B RIEFINGS IN FUNC TIONAL GENOMICS AND P ROTEOMICS . VOL 8. NO 4.

243^249

doi:10.1093/bfgp/elp026

Combinatorial patterning mechanisms

in the Drosophila embryo
Vivek S. Chopra and Mike Levine
Advance Access publication date 3 August 2009

Abstract

Keywords: DV patterning; cis-regulatory elements; Drosophila; enhancers; repressors

FORMATION OF THE DORSAL

GRADIENT
The Dorsal protein is related to mammalian NF-kB,
a critical transcription factor implicated in a variety
of processes including innate immunity, inflammation and B-lymphocyte differentiation [13]. The
Dorsal mRNA is maternally expressed in the developing oocyte, and it is possible that low levels of
the protein are also synthesized within the ovary.
However, an inhibitory protein, Cactus (related
to mammalian IkB), retains Dorsal within the cytoplasm of mature oocytes and early embryos [46].
After fertilization, an extracellular serine protease
cascade leads to the proteolytic processing of proSpaetzle into an active ligand [79]. Pro-Spaetzle
is distributed throughout the perivitelline matrix
surrounding the unfertilized egg and early embryo.

This processing event is probably triggered by the

synthesis of one or more serine proteases [9]. These
enzymes are encoded by maternal mRNAs present
in the unfertilized egg. After fertilization they produce proteins with signal peptides that result in their
secretion into the perivitelline matrix.
Although distributed throughout the extracellular matrix, the resulting serine proteases
(Gastrulation defective, Snake and Easter) are active
only in ventral regions of the matrix, where there
is a presumed modification mark produced
by the prior action of a localized extracellular
heparan sulfotransferase encoded by Pipe [9, 10].
The activation of the serine protease cascade at the
extracellular mark results in the localized cleavage
of Pro-Spaetzle into the active Spaetzle ligand
[7, 1114].

Corresponding author. Mike Levine, Department Molecular & Cell Biology, Division of Genetics, Genomics, and Development,
Center for Integrative Genomics, University of California, Berkeley, CA 94720, USA. Tel: 510-642-5007; Fax: 510-642-9937;
E-mail: mlevine@berkeley.edu
Vivek S. Chopra is the postdoctoral fellow of Department of Molecular and Cell Biology, University of California, Berkeley. His
current research interest is to explore various mechanisms by which cis-regulatory elements control gene regulation during
embryogenesis.
Michael Levine is the professor of Genetics Genomics and Development, Department of Molecular and Cell Biology, University of
California, Berkeley. Research in the Levine lab focuses on studying gene networks that control animal development and disease.
Particularly, the control of segmentation and gastrulation in the early Drosophila embryo, the immune response in Drosophila larvae, and
the differentiation of the notochord and heart in the sea squirt, Ciona intestinalis.
The Author 2009. Published by Oxford University Press. For permissions, please email: journals.permissions@oxfordjournals.org

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The classical concept of the morphogen gradient proposes that small differences in the levels of a signalling molecule
or transcription factor are responsible for producing a continuous spectrum of distinctive cellular identities across
a na| ve field of cells. In this review, we discuss how the Dorsal gradient controls the dorsal-ventral patterning of
the early Drosophila embryo. This gradient extends from the ventral midline of the embryo into dorso-lateral regions,
encompassing a cross-sectional field of approximately 20 cells. There is no evidence that these cells acquire distinctive identities due to subtle changes in the nuclear concentrations of the Dorsal protein. Rather, a variety of evidence
suggests that the Dorsal gradient generates just three primary thresholds of gene activity. High levels activate
gene expression in the presumptive mesoderm, while intermediate and low levels activate gene expression in the
ventral and dorsal neurogenic ectoderm, respectively. We discuss how these primary readouts of the gradient establish localized domains of cell signalling, which work in a combinatorial manner with transcriptional networks to
produce complex patterns of gene expression and tissue differentiation.

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Chopra and Levine

DORSAL GRADIENT
THRESHOLDS
The Dorsal nuclear gradient is first formed 90 min
after fertilization, shortly after cleavage nuclei enter
the cortical regions of the syncitial embryo. The
gradient persists during cellularization, but rapidly
disappears during gastrulation and the initial phases
of germ band elongation. Thus, the Dorsal gradient
is active for <2 h interval during embryogenesis,
but nonetheless plays a decisive role in launching
an elaborate gene regulatory network that governs
dorsalventral (DV) patterning [25, 26].
A combination of genetic methods and wholegenome assays suggests that the Dorsal gradient
directly regulates over 50 different target genes in
the cellularizing embryo [27, 28]. It also indirectly
regulates another 1020 genes engaged in the
patterning of the Dorsal ectoderm in response to a
Dpp signalling gradient. Most of the direct Dorsal
target genes are also regulated by Twist and/or
Snail, which are sequence-specific transcription factors encoded by immediate early target genes of the
Dorsal gradient. Whole-genome ChIP-chip assays
have identified most of the regulatory DNAs that

are controlled by Dorsal along with Twist and/or

Snail [2630].
Approximately 50 direct Dorsal target genes display at least six different patterns of gene expression.
However, there is reason to believe that these six
patterns are produced by just three primary threshold readouts of the Dorsal gradient. High levels of
the gradient activate type 1 target genes such as
twist, snail, heartless and NF-Yc. Intermediate levels
activate type 2 genes in ventral regions of the neurogenic ectoderm, while low levels activate type 3
target genes throughout the presumptive neurogenic
ectoderm. In principle, type 2 and type 3 genes can
be activated by high levels of the Dorsal gradient
within the presumptive mesoderm, but the associated regulatory DNAs contain Snail repressor binding sites that inhibit expression in these regions
[2729, 31] (Figure 1).
The computational analysis of approximately 25
direct Dorsal target enhancers identified just two
classes of Dorsal binding sites: high-affinity sites and
low-affinity sites. Most of the type 1 enhancers, which
mediate gene expression in the presumptive mesoderm containing the highest levels of the Dorsal gradient, contain low-affinity binding sites (Figure 2A).
In contrast, at least a subset of the Dorsal binding sites
contained within type 2 and type 3 enhancers correspond to high-affinity sites (Figure 2C and E). There is
no clear difference in the quality of the Dorsal binding sites contained in the type 2 and type 3 enhancers,
even though the type 3 genes are activated by
lower levels of the Dorsal gradient than type 2
genes (Figure 2CF). That is, the dorsal limits of
the type 3 expression patterns extend at least five
cell diameters beyond the limits of the type 2 expression patterns [25, 3134].
What accounts for the distinctive type 2 and
type 3 expression patterns? Conventional views of
morphogen gradients might predict that the binding
affinities of the Dorsal recognition sequences would
be a key determinant of these different thresholds.
However, as mentioned above, there is no significant
difference in the Dorsal binding affinities of type 2
and type 3 enhancers. Instead, distinct type 2 and
type 3 expression patterns appear to depend on
different combinatorial codes of gene expression.
All of the type 2 enhancers contain linked binding
sites for Dorsal and Twist [25, 31, 33] (Figure 2C).
Twist is a bHLH activator that probably forms
heterodimeric complexes with Daughterless (Da),
a ubiquitous, maternal bHLH activator related to

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During the first 90 min after fertilization, activated

Spaetzle interacts with the transmembrane Toll
receptor distributed throughout the plasma membrane of the early embryo. It is thought that
Spaetzle is distributed in an extracellular gradient,
with peak levels of the active ligand located in ventral regions and lower levels in more lateral regions
[7, 14, 15]. Several models have been suggested to
account for the formation of this gradient, but the
exact details remain elusive.
The localized activation of the Toll receptor leads
to the translocation of the Dorsal protein from the
cytoplasm to the nucleus of syncitial-stage embryos.
The activated receptor leads to the induction of the
cytoplasmic Pelle kinase, which in turn results in the
modification and proteasome-mediated degradation
of the Cactus inhibitor [1620]. This degradation
releases Dorsal from the cytoplasm to the nucleus.
Thus, an extracellular gradient of the Toll ligand
Spaetzle leads to the formation of a Dorsal nuclear
gradient [18, 2123]. There is evidence for a linear
correlation between the levels of activated Toll
receptor and the amount of Dorsal protein that
enters nuclei [18, 24].

Combinatorial patterning mechanisms in Drosophila embryo

mammalian E12/E47 [3537]. Twist (and Twist/Da)

is distributed in a steep and narrow gradient, with a
rapid loss of protein across several cell diameters in
the ventral-most regions of the presumptive neurogenic ectoderm [36, 38, 39]. Linked Dorsal and
Twist binding sites appear to foster cooperative
proteinprotein interactions leading to efficient
occupancy of the sites in the ventral neurogenic
ectoderm, where there are low levels of the Dorsal

Figure 2: Cis-regulatory code for DV patterning.

(A) High levels of Dorsal activate type 1 genes such as
sna and restrict the pattern to presumptive mesoderm.
The type 1 enhancers contain low affinity Dorsal
and/or Twist binding sites. (B) The type 1A enhancers
containing gene sim contain high affinity Dorsal site
along with Su(H) site that depends on Notch signalling.
This is expressed in the mesectoderm regions.
(C) Type 2 enhancers containing genes such as vnd, are
activated by intermediate levels of Dorsal gradient and
low levels of Twist. All the known type 2 enhancers
contain fixed arrangement of Dorsal and Twist binding
sites, i.e. an optimal Dorsal site is closely linked to an
asymmetric Twist site that is in convergent orientation
relative to Dorsal site (black arrow). This cooperative
arrangement of binding sites leads to stable expression
in ventral regions of neurogenic ectoderm. (D) EGF
signalling is induced by intermediate levels of Dorsal
and is important for induction of type 2A gene ind in
the neurogenic ectoderm. The ind enhancer contains
high affinity Dorsal sites and ETS sites that mediate
EGF signalling. Two classes of type 3 enhancers are
activated by low levels of Dorsal gradient (E) activation
and (F) repression. Optimal Dorsal binding sites are
linked to Zelda binding sites within the intronic
enhancer of sog (E) and leads to activation throughout
neurogenic ectoderm. Same low levels of Dorsal also
represses zen and it contains optimal Dorsal binding
sites and an AT-rich motif (type 3A) that binds one
or more ubiquitous factors that converts Dorsal
into a silencer via the recruitment of Groucho
corepressor (F).

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Figure 1: The nuclear Dorsal gradient creates various

thresholds of gene expression. The nuclear gradient of
Dorsal lead to concentration dependent activation of
60 ^70 target genes in the dorsal ^ventral axis of the
early embryo. The expression domains of these genes
are represented in the cross-section through an early
embryo (arranged as dorsal side up and ventral side
down). Blue circles represent different levels of Dorsal
expression in the nuclei from high (filled circle) to intermediate (light blue shades) to no Dorsal (white circles).
At least six different expression patterns are believed
to be generated. High Dorsal in the nucleus activate
type 1 genes such as sna in the presumptive mesoderm.
Type 2 genes like vnd are activated by intermediate
levels of Dorsal gradient in the presumptive neurectoderm. The Type 1A and 2A expression profile shown by
sim and ind depend on Notch and EGF signalling and
high and intermediate Dorsal levels, respectively. The
lowest level of Dorsal gradient generates two type 3
kind of responses as depicted for sog and zen. The
similar low levels of Dorsal that is required for expression of sog represses zen. Thus, nuclear Dorsal gradient
generates three basic transcription responses that
produces a total of six different patterns of gene
expression along the DV axis. This figure was adapted
from review by Stathopoulos and Levine [44].

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Chopra and Levine

CONVERSION OF TYPE 3
ENHANCERS INTO SILENCERS
The preceding arguments account for just three of
the six DV expression patterns seen for Dorsal target
genes. A fourth pattern, localized expression in the
presumptive dorsal ectoderm, employs the same
logic as that described for the type 3 response.
Dorsal target genes like dpp and zen are repressed
by the same low levels of the Dorsal gradient that
activate sog and other type 3 genes. However, the
regulatory DNAs associated with dpp and zen do not
contain linked Dorsal and Zelda binding sites.
Instead, they contain linked binding sites for Dorsal
and an AT-rich sequence motif [45] (Figure 2F).
Several proteins might interact with this motif,
including Cut and Dead Ringer [46, 47], but it is

not clear which of these (or other) factors are critical

for the activities of the dpp and zen regulatory DNAs.
There is evidence that protein(s) bound to the
AT-motif interact with Dorsal at neighboring sites,
and cause a conformational change in the Dorsal
protein so that it interacts with the corepressor
protein, Groucho [48, 49] (Figure 2F). Normally,
Dorsal functions as an activator, but when it interacts
with proteins bound at the AT-motif, it works as
a repressor by recruiting Groucho through exposure
of a cryptic repression peptide, FxLxxIL [45, 50].
In principle, the replacement of Zelda activator
sites with AT-rich repressor elements would convert
type 3 enhancers into type 3 silencers, which restrict
gene expression to the Dorsal ectoderm due to
repression in ventral and lateral regions by high and
low levels of the Dorsal gradient.

DV PATTERNING NETWORK
EXPLAINS THE REMAINING
EXPRESSION PATTERNS
The two remaining expression patterns are exemplified by the regulation of two critical neurogenic
genes, sim and ind. sim encodes a bHLH-PAS transcription factor that controls the differentiation of the
specialized ventral midline of the Drosophila ventral
nerve cord [51]. ind encodes a homeodomain transcription factor that controls the development of
intermediate neurons in lateral regions of the nerve
cord [52].
Sim is expressed in a single line of cells on
both sides of the presumptive mesoderm. The
Dorsal gradient establishes this highly refined expression pattern through the localized deployment of the
Notch signalling pathway. Dorsal (and Twist) activate snail expression in the ventral mesoderm. Snail
represses the expression of Tom, a repressor of neuralized, which encodes a ubiquitin ligase required for
the trafficking of the Notch ligand Delta [53, 54].
As a result of this double negative gate (Snail
represses Tom, which inhibits Notch signalling via
repression of neuralized), the mesoderm triggers
Notch signalling and the activation of sim expression
in the ventral-most cells of the presumptive neurogenic ectoderm [55]. The sim enhancer contains
a series of low-affinity Dorsal binding sites, Twist
sites and Suppressor of Hairless [Su(H)] binding
sites, which mediate Notch signalling [25]. Thus,
the combination of Dorsal, Twist and Notch

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gradient and severely limiting levels of Twist [25, 31,

38, 40, 41]. The extensive reliance of type 2 enhancers on critical Twist binding sites is probably responsible for the restricted expression of type 2 genes in
ventral regions of the neurogenic ectoderm.
In contrast, at least some of the type 3 enhancers,
particularly those associated with the sog locus, contain linked binding sites for Dorsal and a ubiquitous,
maternal zinc-finger transcription factor called Zelda
[42] (Figure 2E). In essence, type 3 enhancers have
Zelda binding sites in place of the Twist sites seen in
type 2 enhancers [43]. It is possible, but currently
unproven, that Dorsal and Zelda cooperatively interact to ensure efficient occupancy of the Dorsal binding sites in Dorsal regions of the presumptive
neurogenic ectoderm where there are diminishing
levels of the Dorsal gradient.
Altogether, the current evidence suggests that
there are just two basic classes of Dorsal recognition
sequences, low-affinity sites and high-affinity sites.
These two classes of binding sites distinguish activation by high versus intermediate or low levels of
the Dorsal gradient. Differential patterns of type 2
and type 3 Dorsal target genes result from distinctive
combinatorial codes. Dorsal Twist define the type
2 response while Dorsal Zelda define the type 3
response. Many of the type 1 enhancers contain both
Dorsal and Twist binding sites. However, expression
is restricted to ventral regions containing peak levels
of the Dorsal gradient since type 1 enhancers contain
low-affinity Dorsal binding sites that are not tightly
linked to Twist sites, as seen in type 2 enhancers
[25, 26, 43, 44].

Combinatorial patterning mechanisms in Drosophila embryo

Key Points
 The nuclear concentrations of Dorsal protein controls the
dorsal ^ventral patterning in the early Drosophila embryo.
 The Dorsal gradient generates three primary thresholds of
gene activity via combinatorial action of various transcription
factors.
 High levels of nuclear Dorsal activate gene expression in the
presumptive mesoderm, intermediate levels activate genes in
ventral neurogenic ectoderm and low levels activate gene
expression in the Dorsal neurogenic ectoderm.
 The primary readouts of the gradient establish localized domains
of cell signalling, which work in a combinatorial manner with
inputs from transcriptional networks to produce complex
patterns of gene expression and tissue differentiation.

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CONCLUSIONS
The Dorsal gradient generates six distinct patterns
of gene expression across the DV axis of the early
Drosophila embryo. The in-depth analysis of a large
collection of DV regulatory DNAs suggests that
Dorsal does not function as a classical morphogen
gradient. It is not sufficient to produce a spectrum
of expression patterns, for example, by interacting
with target enhancers containing different numbers,
arrangements or affinities of Dorsal binding sites.
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manner to direct different patterns of gene expression. Dorsal works with Twist, Zelda, Su(H) and
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