene response.
Pioneer TFs are required to move nucleosomes so that morphogens
and these other inputs can act. This combinatorial regulation by
multiple factors modifies the concentration-dependent responses to
morphogen gradients and sets the final expression domain of the
target genes. Furthermore, multiple repressors delineate boundaries
of genes expressed along both the DV and AP axes, and these
factors can also influence timing of gene expression. Broadly
expressed repressors Run and Su(H) can also act to influence the
timing of enhancer action, possibly acting as a counterbalance to
pioneer activators, such as Zld (Koromila and Stathopoulos, 2017).
In this way, gradients of morphogens play a crucial role in activating
expression of target genes across fields of cells such as the DV and
AP axes in a concentration-dependent manner, but this regulation
occurs in the context of multiple other nuanced inputs that ensure
precise and robust outputs in space and time.
Morphogen inputs to multiple co-acting enhancers can vary
The
Drosophila
embryo has been instrumental as a model system
for demonstrating that gene expression patterns are supported
through coordinate action of enhancer sequences. Some cis-
regulatory systems are composed of multiple, distinct enhancers
that function in an apparently additive manner to generate gene
expression patterns. For example, five enhancers combine to
support the expression of seven stripes of expression of the gene
even skipped
(
eve
) (Fujioka et al., 1999; Small et al., 1992)
(Fig. 4D). Even though these enhancers support predominantly
different patterns, surprisingly, they also can interact; for example,
deletion of the
eve
stripe 1 enhancer leads to precocious and
expanded expression of
eve
stripe 2 (Lim et al., 2018), possibly due
to sharing of repressor input.
In contrast, it also has become clear that some genes are regulated
by several enhancers that support similar spatiotemporal activity
(Barolo, 2012; Hong et al., 2008a; McGregor et al., 2007). This is
true even in the short period of time before embryos undergo
gastrulation. Owing to the similarity in expression output, the first
enhancer identified at a locus was called the
‘
primary enhancer
’
and
the subsequent enhancers that exhibited similar spatio-temporal
expression were named
‘
shadow enhancers
’
. It was initially thought
that the functions of
Drosophila
shadow enhancers, typically
located at a distance from the promoter, are redundant to counterpart
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Development (2021) 148, dev196113. doi:10.1242/dev.196113
DEVELOPMENT
primary enhancers, as is commonly the case in vertebrates (Xiong
et al., 2002). More recently, it has been suggested that the
Drosophila
shadow enhancers that act to support patterning in
embryos have similar, but not identical, functions compared with
the associated primary enhancers (Dunipace et al., 2011; Dunipace
et al., 2019; El-Sherif and Levine, 2016; Perry et al., 2011).
Contributions from multiple enhancers to a single target gene
complicate the issue of how concentration-dependent input from
morphogens and regulation by other transcription factors translate
into observed gene expression outputs.
Dominant repression of multiple enhancers promotes dynamic shifts
during patterning
During patterning, accumulating evidence suggests that refinement
of the expression domain is predominantly controlled by distal
(
‘
shadow
’
) enhancers (Dunipace et al., 2011; Perry et al., 2011). At
the
sna
locus, the proximal enhancer supports a slightly expanded
domain, whereas the distal enhancer supports a domain similar to
that supported by the full
sna
locus (Dunipace et al., 2011). This is
because the distal enhancer is responsive to input from repressors
(Ozdemir et al., 2014). Furthermore, the distal enhancer limits the
proximal enhancer activity in the region where the gene is repressed.
In this manner, the distal enhancer can dominantly affect the final
expression domain of the gene (Fig. 4A). It is possible that distal
enhancers are associated, more generally, with dominant repression
inputs and this allows genes to be silenced conditionally only when
the enhancer is active.
Non-additive expression output is not only a feature of genes
expressed along the DV axis, but is also a crucial mode of regulation
in genes expressed along the AP axis. For example, the proximal
and distal enhancers at the
hb
locus share similar spatial and
temporal activity in the anterior region in early embryos. However,
unlike the proximal enhancer, the distal enhancer does not support
expression at the anterior pole. At the anterior pole, Torso signaling
represses
hb
expression, and only the distal enhancer is responsive
to input from Torso signaling. To set the
hb
anterior boundary, the
distal enhancer interferes with the proximal enhancer activity,
resulting in
hb
repression at the anterior pole (Fig. 4A) (Perry et al.,
2011). Furthermore, non-additive effects are also observed in the
cis-regulatory systems associated with other genes expressed along
the AP axis, including
kni
and
Kr
(El-Sherif and Levine, 2016). In
this way, coordinate input from multiple enhancers is necessary to
set correct boundaries for a number of AP genes, even in the cases
where individual enhancer outputs appear similar (Barolo and
Levine, 1997; El-Sherif and Levine, 2016; Perry et al., 2011).
In addition, enhancer interactions not only help to refine spatial
patterns, but can also function to modulate levels of gene
expression. For example, some genes, such as
kni, sna
and
Kr
,
require balanced input from two enhancers (Bothma et al., 2015;
Scholes et al., 2019). In the case of
kni
, two enhancers act in an
C Regulation of level: non-additive
Proximal enhancer only
Distal enhancer only
A Regulation of spatial outputs: non-additive
12345 6 7
St 3+7
St 2
St 4+6 St 1+5
Low morphogen levels
Target gene level
High morphogen levels
E
Time
D Regulation of spatial outputs: additive
B Regulation of level: additive
On
Proximal and distal enhancers
Off
Off
f
f
f
Low
High
Intermediate
Intermediate
Low
High
On
On
On
Bcd
Proximal enhancer
Repressor
Distal enhancer
Morphogen concentration and/or
‘X’ timing factors
High morphogen
Low morphogen
hb
Key
kni
sna
Fig. 4. Multiple enhancers coordinate to support precise gene expression outputs.
(A) The proximal (yellow) and distal (gold) enhancers that are bound by
Bcd to drive
hb
gene expression (pink) are active in similar domains, but the distal enhancer expression, specifically, has a repressive input that acts in a
dominant fashion (Perry et al., 2012). (B)
kni
levels (teal) are supported by both proximal and distal enhancers, and each enhancer equally contributes to support
kni
levels (Bothma et al., 2015). (C) Proximal and distal enhancers at the
sna
locus act together to regulate levels of expression (brown) in a non-additive
(or sub-additive) manner (Bothma et al., 2015; Dunipace et al., 2011). This response presumably relates to dominant repression by the proximal enhancer that
acts to limit the distal enhancer activity. (D)
eve
expression (purple shades) is driven by multiple enhancers that support expression in predominantly distinct
domains (reviewed by Borok et al., 2010). St, stripe. (E) Schematic showing how morphogen concentration may differentially control individual enhancer action
over time in response to rising morphogen levels, as associated with Dorsal, and/or through input from timing factors that provide temporal information (
‘
X
’
).
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DEVELOPMENT
additive way; generating higher levels of
kni
expression than is
produced by either enhancer alone (Fig. 4B), whereas the distal and
proximal
sna
enhancers act in a non-additive (sub-additive) manner
to support proper levels of endogenous
sna
gene expression
(Bothma et al., 2015; Dunipace et al., 2011) (Fig. 4C). Although
each
sna
enhancer is responsive to Dl levels during early DV
patterning (Irizarry et al., 2020), these two enhancers support
sna
with different strengths: the distal enhancer drives high expression
levels (
‘
strong enhancer
’
), whereas the proximal enhancer supports
low expression levels (
‘
weak enhancer
’
) (Bothma et al., 2015;
Dunipace et al., 2011). Presumably, frequent interactions between
sna
distal enhancer and the
sna
promoter mediate RNA polymerase
II binding and release at the maximal level, so additional input from
the weak proximal enhancer does not further increase
sna
overall
levels (Bothma et al., 2015). Instead, when the proximal enhancer is
deleted,
sna
levels increase relative to when both enhancers are
present. In contrast, when the distal enhancer is deleted,
sna
levels
decrease in comparison with when both enhancers are present.
Thus, these results suggest that the proximal enhancer attenuates the
activity of the distal enhancer and the action of both enhancers is
required to support correct
sna
levels (Fig. 4C) (Dunipace et al.,
2011). Similar studies at the
Kr
locus have also identified enhancers
that act in a sub-additive way to support
Kr
expression (see Scholes
et al., 2019). However, the molecular mechanism by which the
proximal enhancer limits the distal enhancer activity is unknown.
Perhaps these co-acting enhancers respond to different morphogen
levels or with temporally distinct activities to support dynamic
regulation of gene expression levels (Fig. 4E).
It is clear that the
cis
-regulatory logic inherent in target gene
enhancer sequences serves to interpret morphogen inputs to support
spatial as well as temporal-regulated outputs (Datta et al., 2018;
Koromila et al., 2020; Yuh et al., 1998). Using diverse mechanisms,
co-acting enhancer pairs likely act to support precise and accurate
gene expression (Bentovim et al., 2017), even when the dose of Bcd
and Dl morphogens varies in response to genetic and environmental
conditions.
Conclusions
In summary, by dissecting the role of morphogen inputs directly as
well as the ability of cis-regulatory modules to work in a coordinate
manner, the field is making progress towards understanding the
process of patterning. As highlighted above, significant similarities
exist between the mechanisms of target gene activation mediated by
the two morphogens Bcd and Dl: combinatorial activation, boundary
positioning by repressors and contribution from multiple/shadow
enhancers (Fig. 5B). However, several differences suggest these
morphogens have distinct attributes.
For example, the differential dynamics associated with
morphogens likely impact temporal processing of target gene
threshold responses. Interestingly, Dl and Bcd appear to have
opposite spatiotemporal trends. At the ventral-most region of the
Normalized nuclear Dl intensity
A
0.2
0.4
0.6
0.8
1
20
40
60
80
100
(min)
nc
11
12
13
14
0%
20%
33%
50%
C Space-dependent differences in temporal dynamics
D
V
Dl
Bcd
P
A
B Coordinate system driven by dynamic inputs
D Opposite trends for Dl and
Bcd
DV position
Normalized
nuclear intensity
0
1
50
100
(min)
nc 11 12 13
14
50
100
(min)
nc 11 12 13
14
Normalized
nuclear intensity
1
0
Fig. 5. Morphogen dynamics in time and space.
(A) A representation of
morphogen Dorsal (Dl) nuclear levels at four different positions along the DV
axis from nuclear cycle (nc) 11 to 14. Fluorescence intensity was measured by
monitoring Dl-Venus (Reeves et al., 2012). (B) Nuclei (brown) in different
positions along the DV or AP axes exhibit different levels of Dl (red) and Bcd
(blue) morphogens. (C) Dynamics of morphogen inputs over time may vary
along the respective axes. This is measured for Dl (colored circles along the
DV axis, top, correspond to A) and hypothesized for Bcd, which may relate to
increased protein production over time or to diffusion. (D) Dual input by two
morphogens exhibiting opposite dynamic trends. Nuclei along the DV axis
exhibit progressively smaller changes in levels of nuclear Dl from one nuclear
cycle to the next (see A), whereas it is hypothesized that Bcd exhibits the
opposite trend over time such that a nucleus located more posteriorly may have
a larger relative increase in Bcd levels compared with a more anterior nucleus
(compare graphs).
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DEVELOPMENT
embryo where peak Dl levels are present, a nucleus experiences a
relatively large increase in nuclear levels of Dl morphogen from one
nuclear cycle to the next (e.g. 0% DV position) (Fig. 5A), whereas
nuclei located at a distance along the DV axis perceive a smaller
increase (e.g. 50% DV position) (Fig. 5A). Therefore, along the DV
axis, ventral nuclei (see plot for trace) see the largest change in Dl
exposure over time, and nuclei located along the DV axis experience
a decreasing differential the more dorsal their position in the embryo
(Fig. 5C). By contrast, Bcd exhibits the opposite trend (Fig. 5C).
Nuclei positioned more posteriorly are likely exposed to a relatively
larger differential in Bcd levels during patterning, owing to
increased Bcd protein production over time, as well as to
diffusion of Bcd from the anterior (Fig. 5D). These different
dynamics likely impact target gene threshold responses; for
example, by influencing transcriptional bursting (Sanchez and
Golding, 2013). Despite the variety of cis-regulatory elements
driving gap gene expression downstream of Bcd, these genes exhibit
similar bursting kinetics (Zoller et al., 2018). However, bursting
frequency associated with individual enhancers has been shown to
vary (Fukaya et al., 2016). Whereas the bursting parameters
regulated by Bcd and Dl morphogens remain unclear, the BMP
morphogen gradient has been shown to control burst frequency by
differentially regulating promoter activation rate (Hoppe et al.,
2020). In addition to further study of the influence of Bcd and Dl on
bursting kinetics, recent advances in single-cell sequencing coupled
to ATAC-seq analyses, as well as the use of optogenetic approaches
to inactivate these TFs with temporal precision, should also provide
further insight into Bcd and Dl morphogen responses.
In addition, absolute morphogen levels may not necessarily act
only to regulate the spatial domain of target gene expression. In the
case of Dl at the late blastoderm stage, many target genes maintain
constant expression domains, whereas Dl levels continue to
increase. During this late stage of the patterning process, Dl may
no longer impact patterning of some target genes because other
transcription factors, activators and/or repressors have taken over
this role. A recent study of temporal regulation of
sna
expression
identified that the Dl target Twi can suffice to activate late
expression through one particular enhancer,
sna_Distal,
and serves
as a molecular example of the phenomenon known as hysteresis
(Huang et al., 2017; Irizarry et al., 2020). In various contexts,
hysteresis, a behavior of systems that depends on memory, has been
proposed as a mechanism to support robust patterning under
inherent noisiness during development (Balaskas et al., 2012;
Bollenbach et al., 2008; Manu et al., 2009). It will be of interest to
determine whether these maternal transcription factors play any role
in gene regulation after cellularization, when their nuclear gradients
are still building, but both Dl and Bcd have been shown to be
expendable for the expression of particular target genes (i.e.
sna
and
Kr
, respectively) (Huang et al., 2017; Irizarry et al., 2020).
Furthermore, how scaling of gene expression patterns along body
axes is controlled is not completely understood. Several studies have
focused on how scaling across the AP axis is achieved by deposition
of varying amounts of
bcd
mRNA into eggs that is dependent on
their volume (Cheung et al., 2011). This maternally derived size-
dependent information can propagate in space and time due to the
dynamics of the gene regulatory network (Wu et al., 2015). In DV
patterning, the shape of the Dl nuclear gradient scales with size of
the DV axis but, surprisingly, only a subset of target genes scale
correspondingly (Chahda et al., 2013; Garcia et al., 2013).
Furthermore, how morphogen input affects scaling of target genes
is not completely clear. However, studies of Bcd input to
hb
gene
expression have suggested that Bcd provides input to early-acting
enhancers and, subsequently, other Bcd-independent late-acting
enhancers take over (Liu and Ma, 2013). It has been suggested that
this
‘
hand-off
’
mechanism allows a window of opportunity during
which
hb
gene expression can benefit from early Bcd gradient input
in order to process gradient properties, including information
pertaining to scaling. Whether scaling impacts one or more
enhancers acting in cis-regulatory systems is an interesting issue
that remains to be investigated.
Patterning requires that cells integrate inputs from graded
morphogens as well as from a variety of other transcription factors
present as a result of the extant gene regulatory network. Furthermore,
the complex enhancer properties described above (i.e. enhancer
dominance, long-range interactions and hysteresis/memory) likely
include spatiotemporal control mechanisms present in many
metazoan genes and not just limited to patterning outputs (Katikala
et al., 2013). Nevertheless, patterning by morphogens in early
Drosophila
embryos provides an excellent system with which to
explore how spatiotemporal gene expression is controlled, and
thereby provide insight into developmental gene expression more
broadly. For example, stage-specific changes in responsiveness of
target genes to morphogen levels is also observed in other
Drosophila
developmental processes, as well as in vertebrate systems (Balaskas
et al., 2012; Nahmad and Stathopoulos, 2009). Cells become
desensitized with increased exposure to morphogen input over
time; furthermore, hysteresis allows the cells to maintain proper
identities regardless of fluctuation of morphogen input (Balaskas
et al., 2012; Dessaud et al., 2010). It will be of interest to determine
whether specific enhancer action(s) supports these and other
examples of gene expression dynamics described across
developmental systems.
Acknowledgements
We are grateful to Leslie Dunpiace, Susie Newcomb and Ellen Rothenberg for
comments on the manuscript.
Competing interests
The authors declare no competing or financial interests.
Funding
The authors
’
research is funded by the National Institutes of Health (R21HD095639
and R35GM118146 to A.S.). Deposited in PMC for release after 12 months.
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